Effects of Culling on Leptospira interrogans Carriage by Rats

Science.gov (United States)

Byers, Kaylee A.; Donovan, Christina M.; Bidulka, Julie J.; Stephen, Craig; Patrick, David M.; Himsworth, Chelsea G.

2018-01-01

We found that lethal, urban rat control is associated with a significant increase in the odds that surviving rats carry Leptospira interrogans. Our results suggest that human interventions have the potential to affect and even increase the prevalence of zoonotic pathogens within rat populations. PMID:29350160

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

NARCIS (Netherlands)

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

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

Science.gov (United States)

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

2016-09-08

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. Copyright © 2016 Amran et al.

Leptospira Interrogans Induces Fibrosis in the Mouse Kidney through Inos-Dependent, TLR- and NLR-Independent Signaling Pathways

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Fanton d'Andon, Martine; Quellard, Nathalie; Fernandez, Béatrice; Ratet, Gwenn; Lacroix-Lamandé, Sonia; Vandewalle, Alain; Boneca, Ivo G.; Goujon, Jean-Michel; Werts, Catherine

2014-01-01

Background Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Rodents carry L. interrogans asymptomatically in their kidneys and excrete bacteria in the urine, contaminating the environment. Humans get infected through skin contact and develop a mild or severe leptospirosis that may lead to renal failure and fibrosis. L. interrogans provoke an interstitial nephritis, but the induction of fibrosis caused by L. interrogans has not been studied in murine models. Innate immune receptors from the TLR and NLR families have recently been shown to play a role in the development and progression of tissue fibrosis in the lung, liver and kidneys under different pathophysiological situations. We recently showed that TLR2, TLR4, and NLRP3 receptors were crucial in the defense against leptospirosis. Moreover, infection of a human cell line with L. interrogans was shown to induce TLR2-dependent production of fibronectin, a component of the extracellular matrix. Therefore, we thought to assess the presence of renal fibrosis in L. interrogans infected mice and to analyze the contribution of some innate immune pathways in this process. Methodology/principal findings Here, we characterized by immunohistochemical studies and quantitative real-time PCR, a model of Leptospira-infected C57BL/6J mice, with chronic carriage of L. interrogans inducing mild renal fibrosis. Using various strains of transgenic mice, we determined that the renal infiltrates of T cells and, unexpectedly, TLR and NLR receptors, are not required to generate Leptospira-induced renal fibrosis. We also show that the iNOS enzyme, known to play a role in Leptospira-induced interstitial nephritis, also plays a role in the induction of renal fibrosis. Conclusion/significance To our knowledge, this work provides the first experimental murine model of sustained renal fibrosis induced by a chronic bacterial infection that may be peculiar, since it does not rely on TLR or NLR receptors

Cloning and Sequencing of Gene Encoding Outer Membrane Lipoprotein LipL41 of Leptospira Interrogans Serovar Grippotyphosa

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M.S. Soltani

2014-12-01

Full Text Available Background: Leptospirosis is an infectious bacterial disease caused by pathogenic serovars of Leptospira. Development of reliable and applicable diagnostic test and also recombinant vaccine for this disease require specific antigens that are highly conserved among diverse pathogenic leptospiral serovars. Outer membrane proteins(OMPs of leptospira are effective antigens which can stimulate remarkable immune responses during infection, among them LipL41 is an immunogenic lipoprotein which is present only in pathogenic serovars so it could be regarded as a good candidate for vaccine development and diagnostic method. In order to identify genetic conservation of the lipL41 gene, we cloned and sequenced this gen from Leptospira interrogans serovar vaccinal and field of Grippotyphosa. Materials and Methods: Leptospira interrogans serovar vaccinal Grippotyphosa (RTCC2808 and serovar field Grippotyphosa (RTCC2825were used to inoculate into the selective culture medium(EMJH. The genomic DNA was extracted by standard phenol-chloroform method. The lipL41 gene were amplified by specific primers and cloned into pTZ57R/T vector and transformed into the competent E. coli (Top10 cells. the extracted recombinant plasmid were sequenced. And the related sequences were subjected to homology analysis by comparing them to sequences in the Genbank database. Results: PCR amplification of the lipL41 gene resulted in the 1065 bp PCR product. DNA sequence analysis revealed that lipL41 gene between serovar vaccinal Grippotyphosa (RTCC2808and serovar field Grippotyphosa (RTCC2825 in Iran was 100%. It was also showed that the lipL41 gene had high identity (96%-100% with other pathogenic serovars submitted in Genbank database. Conclusion: The results of this study showed that the lipL41 gene was highly conserved among various pathogenic Leptospira serovars( >95.9 % identity. Hence the cloned gene could be further used for expression of recombinant protein for serodiagnosis

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

International Nuclear Information System (INIS)

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

2006-01-01

Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP + reductase. Ferredoxin-NADP + 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

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

Energy Technology Data Exchange (ETDEWEB)

Nascimento, Alessandro S.; Ferrarezi, Thiago [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil); Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A. [Facultad de Ciencias Bioquímicas y Farmacéuticas, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario (Argentina); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil)

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.

Characterization of Leptospira interrogans serovar Pomona isolated from swine in Brazil

NARCIS (Netherlands)

Miraglia, Fabiana; Moreno, Luisa Z.; Morais, Zenaide M.; Langoni, Helio; Shimabukuro, Fabio H.; Dellagostin, Odir A.; Hartskeerl, Rudy; Vasconcellos, Silvio A.; Moreno, Andrea Micke

2015-01-01

Leptospira interrogans swine infection is a cause of serious economic loss and a potential human health hazard. In Brazil, the most common serovars associated with swine infections are Pomona, Icterohaemorrhagie and Tarassovi. Cross-reactions among serovars and the failure of infected animals to

Complete genome sequence of Leptospira interrogans serovar Bratislava, strain PigK151

Science.gov (United States)

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 ...

Novel Leptospira interrogans protein Lsa32 is expressed during infection and binds laminin and plasminogen.

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Domingos, Renan F; Fernandes, Luis G; Romero, Eliete C; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2015-04-01

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease of human and veterinary concern. The quest for novel antigens that could mediate host-pathogen interactions is being pursued. Owing to their location, these antigens have the potential to elicit numerous activities, including immune response and adhesion. This study focuses on a hypothetical protein of Leptospira, encoded by the gene LIC11089, and its three derived fragments: the N-terminal, intermediate and C terminus regions. The gene coding for the full-length protein and fragments was cloned and expressed in Escherichia coli BL21(SI) strain by using the expression vector pAE. The recombinant protein and fragments tagged with hexahistidine at the N terminus were purified by metal affinity chromatography. The leptospiral full-length protein, named Lsa32 (leptospiral surface adhesin, 32 kDa), adheres to laminin, with the C terminus region being responsible for this interaction. Lsa32 binds to plasminogen in a dose-dependent fashion, generating plasmin when an activator is provided. Moreover, antibodies present in leptospirosis serum samples were able to recognize Lsa32. Lsa32 is most likely a new surface protein of Leptospira, as revealed by proteinase K susceptibility. Altogether, our data suggest that this multifaceted protein is expressed during infection and may play a role in host-L. interrogans interactions. © 2015 The Authors.

The classification of Sejroe group serovars of Leptospira interrogans with monoclonal antibodies

NARCIS (Netherlands)

Terpstra, W. J.; Korver, H.; van Leeuwen, J.; Klatser, P. R.; Kolk, A. H.

1985-01-01

Using the hybridoma technique we produced monoclonal antibodies to serovars of Leptospira interrogans. We focussed on serovar hardjo which is an important pathogen for humans and animals, and on other serovars of the Sejroe group. With combinations of monoclonals, characteristic patterns of

Estudos estruturais de proteínas de Leptospira interrogans sorovar Copenhageni potencialmente localizadas no envelope celular

OpenAIRE

Priscila Oliveira de Giuseppe

2010-01-01

Resumo: Leptospira interrogans é uma bactéria espiroqueta que causa a leptospirose, uma zoonose de distribuição mundial que afeta mais de 500.000 pessoas anualmente. Pouco se sabe sobre a biologia de leptospiras, o que dificulta a elaboração de novas estratégias de prevenção e de tratamento contra a doença. Cerca de 60 % dos genes de L. interrogans codifica proteínas que não apresentam similaridade de sequência significativa com proteínas de função conhecida. Como a estrutura cristalográfica ...

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

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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.

Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

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Vieira, Monica L; de Morais, Zenaide M; Gonçales, Amane P; Romero, Eliete C; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-01-01

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis. 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Pathogenomic inference of virulence-associated genes in Leptospira interrogans.

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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.

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

Science.gov (United States)

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. Copyright © 2015. Published by Elsevier B.V.

Isolation of Salmonella enterica and serologic reactivity to Leptospira interrogans in opossums (Didelphis virginiana from Yucatán, México Aislamiento de Salmonella enterica y reactividad serológica a Leptospira interrogans en tlacuaches (Didelphis virginiana de Yucatán, México

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Hugo Antonio RUIZ-PIÑA

2002-07-01

Full Text Available 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.La presencia de Salmonella enterica y evidencia serológica de infección por Leptospira interrogans fueron detectadas en tlacuaches de la especie Didelphis virginiana capturados en una localidad semi-urbana del estado de Yucatán, México. Se capturaron 91 marsupiales durante el período de abril de 1996 a mayo de 1998. De un total de 17 muestras de heces, se aislaron cuatro serotipos de Salmonella enterica subsp. enterica (Sandiego, Newport, Anatum y Minnesota y una Salmonella enterica subsp. arizonae serovar O44:Z4,Z23:-. En algunos tlacuaches se registraron infecciones mixtas. De 81 muestras de suero, cuatro (4,9% presentaron reacciones positivas con los serovares pomona y wolffi, ambos pertenecientes al género Leptospira. Los tlacuaches con serología positiva fueron capturados en el hábitat peridomiciliar. Los animales infectados con Salmonella enterica fueron capturados en los períodos de seca y

Leptospira interrogans serotype hardjo in dairy cows

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Vidić Branka M.

2003-01-01

Full Text Available Data on L. hardjo infection of dairy cows in the world pint out its important role in the occurrence of health and economic problem. L. interrogans serotype hardjo has been described as the cause of miscarriages, stillbirts, or the birhs of poorly vital calves, agalactia, mastitis, and low fertility in cows. Two L. hardjo genotypes have been identified in cows, namely, hardjopraitno and hardjobovis. Serological investigations have established a drastic increase in this leptospiral infection in cows. L. hardjo has become adapted to cattle as the primary host, so that an infection is maintained in herds and becomes deeply rooted because of the permanent presence of the source of infection. It was believed that sheep were accidental hosts, but the latest research suggest that they are yet another, transitory, host for maintining this leptospira serotype. L. hardjo is also important from the aspect of human health, especially of persons who are professionally exposed to this infection. L. hardjo infection is detected using serological tests and by proving the presence of leptospira. The medicine of choice in the therapy of leptospiral infections is streptomycin (DSM. Therapy using oxytetracyclines for clinical mastitis was also proven effective. Treatment is most successful in the early stage of the disease. A single dose of streptomycin administered in infected herds reduces the duration period of leptospira excretion through urine, thus preventing the spread of infection thorugh contaminated urine. The basic components of the plan to contain leptospira are the following: serological investigations, sanitary-higiene measures, the elimination of animals which excrete leptospira through urine, therapy, vaccination, quarantine.

Seropositividad a Leptospira interrogans en perros de la ciudad de Rosario, Argentina

OpenAIRE

Seghesso Zabala, Ada; Anthony Omezzolli, Lilian María; Poli Lovagnini, Georgina; Francois Barbagelata, Silvina

2013-01-01

Introducción: la leptospirosis es una de las zoonosis más difundidas mundialmente. La aparición de epidemias urbanas ocasionadas por las inundaciones, la transformaron en un grave problema para la salud pública. Una de las especies animales más afectadas por la leptospirosis es la canina. Los perros se infectan con Leptospira interrogans y padecen la enfermedad, que constituye uno de los factores de riesgo más importante en la transmisión de leptospiras en zonas urbanas. El diagnóstico de cer...

Pathogenomic inference of virulence-associated genes in Leptospira interrogans.

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Jason S Lehmann

Full Text Available 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.

Cloning, high-level expression, purification and crystallization of peptide deformylase from Leptospira interrogans.

Science.gov (United States)

Li, Yikun; Ren, Shuangxi; Gong, Weimin

2002-05-01

A new peptide deformylase (PDF; EC 3.5.1.27) gene from Leptospira interrogans was identified and cloned into expression plasmid pET22b(+) and was highly expressed in Escherichia coli BL21(DE3). With DEAE-Sepharose anion-exchange chromatography followed by Superdex G-75 size-exclusion chromatography, 60 mg of PDF from L. interrogans was purified from 1 l of cell culture. Crystallization screening of the purified enzyme resulted in two crystal forms, from one of which a 3 A resolution X-ray diffraction data set has been collected.

Determination of Leptospira borgpetersenii serovar Javanica and Leptospira interrogans serovar Bataviae as the persistent Leptospira serovars circulating in the urban rat populations in Peninsular Malaysia.

Science.gov (United States)

Benacer, Douadi; Mohd Zain, Siti Nursheena; Sim, Shin Zhu; Mohd Khalid, Mohd Khairul Nizam; Galloway, Renee L; Souris, Marc; Thong, Kwai Lin

2016-03-01

Leptospirosis is an emerging infectious disease of global significance, and is endemic in tropical countries, including Malaysia. Over the last decade, a dramatic increase of human cases was reported; however, information on the primary vector, the rat, and the Leptospira serovars circulating among the rat population is limited. Therefore, the present study was undertaken to isolate Leptospira and characterise the serovars circulating in the urban rat populations from selected main cities in Peninsular Malaysia. Rat trappings were carried out between October 2011 to February 2014 in five urban cities which were chosen as study sites to represent different geographical locations in Peninsular Malaysia. Microscopic agglutination test (MAT) and PCR were carried out to identify the Leptospiral serogroup and determine the pathogenic status of the isolates, respectively while pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD)-PCR were used to characterize the isolates. Three rat species were identified from the three hundred and fifty seven rats captured with Rattus rattus, being the dominant rat species (285, 80 %) followed by Rattus norgevicus (53, 15 %) and Rattus exulans (19, 5 %). Only 39 samples (11.0 %) were positive by culture and further confirmed as pathogenic Leptospira by PCR. Significant associations were shown between host infection with locality, season, host-age and species. Based on MAT, two serogroups were identified in the population namely; L. borgpetersenii serogroup Javanica (n = 16) and L. interrogans serogroup Bataviae (n = 23). Pulsed-field gel electrophoresis (PFGE) distinguished the two serovars in the urban rat populations: L. borgpetersenii serovar Javanica (41 %), and L. interrogans serovar Bataviae (59 %). RAPD-PCR yielded 14 distinct patterns and was found to be more discriminative than PFGE. This study confirms two Leptospira serovars circulating among the urban rats population in Peninsular

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

NARCIS (Netherlands)

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

2013-01-01

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

Imunodiagnóstico da leptospirose humana através do teste ELISA-IgM, empregando-se diferentes preparações antigênicas a partir de sorotipos prevalentes de Leptospira interrogans Immunodiagnostic of human leptospirosis by ELISA-IgM, employing: different antigenic preparations as from prevalent serovars of Leptospira interrogans

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Marcos Vinicius da Silva

1990-08-01

Full Text Available Realizou-se estudo comparativo de diferentes sorotipos de Leptospira interrogans utilizados na preparação de antígenos empregados no teste ELISA, para a detecção de anticorpos da classe IgM, em amostras de soro na fase precoce e tardia da leptospirose humana. Foram utilizados dez sorotipos, escolhidos entre os que apresentaram maior reatividade na soroaglutinação microscópica (SAM, na cidade de São Paulo. Os cinco sorotipos que apresentaram melhores resultados individualmente no teste ELISA-IgM (canicola, hebdomadis, icterohaemorrhagiae, cynopteri e brasiliensis, foram também estudados em mistura antigênica. Os antígenos não tratados apresentaram maior reatividade do que os antígenos tratados com Triton X - 100 (4% à temperatura de 50ºC, durante 4 horas. O teste ELISA-IgM empregando os sorotipos não tratados, isoladamente, e em mistura antigênica, mostrou-se altamente sensível, podendo ser empregado como teste de triagem para o diagnóstico precoce da leptospirose humana. Outra aplicação do teste é permitir a detecção do início de situações epidêmicas ou de surtos, possibilitando acionar medidas de vigilância epidemiológica.A comparative study among different serovars of Leptospira interrogans was performed in order to prepare antigens to detect IgM antibodies by ELISA in early and late phase of human leptospirosis. Ten serovars were chosen among the most prevalent detected by microscopic seroagglutination (SAM in São Paulo city. Using ELISA-IgM five of them showed better results (canicola, hebdomadis, icterohaemorrhagiae, cynopteri and brasiliensis. These ones were also studied in a pool. The non-treated antigens showed higher reactivity than the Triton X-100 (4%/50ºC/4h. ELISA-IgM using individually or pool of non treated antigens proved to be reliable with high sensitivity and should be used for an earlier diagnosis of leptospirosis, as a trial test. Faster diagnostic elucidation can be useful to detect

Characterization of leptospiral proteins that afford partial protection in hamsters against lethal challenge with Leptospira interrogans.

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Atzingen, Marina V; Gonçales, Amane P; de Morais, Zenaide M; Araújo, Eduardo R; De Brito, Thales; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-09-01

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of Leptospira interrogans serovar Copenhageni together with bioinformatic tools allow us to search for novel antigen candidates suitable for improved vaccines against leptospirosis. This study focused on three genes encoding conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from six predominant pathogenic serovars in Brazil. The genes were cloned and expressed in Escherichia coli strain BL21-SI using the expression vector pDEST17. The recombinant proteins tagged with N-terminal 6xHis were purified by metal-charged chromatography. The proteins were recognized by antibodies present in sera from hamsters that were experimentally infected. Immunization of hamsters followed by challenge with a lethal dose of a virulent strain of Leptospira showed that the recombinant protein rLIC12730 afforded statistically significant protection to animals (44 %), followed by rLIC10494 (40 %) and rLIC12922 (30 %). Immunization with these proteins produced an increase in antibody titres during subsequent boosters, suggesting the involvement of a T-helper 2 response. Although more studies are needed, these data suggest that rLIC12730 and rLIC10494 are promising candidates for a multivalent vaccine for the prevention of leptospirosis.

Use of saprophytic leptospira strains in the serodiagnosis of experimental leptospirosis in guinea-pigs (Cavia sp

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Raul J. S. Girio

1988-04-01

Full Text Available The efficiency of four Leptospira biflexa strains (Buenos Aires, Patoc 1, Rufino and São Paulo as single antigen in the serodiagnosis in guinea-pigs experimentally infected with seven Leptospira interrogans serovars (canicola, grippotyphosa, hardjo, icterohaemorrhagiae, pomona, tarassovi and wolffi was evaluated by the microscopic agglutination test. The four saprophytic strains were not able to reveal antibody titres in sera of guinea-pigs experimentally infected with Leptospira interrogans. Serological cross-reactions were observed between strains Patoc 1 and São Paulo and between serovars wolffi and hardjo.

Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

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Boonsathorn, Naphatsawan; Konghom, Ganokrot; Mongkolsiri, Kaveewan; Jirapongwattana, Chanin; Balachandra, Kruavon; Naigowit, Pimjai; Sawanpanyalert, Pathom

2009-01-01

Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

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

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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 ...

MHC class II DRB diversity predicts antigen recognition and is associated with disease severity in California sea lions naturally infected with Leptospira interrogans

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Acevedo-Whitehouse, Karina; Gulland, Frances; Bowen, Lizabeth

2018-01-01

We examined the associations between California sea lion MHC class II DRB (Zaca-DRB) configuration and diversity, and leptospirosis. As Zaca-DRB gene sequences are involved with antigen presentation of bacteria and other extracellular pathogens, we predicted that they would play a role in determining responses to these pathogenic spirochaetes. Specifically, we investigated whether Zaca-DRB diversity (number of genes) and configuration (presence of specific genes) explained differences in disease severity, and whether higher levels of Zaca-DRB diversity predicted the number of specific Leptospira interrogans serovars that a sea lion's serum would react against. We found that serum from diseased sea lions with more Zaca-DRB loci reacted against a wider array of serovars. Specific Zaca-DRB loci were linked to reactions with particular serovars. Interestingly, sea lions with clinical manifestation of leptospirosis that had higher numbers of Zaca-DRB loci were less likely to recover from disease than those with lower diversity, and those that harboured Zaca-DRB.C or –G were 4.5 to 5.3 times more likely to die from leptospirosis, regardless of the infective serovars. We propose that for leptospirosis, a disadvantage of having a wider range of antigen presentation might be increased disease severity due to immunopathology. Ours is the first study to examine the importance of Zaca-DRB diversity for antigen detection and disease severity following natural exposure to infective leptospires.

Leptospira interrogans serovar copenhageni harbors two lexA genes involved in SOS response.

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Luciane S Fonseca

Full Text Available Bacteria activate a regulatory network in response to the challenges imposed by DNA damage to genetic material, known as the SOS response. This system is regulated by the RecA recombinase and by the transcriptional repressor lexA. Leptospira interrogans is a pathogen capable of surviving in the environment for weeks, being exposed to a great variety of stress agents and yet retaining its ability to infect the host. This study aims to investigate the behavior of L. interrogans serovar Copenhageni after the stress induced by DNA damage. We show that L. interrogans serovar Copenhageni genome contains two genes encoding putative LexA proteins (lexA1 and lexA2 one of them being potentially acquired by lateral gene transfer. Both genes are induced after DNA damage, but the steady state levels of both LexA proteins drop, probably due to auto-proteolytic activity triggered in this condition. In addition, seven other genes were up-regulated following UV-C irradiation, recA, recN, dinP, and four genes encoding hypothetical proteins. This set of genes is potentially regulated by LexA1, as it showed binding to their promoter regions. All these regions contain degenerated sequences in relation to the previously described SOS box, TTTGN 5CAAA. On the other hand, LexA2 was able to bind to the palindrome TTGTAN10TACAA, found in its own promoter region, but not in the others. Therefore, the L. interrogans serovar Copenhageni SOS regulon may be even more complex, as a result of LexA1 and LexA2 binding to divergent motifs. New possibilities for DNA damage response in Leptospira are expected, with potential influence in other biological responses such as virulence.

Vaccination with leptospiral outer membrane lipoprotein LipL32 reduces kidney invasion of Leptospira interrogans serovar canicola in hamsters.

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Humphryes, P C; Weeks, M E; AbuOun, M; Thomson, G; Núñez, A; Coldham, N G

2014-04-01

The Leptospira interrogans vaccines currently available are serovar specific and require regular booster immunizations to maintain protection of the host. In addition, a hamster challenge batch potency test is necessary to evaluate these vaccines prior to market release, requiring the use of a large number of animals, which is ethically and financially undesirable. Our previous work showed that the N terminus of the outer membrane protein LipL32 was altered in Leptospira interrogans serovar Canicola vaccines that fail the hamster challenge test, suggesting that it may be involved in the protective immune response. The aim of this study was to determine if vaccination with LipL32 protein alone could provide a protective response against challenge with L. interrogans serovar Canicola to hamsters. Recombinant LipL32, purified from an Escherichia coli expression system, was assessed for protective immunity in five groups of hamsters (n = 5) following a challenge with the virulent L. interrogans serovar Canicola strain Kito as a challenge strain. However, no significant survival against the L. interrogans serovar Canicola challenge was observed compared to that of unvaccinated negative controls. Subsequent histological analysis revealed reduced amounts of L. interrogans in the kidneys from the hamsters vaccinated with recombinant LipL32 protein prior to challenge; however, no significant survival against the L. interrogans serovar Canicola challenge was observed compared to that of unvaccinated negative controls. This finding corresponded to a noticeably reduced severity of renal lesions. This study provides evidence that LipL32 is involved in the protective response against L. interrogans serovar Canicola in hamsters and is the first reported link to LipL32-induced protection against kidney invasion.

Development of Transcriptional Fusions to Assess Leptospira interrogans Promoter Activity

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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

Development of transcriptional fusions to assess Leptospira interrogans promoter activity.

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Gustavo M Cerqueira

Full Text Available 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 Sphingomyelinase 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.

[Reconstruction of Leptospira interrogans lipL21 gene and characteristics of its expression product].

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Luo, Dong-jiao; Hu, Ye; Dennin, R H; Yan, Jie

2007-09-01

To reconstruct the nucleotide sequence of Leptospira interrogans lipL21 gene for increasing the output of prokaryotic expression and to understand the changes on immunogenicity of the expression products before and after reconstruction, and to determine the position of envelope lipoprotein LipL21 on the surface of leptospiral body. According to the preferred codons of E.coli, the nucleotide sequence of lipL21 gene was designed and synthesized, and then its prokaryotic expression system was constructed. By using SDS-PAGE plus BioRad agarose image analysor, the expression level changes of lipL21 genes before and after reconstruction were measured. A Western blot assay using rabbit anti-TR/Patoc I serum as the first antibody was performed to identify the immunoreactivity of the two target recombinant proteins rLipL21s before and after reconstruction. The changes of cross agglutination titers of antisera against two rLipL21s before and after reconstruction to the different leptospiral serogroups were demonstrated using microscope agglutination test (MAT). Immuno-electronmicroscopy was applied to confirm the location of LipL21s. The expression outputs of original and reconstructed lipL21 genes were 8.5 % and 46.5 % of the total bacterial proteins, respectively. Both the two rLipL21s could take place immune conjugation reaction with TR/Patoc I antiserum. After immunization with each of the two rLipL21s in rabbits, the animals could produce specific antibody. Similar MAT titers with 1:80 - 1:320 of the two antisera against rLipL21s were present. LipL21 was confirmed to locate on the surface of leptospiral envelope. LipL21 is a superficial antigen of Leptospira interrogans. The expression output of the reconstructed lipL21 gene is remarkably increased. The expression rLipL21 maintains fine antigenicity and immunoreactivity and its antibody still shows an extensive cross immunoagglutination activity. The high expression of the reconstructed lipL21 gene will offer a

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection.

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Gómez, Ricardo M; Vieira, Monica L; Schattner, Mirta; Malaver, Elisa; Watanabe, Monica M; Barbosa, Angela S; Abreu, Patricia A E; de Morais, Zenaide M; Cifuente, Javier O; Atzingen, Marina V; Oliveira, Tatiane R; Vasconcellos, Silvio A; Nascimento, Ana L T O

2008-01-01

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L. interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L. interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira.

Molecular identification of the ompL1 gene within Leptospira interrogans standard serovars.

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Dezhbord, Mehrangiz; Esmaelizad, Majid; Khaki, Pejvak; Fotohi, Fariba; Zarehparvar Moghaddam, Athena

2014-06-11

Leptospirosis, caused by infection with pathogenic Leptospira species, is one of the most prevalent zoonotic diseases in the world. Current leptospiral vaccines are mainly multivalent dead whole-cell mixtures made of several local dominant serovars. Therefore, design and construction of an efficient recombinant vaccine for leptospirosis control is very important. OmpL1 is an immunogenic porin protein that could be of special significance in vaccination and serodiagnosis for leptospirosis. Three strains belonging to pathogenic L. interrogans were analyzed. The specific primers for proliferation of the ompL1 gene were designed. The amplified gene was cloned. In order to investigate the ompL1 nucleotide sequence and homological analysis of this gene, ompL1 genes cloned from standard vaccinal Leptospira serovars prevalent in Iran were sequenced and cloned. PCR amplification of the ompL1 gene using the designed primers resulted in a 963 bp ompL1 gene product. The PCR based on the ompL1 gene detected all pathogenic reference serovars of Leptospira spp. tested. Based on alignment and phylogenetic analysis, although the ompL1 nucleotide sequence was slightly different within three vaccinal serovars (100%-85% identity), amino acid alignment of the OmpL1 proteins revealed that there would be inconsiderable difference among them. The ompL1 gene of the three isolates was well conserved, differing only by a total of 6 bp and the proteins by 2 amino acids. The cloned gene could be further used for expression and recombinant OmpL1 as an efficient and conserved antigen, and may be a useful vaccine candidate against leptospirosis in our region.

Interleukin 12 in part regulates gamma interferon release in human whole blood stimulated with Leptospira interrogans

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de Fost, Maaike; Hartskeerl, Rudy A.; Groenendijk, Martijn R.; van der Poll, Tom

2003-01-01

Heat-killed pathogenic Leptospira interrogans serovar rachmati induced the production of gamma interferon (IFN-gamma) and the IFN-gamma-inducing cytokines interleukin-12p40 (IL-12p40) and tumor necrosis factor alpha in human whole blood in vitro. The production of IFN-gamma was largely dependent on

[Sequences and expression pattern of mce gene in Leptospira interrogans of different serogroups].

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Zhang, Lei; Xue, Feng; Yan, Jie; Mao, Ya-fei; Li, Li-wei

2008-11-01

To determine the frequency of mce gene in Leptospira interrogans, and to investigate the gene transcription levels of L. interrogans before and after infecting cells. The segments of entire mce genes from 13 L.interrogans strains and 1 L.biflexa strain were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of mce gene was constructed; the expression and output of the target recombinant protein rMce were examined by SDS-PAGE and Western Blot assay. Rabbits were intradermally immunized with rMce to prepare the antiserum, the titer of antiserum was measured by immunodiffusion test. The transcription levels of mce gene in L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 before and after infecting J774A.1 cells were monitored by real-time fluorescence quantitative RT-PCR. mce gene was carried in all tested L.interrogans strains, but not in L.biflexa serogroup Semaranga serovar patoc strain Patoc I. The similarities of nucleotide and putative amino acid sequences of the cloned mce genes to the reported sequences (GenBank accession No: NP712236) were 99.02%-100% and 97.91%-100%, respectively. The constructed prokaryotic expression system of mce gene expressed rMce and the output of rMce was about 5% of the total bacterial proteins. The antiserum against whole cell of L.interrogans strain 56601 efficiently recognized rMce. After infecting J774A.1 cells, transcription levels of the mce gene in L.interrogans strain 56601 were remarkably up-regulated. The constructed prokaryotic expression system of mce gene and the prepared antiserum against rMce provide useful tools for further study of the gene function.

Pesquisa de leptospiras e de anticorpos contra leptospiras em animais e humanos de propriedades rurais nos biomas brasileiros Pantanal e Caatinga

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Felipe Jorge da Silva

2015-09-01

Full Text Available The occurrence of Leptospira and of seroreactivity against Leptospira was investigated in animals and humans from six farms located in two Brazilian biomes that have different geoclimatic conditions: Pantanal – municipalities of Miranda (MS, Itiquira (MT and Pocone (MT and Caatinga – municipalities of Sobradinho (BA, Garanhuns (PE and Sobral (BA. Blood and urine samples of wildlife, domestic animals and humans were collected at each property. The samples were collected from February to April 2012 in Caatinga and from July to September 2012 in Pantanal. The serological reactivity against Leptospira spp. was verified by microscopic agglutination technique (MAT made with a collection consisting by 24 antigens of Leptospira spp. The leptospires research was carried out by urine samples crop sown in Fletcher resources and Ellinghausen – McCullough – Johnson – Harris (EMJH. Crops with growth of leptospires were referred to the Leptospirosis Laboratory of the Institute of Pathobiology, National Institute of Agricultural Technology, Buenos Aires, Argentina and isolated Leptospira strains were genotyped with the technique of Multiple Locus Variable Number Tandem Repeat Analysis (MLVA. The classification procedure employed the VNTR 4, 7, 9, 10, 19, 23, 31, LB4 and LB5, which discriminate strains of L. interrogans and L. borgpetersenii. In Pantanal, 17 wildlife, 65 domestic animals and two humans were examined. In Caatinga, seven wild animals were examined, along with 100 domestic animals and 26 humans. Of 84 blood samples tested in Pantanal, 47 (55.95% were positive and, of 133 in Caatinga, 59 (44.36% were reactant. By Fisher’s exact test, considering a 0.05 significance level, there was no difference between the proportions of serum reagent animals against Leptospira spp. in two biome reviews (p = 0.063. The predominant serovars in SAM reactions were: 1 Pantanal – Bratislava (wildlife, dogs and humans, Grippotyphosa (horses and cattle; 2

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

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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...

Comparative analysis of lipopolysaccharides of pathogenic and intermediately pathogenic Leptospira species.

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Patra, Kailash P; Choudhury, Biswa; Matthias, Michael M; Baga, Sheyenne; Bandyopadhya, Keya; Vinetz, Joseph M

2015-10-30

Lipopolysaccharides (LPS) are complex, amphipathic biomolecules that constitute the major surface component of Gram-negative bacteria. Leptospira, unlike other human-pathogenic spirochetes, produce LPS, which is fundamental to the taxonomy of the genus, involved in host-adaption and also the target of diagnostic antibodies. Despite its significance, little is known of Leptospira LPS composition and carbohydrate structure among different serovars. LPS from Leptospira interrogans serovar Copenhageni strain L1-130, a pathogenic species, and L. licerasiae serovar Varillal strain VAR 010, an intermediately pathogenic species, were studied. LPS prepared from aqueous and phenol phases were analyzed separately. L. interrogans serovar Copenhageni has additional sugars not found in L. licerasiae serovar Varillal, including fucose (2.7%), a high amount of GlcNAc (12.3%), and two different types of dideoxy HexNAc. SDS-PAGE indicated that L. interrogans serovar Copenhageni LPS had a far higher molecular weight and complexity than that of L. licerasiae serovar Varillal. Chemical composition showed that L. interrogans serovar Copenhageni LPS has an extended O-antigenic polysaccharide consisting of sugars, not present in L. licerasiae serovar Varillal. Arabinose, xylose, mannose, galactose and L-glycero-D-mannoheptose were detected in both the species. Fatty acid analysis by gas chromatography-mass spectrometry (GC-MS) showed the presence of hydroxypalmitate (3-OH-C16:0) only in L. interrogans serovar Copenhageni. Negative staining electron microscopic examination of LPS showed different filamentous morphologies in L. interrogans serovar Copenhageni vs. L. licerasiae serovar Varillal. This comparative biochemical analysis of pathogenic and intermediately pathogenic Leptospira LPS reveals important carbohydrate and lipid differences that underlie future work in understanding the mechanisms of host-adaptation, pathogenicity and vaccine development in leptospirosis.

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

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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. Copyright © 2014 Elsevier Ltd. All rights reserved.

Crystal structure of homoserine O-acetyltransferase from Leptospira interrogans

International Nuclear Information System (INIS)

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

2007-01-01

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 α/β domain containing the catalytic site and a helical bundle called the lid domain. Overall, the structure fold belongs to α/β 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

A LigA three-domain region protects hamsters from lethal infection by Leptospira interrogans.

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Mariana L Coutinho

2011-12-01

Full Text Available The leptospiral LigA protein consists of 13 bacterial immunoglobulin-like (Big domains and is the only purified recombinant subunit vaccine that has been demonstrated to protect against lethal challenge by a clinical isolate of Leptospira interrogans in the hamster model of leptospirosis. We determined the minimum number and location of LigA domains required for immunoprotection. Immunization with domains 11 and 12 was found to be required but insufficient for protection. Inclusion of a third domain, either 10 or 13, was required for 100% survival after intraperitoneal challenge with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. As in previous studies, survivors had renal colonization; here, we quantitated the leptospiral burden by qPCR to be 1.2×10(3 to 8×10(5 copies of leptospiral DNA per microgram of kidney DNA. Although renal histopathology in survivors revealed tubulointerstitial changes indicating an inflammatory response to the infection, blood chemistry analysis indicated that renal function was normal. These studies define the Big domains of LigA that account for its vaccine efficacy and highlight the need for additional strategies to achieve sterilizing immunity to protect the mammalian host from leptospiral infection and its consequences.

Expression and characterization of an iron-regulated hemin-binding protein, HbpA, from Leptospira interrogans serovar Lai.

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Asuthkar, Swapna; Velineni, Sridhar; Stadlmann, Johannes; Altmann, Friedrich; Sritharan, Manjula

2007-09-01

In an earlier study, based on the ferric enterobactin receptor FepA of Escherichia coli, we identified and modeled a TonB-dependent outer membrane receptor protein (LB191) from the genome of Leptospira interrogans serovar Lai. Based on in silico analysis, we hypothesized that this protein was an iron-dependent hemin-binding protein. In this study, we provide experimental evidence to prove that this protein, termed HbpA (hemin-binding protein A), is indeed an iron-regulated hemin-binding protein. We cloned and expressed the full-length 81-kDa recombinant rHbpA protein and a truncated 55-kDa protein from L. interrogans serovar Lai, both of which bind hemin-agarose. Assay of hemin-associated peroxidase activity and spectrofluorimetric analysis provided confirmatory evidence of hemin binding by HbpA. Immunofluorescence studies by confocal microscopy and the microscopic agglutination test demonstrated the surface localization and the iron-regulated expression of HbpA in L. interrogans. Southern blot analysis confirmed our earlier observation that the hbpA gene was present only in some of the pathogenic serovars and was absent in Leptospira biflexa. Hemin-agarose affinity studies showed another hemin-binding protein with a molecular mass of approximately 44 kDa, whose expression was independent of iron levels. This protein was seen in several serovars, including nonpathogenic L. biflexa. Sequence analysis and immunoreactivity with specific antibodies showed this protein to be LipL41.

Ecology of Leptospira interrogans in Norway rats (Rattus norvegicus in an inner-city neighborhood of Vancouver, Canada.

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Chelsea G Himsworth

Full Text Available Leptospira interrogans is a bacterial zoonosis with a worldwide distribution for which rats (Rattus spp. are the primary reservoir in urban settings. In order to assess, monitor, and mitigate the risk to humans, it is important to understand the ecology of this pathogen in rats. The objective of this study was to characterize the ecology of L. interrogans in Norway rats (Rattus norvegicus in an impoverished inner-city neighborhood of Vancouver, Canada.Trapping was performed in 43 city blocks, and one location within the adjacent port, over a 12 month period. Kidney samples were tested for the presence of L. interrogans using PCR and sequencing. A multivariable model was built to predict L. interrogans infection status in individual rats using season and morphometric data (e.g., weight, sex, maturity, condition, etc. as independent variables. Spatial analysis was undertaken to identify clusters of high and low L. interrogans prevalence. The prevalence of L. interrogans varied remarkably among blocks (0-66.7%, and spatial clusters of both high and low L. interrogans prevalence were identified. In the final cluster-controlled model, characteristics associated with L. interrogans-infection in rats included weight (OR = 1.14, 95% CI = 1.07-1.20, increased internal fat (OR = 2.12, 95% CI = 1.06-4.25, and number of bite wounds (OR = 1.20, 95% CI = 0.96-1.49.Because L. interrogans prevalence varied with weight, body fat, and bite wounds, this study suggests that social structure and interactions among rats may influence transmission. The prevalence and distribution of L. interrogans in rats was also highly variable even over a short geographic distance. These factors should be considered in future risk management efforts.

Frequency of exposure of endangered Caspian seals to Canine distemper virus, Leptospira interrogans, and Toxoplasma gondii.

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Somayeh Namroodi

Full Text Available Canine distemper virus (CDV, Leptospira interrogans, and Toxoplasma gondii are potentially lethal pathogens associated with decline in marine mammal populations. The Caspian Sea is home for the endangered Caspian seal (Pusa caspica. In the late 1990s and early 2000s, CDV caused a series of mortality events involving at least several thousand Caspian seals. To assess current infection status in Caspian seals, we surveyed for antibodies to three pathogens with potential to cause mortality in marine mammals. During 2015-2017, we tested serum samples from 36, apparently healthy, Caspian seals, accidentally caught in fishing nets in the Caspian Sea off Northern Iran, for antibodies to CDV, L. interrogans, and T. gondii, by virus neutralization, microscopic agglutination, and modified agglutination, respectively. Twelve (33%, 6 (17%, and 30 (83% samples were positive for CDV, L. interrogans and T. gondii antibodies, respectively. The highest titers of CDV, L. interrogans, and T. gondii antibodies were 16, 400, and 50, respectively. Frequencies of antibody to these pathogens were higher in seals >1 year old compared to seals <1 year old. Two serovars of L. interrogans (Pomona and Canicola were detected. Our results suggest a need for additional studies to clarify the impact of these pathogens on Caspian seal population decline and the improvement of management programs, including systematic screening to detect and protect the remaining population from disease outbreaks.

Frequency of exposure of endangered Caspian seals to Canine distemper virus, Leptospira interrogans, and Toxoplasma gondii.

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Namroodi, Somayeh; Shirazi, Amir S; Khaleghi, Seyyed Reza; N Mills, James; Kheirabady, Vahid

2018-01-01

Canine distemper virus (CDV), Leptospira interrogans, and Toxoplasma gondii are potentially lethal pathogens associated with decline in marine mammal populations. The Caspian Sea is home for the endangered Caspian seal (Pusa caspica). In the late 1990s and early 2000s, CDV caused a series of mortality events involving at least several thousand Caspian seals. To assess current infection status in Caspian seals, we surveyed for antibodies to three pathogens with potential to cause mortality in marine mammals. During 2015-2017, we tested serum samples from 36, apparently healthy, Caspian seals, accidentally caught in fishing nets in the Caspian Sea off Northern Iran, for antibodies to CDV, L. interrogans, and T. gondii, by virus neutralization, microscopic agglutination, and modified agglutination, respectively. Twelve (33%), 6 (17%), and 30 (83%) samples were positive for CDV, L. interrogans and T. gondii antibodies, respectively. The highest titers of CDV, L. interrogans, and T. gondii antibodies were 16, 400, and 50, respectively. Frequencies of antibody to these pathogens were higher in seals >1 year old compared to seals <1 year old. Two serovars of L. interrogans (Pomona and Canicola) were detected. Our results suggest a need for additional studies to clarify the impact of these pathogens on Caspian seal population decline and the improvement of management programs, including systematic screening to detect and protect the remaining population from disease outbreaks.

Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells

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Sato, Hiromi

2017-01-01

Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways

Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells.

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Sato, Hiromi; Coburn, Jenifer

2017-07-01

Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways

Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells.

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Hiromi Sato

2017-07-01

Full Text Available Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1 extracellular matrix, 2 intercellular adhesion molecules and cell surface receptors, 3 intracellular proteins, 4 cell-cell junction proteins, and 5 a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or

The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment

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Hu, Wei-Lin; Pappas, Christopher J.; Zhang, Jun-Jie; Yang, You-Yun; Yan, Jie

2016-01-01

ABSTRACT Leptospira interrogans is the agent of leptospirosis, a reemerging zoonotic disease. It is transmitted to humans through environmental surface waters contaminated by the urine of mammals chronically infected by pathogenic strains able to survive in water for long periods. Little is known about the regulatory pathways underlying environmental sensing and host adaptation of L. interrogans during its enzootic cycle. This study identifies the EbpA-RpoN regulatory pathway in L. interrogans. In this pathway, EbpA, a σ54 activator and putative prokaryotic enhancer-binding protein (EBP), and the alternative sigma factor RpoN (σ54) control expression of at least three genes, encoding AmtB (an ammonium transport protein) and two proteins of unknown function. Electrophoresis mobility shift assay demonstrated that recombinant RpoN and EbpA bind to the promoter region and upstream of these three identified genes, respectively. Genetic disruption of ebpA in L. interrogans serovar Manilae virtually abolished expression of the three genes, including amtB in two independent ebpA mutants. Complementation of the ebpA mutant restored expression of these genes. Intraperitoneal inoculation of gerbils with the ebpA mutant did not affect mortality. However, the ebpA mutant had decreased cell length in vitro and had a significantly lowered cell density at stationary phase when grown with l-alanine as the sole nitrogen source. Furthermore, the ebpA mutant has dramatically reduced long-term survival ability in water. Together, these studies identify a regulatory pathway, the EbpA-RpoN pathway, that plays an important role in the zoonotic cycle of L. interrogans. IMPORTANCE Leptospirosis is a reemerging disease with global importance. However, our understanding of gene regulation of the spirochetal pathogen Leptospira interrogans is still in its infancy, largely due to the lack of robust tools for genetic manipulation of this spirochete. Little is known about how the pathogen

The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment.

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Hu, Wei-Lin; Pappas, Christopher J; Zhang, Jun-Jie; Yang, You-Yun; Yan, Jie; Picardeau, Mathieu; Yang, X Frank

2017-02-01

Leptospira interrogans is the agent of leptospirosis, a reemerging zoonotic disease. It is transmitted to humans through environmental surface waters contaminated by the urine of mammals chronically infected by pathogenic strains able to survive in water for long periods. Little is known about the regulatory pathways underlying environmental sensing and host adaptation of L. interrogans during its enzootic cycle. This study identifies the EbpA-RpoN regulatory pathway in L. interrogans In this pathway, EbpA, a σ 54 activator and putative prokaryotic enhancer-binding protein (EBP), and the alternative sigma factor RpoN (σ 54 ) control expression of at least three genes, encoding AmtB (an ammonium transport protein) and two proteins of unknown function. Electrophoresis mobility shift assay demonstrated that recombinant RpoN and EbpA bind to the promoter region and upstream of these three identified genes, respectively. Genetic disruption of ebpA in L. interrogans serovar Manilae virtually abolished expression of the three genes, including amtB in two independent ebpA mutants. Complementation of the ebpA mutant restored expression of these genes. Intraperitoneal inoculation of gerbils with the ebpA mutant did not affect mortality. However, the ebpA mutant had decreased cell length in vitro and had a significantly lowered cell density at stationary phase when grown with l-alanine as the sole nitrogen source. Furthermore, the ebpA mutant has dramatically reduced long-term survival ability in water. Together, these studies identify a regulatory pathway, the EbpA-RpoN pathway, that plays an important role in the zoonotic cycle of L. interrogans IMPORTANCE: Leptospirosis is a reemerging disease with global importance. However, our understanding of gene regulation of the spirochetal pathogen Leptospira interrogans is still in its infancy, largely due to the lack of robust tools for genetic manipulation of this spirochete. Little is known about how the pathogen achieves its

Chemogenomics profiling of drug targets of peptidoglycan biosynthesis pathway in Leptospira interrogans by virtual screening approaches.

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Bhattacharjee, Biplab; Simon, Rose Mary; Gangadharaiah, Chaithra; Karunakar, Prashantha

2013-06-28

Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. The availability of ligand libraries has facilitated the search for novel drug targets using chemogenomics approaches, compared with the traditional method of drug discovery, which is time consuming and yields few leads with little intracellular information for guiding target selection. Recent subtractive genomics studies have revealed the putative drug targets in peptidoglycan biosynthesis pathways in Leptospira interrogans. Aligand library for the murD ligase enzyme in the peptidoglycan pathway has also been identified. Our approach in this research involves screening of the pre-existing ligand library of murD with related protein family members in the putative drug target assembly in the peptidoglycan biosynthesis pathway. A chemogenomics approach has been implemented here, which involves screening of known ligands of a protein family having analogous domain architecture for identification of leads for existing druggable protein family members. By means of this approach, one murC and one murF inhibitor were identified, providing a platform for developing an antileptospirosis drug targeting the peptidoglycan biosynthesis pathway. Given that the peptidoglycan biosynthesis pathway is exclusive to bacteria, the in silico identified mur ligase inhibitors are expected to be broad-spectrum Gram-negative inhibitors if synthesized and tested in in vitro and in vivo assays.

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.

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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

2016-03-09

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.

A model system for studying the transcriptomic and physiological changes associated with mammalian host-adaptation by Leptospira interrogans serovar Copenhageni.

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Melissa J Caimano

2014-03-01

Full Text Available 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

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

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Li, Xu; Huang, Hua [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Song, Xiaomin [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Wang, Yanli; Xu, Hang [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Teng, Maikun [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Gong, Weimin, E-mail: wgong@sun5.ibp.ac.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

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.

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

International Nuclear Information System (INIS)

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

2006-01-01

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 M is calculated to be 2.4 Å 3 Da −1 , assuming there to be 12 protein molecules in the asymmetric unit

Comparative genomics of pathogenic Leptospira interrogans serovar Canicola isolated from swine and human in Brazil

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Luisa Z Moreno

Full Text Available Leptospira interrogans serovar Canicola is one of the most important pathogenic serovars for the maintenance of urban leptospirosis. Even though it is considered highly adapted to dogs, serovar Canicola infection has already been described in other animals and even a few human cases. Here, we present the genomic characterisation of two Brazilian L. interrogans serovar Canicola strains isolated from slaughtered sows (L0-3 and L0-4 and their comparison with human strain Fiocruz LV133. It was observed that the porcine serovar Canicola strains present the genetic machinery to cause human infection and, therefore, represent a higher risk to public health. Both human and porcine serovar Canicola isolates also presented sequences with high identity to the Chinese serovar Canicola published plasmids pGui1 and pGui2. The plasmids identification in the Brazilian and Chinese serovar Canicola strains suggest that extra-chromosomal elements are one more feature of this serovar that was previously unnoticed.

A putative regulatory genetic locus modulates virulence in the pathogen Leptospira interrogans.

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Eshghi, Azad; Becam, Jérôme; Lambert, Ambroise; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Wunder, Elsio A; Ko, Albert I; Coppee, Jean-Yves; Goarant, Cyrille; Picardeau, Mathieu

2014-06-01

Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139(-) mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.

Radiometric method for the rapid detection of Leptospira organisms

International Nuclear Information System (INIS)

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

1986-01-01

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 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

Radiometric method for the rapid detection of Leptospira organisms

Energy Technology Data Exchange (ETDEWEB)

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.

Seropositivity to Leptospira interrogans serovar Bratislava associated to reproductive problems without significant biochemical or hematological alterations in horses Soropositividade para Leptospira interrogans serovar Bratislava associada a falhas reprodutivas sem alterações hematológicas e bioquímicas significativas em cavalos

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Melissa Pinna

2010-10-01

Full Text Available The objective was to study haematological and biochemical alterations associated to seropositivity to Leptospira interrogans serovar Bratislava infection in horses with reproductive alterations, such as neonatal deaths, embryonic deaths and abortions. A flock of mares with poor reproductive performance was studied. Eighty-two (58.6% were seropositive (titre 200; 72 of those (87.8% for Bratislava. Slight haematological and biochemical alterations were observed, being more frequent (PO objetivo deste trabalho foi estudar alterações hematológicas e bioquímicas associadas à soropositividade para Leptospira interrogans sorovar Bratislava em cavalos com alterações reprodutivas, tais como mortes neonatais, absorção embrionária e abortamentos. Um rebanho de éguas com baixos índices reprodutivos foi estudado. Oitenta e duas (58,6% foram soropositivas (títulos 200, sendo 72 destas (87,8% para Bratislava. Foram observadas poucas alterações hematológicas e bioquímicas, mais frequentes (P<0,05 em éguas soropositivas do que soronegativas. Cavalos soropositivos para Bratislava não tinham alterações graves nos valores hematológicos e bioquímicos. Esses achados reforçam que esse sorovar seja adaptado de cavalos e cause apenas sintomas brandos, associados a falhas reprodutivas.

Effect of Leptospira interrogans outer membrane proteins LipL32 on HUVEC.

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Sun, Zhan; Bao, Lang; Li, DaoKun; Huang, Bi; Wu, Bingting

2010-09-01

Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)-LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx-LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions. Copyright 2010 Elsevier Ltd. All rights reserved.

Isolation and characterization of Leptospira interrogans from pigs slaughtered in São Paulo State, Brazil Isolamento e caracterização de Leptospira interrogans de suínos abatidos no Estado de São Paulo, Brasil

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Fabiana Miraglia

2008-09-01

Full Text Available 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.Amostras de soro sanguíneo, rim, fígado e trato genital de 137 fêmeas suínas (40 matrizes e 97 marrãs e de urina de 22 matrizes foram colhidas em abatedouro no Estado de São Paulo, no período de abril de 2003 a agosto de 2004 tendo como objetivo o isolamento de Leptospira spp. Quatro estirpes foram isoladas de animais que apresentaram títulos, no teste de soroaglutinação microscópica (SAM > 100, para o sorovar Pomona e de um animal, não reagente na

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

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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.

ChpK and MazF of the toxin-antitoxin modules are involved in the virulence of Leptospira interrogans during infection.

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Komi, Komi Koukoura; Ge, Yu-Mei; Xin, Xiao-Yang; Ojcius, David M; Sun, Dexter; Hu, Wei-Lin; Zhao, Xin; Lin, Xu'ai; Yan, Jie

2015-01-01

Pathogenic Leptospira species are the causative agents of leptospirosis, a global zoonotic infectious disease. Toxin-antitoxin (TA) modules have been confirmed as stress-response elements that induce prokaryotic and eukaryotic cell-growth arrest or death, but their role in the virulence of Leptospira has not been reported. Here, we confirmed that all the tested leptospiral strains had the chpIK and mazEF TA modules with highly-conserved sequences. The transcription and expression of the chpI, chpK, mazE, and mazF genes of Leptospira interrogans strain Lai were significantly increased during infection of phorbol 12-myristate 13-acetate-induced human THP-1 macrophages. The toxic ChpK and MazF but not the antitoxic ChpI and MazE proteins were detectable in the cytoplasmic fraction of leptospire-infected THP-1 cells, indicating the external secretion of ChpK and MazF during infection. Transfection of the chpK or mazF gene caused decreased viability and necrosis in THP-1 cells, whereas the chpI or mazE gene transfection did not affect the viability of THP-1 cells but blocked the ChpK or MazF-induced toxicity. Deletion of the chpK or mazF gene also decreased the late-apoptotic and/or necrotic ratios of THP-1 cells at the late stages of infection. The recombinant protein MazF (rMazF) cleaved the RNAs but not the DNAs from Leptospira and THP-1 cells, and this RNA cleavage was blocked by rMazE. However, the rChpK had no RNA or DNA-degrading activity. All these findings indicate that the ChpK and MazF proteins in TA modules are involved in the virulence of L. interrogans during infection. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

[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].

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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.

Whole genome analysis of Leptospira licerasiae provides insight into leptospiral evolution and pathogenicity.

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Jessica N Ricaldi

Full Text Available The whole genome analysis of two strains of the first intermediately pathogenic leptospiral species to be sequenced (Leptospira licerasiae strains VAR010 and MMD0835 provides insight into their pathogenic potential and deepens our understanding of leptospiral evolution. Comparative analysis of eight leptospiral genomes shows the existence of a core leptospiral genome comprising 1547 genes and 452 conserved genes restricted to infectious species (including L. licerasiae that are likely to be pathogenicity-related. Comparisons of the functional content of the genomes suggests that L. licerasiae retains several proteins related to nitrogen, amino acid and carbohydrate metabolism which might help to explain why these Leptospira grow well in artificial media compared with pathogenic species. L. licerasiae strains VAR010(T and MMD0835 possess two prophage elements. While one element is circular and shares homology with LE1 of L. biflexa, the second is cryptic and homologous to a previously identified but unnamed region in L. interrogans serovars Copenhageni and Lai. We also report a unique O-antigen locus in L. licerasiae comprised of a 6-gene cluster that is unexpectedly short compared with L. interrogans in which analogous regions may include >90 such genes. Sequence homology searches suggest that these genes were acquired by lateral gene transfer (LGT. Furthermore, seven putative genomic islands ranging in size from 5 to 36 kb are present also suggestive of antecedent LGT. How Leptospira become naturally competent remains to be determined, but considering the phylogenetic origins of the genes comprising the O-antigen cluster and other putative laterally transferred genes, L. licerasiae must be able to exchange genetic material with non-invasive environmental bacteria. The data presented here demonstrate that L. licerasiae is genetically more closely related to pathogenic than to saprophytic Leptospira and provide insight into the genomic bases for

Alterações espermáticas e dos níveis plasmáticos de testosterona em cães experimentalmente infectados por Leptospira interrogans sorovar Canicola

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Santana, Lucas Alves de Souza [UNESP

2008-01-01

Conhecendo-se a predileção das leptospiras pelo aparelho urogenital, e a crescente utilização de técnicas de reprodução assistida na espécie canina, o presente trabalho objetivou pesquisar a presença e a ação da Leptospira no sêmen e testículo de cães. Foram utilizados 32 animais, dos quais 20 foram inoculados com uma cepa patogênica de Leptospira interrogans sorovar Canicola e 12 não receberam inóculo algum, sendo considerados animais-controle. Assim, os 32 animais experimentais foram reunid...

Leptospira diversity in animals and humans in Tahiti, French Polynesia.

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Guernier, Vanina; Richard, Vaea; Nhan, Tuxuan; Rouault, Eline; Tessier, Anita; Musso, Didier

2017-06-01

Leptospirosis is a highly endemic bacterial zoonosis in French Polynesia (FP). Nevertheless, data on the epidemiology of leptospirosis in FP are scarce. We conducted molecular studies on Leptospira isolated from humans and the potential main animal reservoirs in order to identify the most likely sources for human infection. Wild rats (n = 113), farm pigs (n = 181) and domestic dogs (n = 4) were screened for Leptospira infection in Tahiti, the most populated island in FP. Positive samples were genotyped and compared to Leptospira isolated from human cases throughout FP (n = 51), using secY, 16S and LipL32 sequencing, and MLST analysis. Leptospira DNA was detected in 20.4% of rats and 26.5% of pigs. We identified two Leptospira species and three sequence types (STs) in animals and humans: Leptospira interrogans ST140 in pigs only and L. interrogans ST17 and Leptospira borgpetersenii ST149 in humans and rats. Overall, L. interrogans was the dominant species and grouped into four clades: one clade including a human case only, two clades including human cases and dogs, and one clade including human cases and rats. All except one pig sample showed a unique L. interrogans (secY) genotype distinct from those isolated from humans, rats and dogs. Moreover, LipL32 sequencing allowed the detection of an additional Leptospira genotype in pigs, clearly distinct from the previous ones. Our data confirm rats as a major potential source for human leptospirosis in FP. By contrast to what was expected, farm pigs did not seem to be a major reservoir for the Leptospira genotypes identified in human patients. Thus, further investigations will be required to determine their significance in leptospirosis transmission in FP.

Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

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Oliveira, Rosane; de Morais, Zenaide Maria; Gonçales, Amane Paldes; Romero, Eliete Caló; Vasconcellos, Silvio Arruda; Nascimento, Ana L T O

2011-01-01

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D) of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

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Rosane Oliveira

Full Text Available Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30 and LIC12238. We have employed Escherichia coli BL21 (SI strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D, 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively. In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa. Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

Heterologous Expression, Purification and Characterization of an Oligopeptidase A from the Pathogen Leptospira interrogans.

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Anu, Prasannan V; Madanan, Madathiparambil G; Nair, Ananthakrishnan J; Nair, Gangaprasad A; Nair, Govinda Pillai M; Sudhakaran, Perumana R; Satheeshkumar, Padikara K

2018-04-01

Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn 2+ at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified.

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

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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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

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Lilian Terezinha de Queiroz Leite

1996-10-01

Full Text Available Monoclonal antibodies (MABs ivere produced against an etbylenediaminetetraacetate (EDTA extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b. The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.Anticorpos monoclonais (AcM foram produzidos contra o extrato EDTA obtido de Leptospira interrogans, sorovar icterohaemorrhagiae. Pelo teste de precipitação foram caracterizados como IgM e IgG (IgGl e IgG2. A eletroforese em gel de poliacrilamida do extrato EDTA revelou diversas bandas quando corada pela prata. No "Western blot", as bandas em torno de 20 kDa reagiram com o AcM 47B4D6, foram oxidadas pelo periodato e não digeridas pela pronase, sugerindo que o determinante é de natureza carboidrato. O determinante reconhecido pelo AcM 47B4D6 estã localizado sob o envelope externo como revelado pela imunocitoquímica usando marcação com ouro coloidal. O AcM contra extrato EDTA do sorovar icterohaemorrahagiae não protegeu hamsters quando inoculados com lepstopira homóloga virulenta.

Identification and Characterization of c-di-GMP Metabolic Enzymes of Leptospira interrogans and c-di-GMP Fluctuations After Thermal Shift and Infection

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Guohui Xiao

2018-04-01

Full Text Available Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira species. The most common species, Leptospira interrogans, can transfer from contaminated soil or water to the human body. It is able to survive these changing environments through sensing and responding to the changes of environmental cues. Cyclic di-GMP (c-di-GMP is a special secondary messenger in bacteria, which can respond to the environment and regulate diverse bacterial behaviors. The c-di-GMP levels in bacterial cells are regulated by diguanylatecyclases (DGC and phosphodiesterases (PDE, which are responsible for synthesizing or hydrolyzing c-di-GMP, respectively. In this study, distribution and phylogenetics of c-di-GMP metabolic genes among 15 leptospiral species were systematically analyzed. Bioinformatics analysis revealed that leptospiral species contain a multitude of c-di-GMP metabolic genes. C-di-GMP metabolic genes in L. interrogans strain Lai 56601 were further analyzed and the results showed that these genes have very diverse expression patterns. Most of the putative DGCs and PDEs possess enzymatic activities, as determined by riboswitch-based dual-fluorescence reporters in vivo or HPLC in vitro. Furtherer analysis of subdomains from GGDEF-containing proteins revealed that the ability to synthesize c-di-GMP was lost when the GAF domain from LA1483 and PAS domain from LA2932 were deleted, while deletion of the REC domain from LA2528 did not affect its ability to synthesize c-di-GMP. Furthermore, high temperatures generally resulted in low c-di-GMP concentrations in L. interrogans and most of the c-di-GMP metabolic genes exhibited differential temperature regulation. Also, infection of murine J774A.1 cells resulted in reduced c-di-GMP levels, while no significant change of c-di-GMP metabolic genes on transcriptional levels were observed during the infection of J774A.1 cells. Taken together, these results provide a basic platform for future studies of c

Three case studies involving Leptospira interrogans serovar pomona infection in mixed farming units : case report

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B. Gummow

1999-07-01

Full Text Available Three case studies involving Leptospira interrogans serovar pomona outbreaks within mixed farming systems in South Africa are described. On 2 farms, pigs constituted the main enterprise with cattle and sheep of secondary importance. On each of these 2 farms, abortion due to L. pomona in sows was confirmed by culture, and antibody titres to pomona were detected in cattle, sheep, horses and dogs. On the 3rd farm, a piggery was ofsecondary importance to cattle farming. Abortion and death in cows occurred on this farmand serology showed titres to various serovars, including pomona. L. pomona was also isolated from bovine urine, an aborted bovine foetus and kidneys from slaughtered pigs. This particular case study was regarded as clinically atypical in that adult Jersey cattle died of acute leptospirosis in a semiarid region of South Africa. In all 3 case studies, the poor management of pig effluent and of the drinking water and its sources played a pivotal role in the transmission of the disease. Inadequate vaccination of animals against Leptospira and poor record-keeping within the secondary farming enterprises were also contributing factors to the spread of leptospirosis.

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

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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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

[Prokaryotic expression of trigeminy artificial fusion gene of Leptospira interrogans and the immunogenicity of its products].

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Luo, Dong-jiao; Qiu, Xiao-feng; Wang, Jiang; Yan, Jin; Wang, Hai-bin; Zhou, Jin-cheng; Yan, Jie

2008-11-01

To construct lipL32/1-lipL21-OmpL1/2 fusion gene of Leptospira interrogans and its prokaryotic expression system, and to identify the immunogenicity of its products. PCR using linking primers was applied to construct lipL32/1-lipL21-OmpL1/2 fusion gene and a prokaryotic expression system of the fusion gene was then established using routine genetic engineering technique. SDS-PAGE was used to examine output of the target recombinant protein rLipL32/1-LipL21-OmpL1/2. Double immunodiffusion and Western Blot assay were applied to identify immunogenicity of rLipL32/1-LipL21-OmpL1/2. lipL32/1-lipL21-OmpL1/2 fusion gene with correct sequence and its prokaryotic expression system E.coli BL21DE3pET42a-lipL32/1-lipL21-ompL1/2 was obtained in this study. The output of rLipL32/1-LipL21- OmpL1/2 after optimisation was 37.78 mg/L. The immunodiffusion titer of rabbit antiserum against rLipL32/1-LipL21-OmpL1/2 was 1:4. The rLipL32/1-LipL21-OmpL1/2 antiserum was able to recognize rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 and rOmpL1/2. Positive Western hybridization signals were found among rLipL32/1-LipL21-OmpL1/2 and rabbit antiserum against whole cell of strain 56601 and serum from patients infected with L.interrogans serogroups Icterohaemorrhagiae, Grippotyphosa, Autumnalis and Pomona. The fusion gene lipL32/1-lipL21-OmpL1/2 and its prokaryotic expression system were successfully constructed in this study. The expressed fusion protein can be used as the antigen for developing universal genetic engineering vaccine and universal serological tests of leptospirosis.

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

International Nuclear Information System (INIS)

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

1988-01-01

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

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

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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.

Kinetics of Leptospira interrogans infection in hamsters after intradermal and subcutaneous challenge.

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Mariana L Coutinho

2014-11-01

Full Text Available 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.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.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.

Characteristic features of intracellular pathogenic Leptospira in infected murine macrophages.

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Toma, Claudia; Okura, Nobuhiko; Takayama, Chitoshi; Suzuki, Toshihiko

2011-11-01

Leptospira interrogans is a spirochaete responsible for a zoonotic disease known as leptospirosis. Leptospires are able to penetrate the abraded skin and mucous membranes and rapidly disseminate to target organs such as the liver, lungs and kidneys. How this pathogen escape from innate immune cells and spread to target organs remains poorly understood. In this paper, the intracellular trafficking undertaken by non-pathogenic Leptospira biflexa and pathogenic L. interrogans in mouse bone marrow-derived macrophages was compared. The delayed in the clearance of L. interrogans was observed. Furthermore, the acquisition of lysosomal markers by L. interrogans-containing phagosomes lagged behind that of L. biflexa-containing phagosomes, and although bone marrow-derived macrophages could degrade L. biflexa as well as L. interrogans, a population of L. interrogans was able to survive and replicate. Intact leptospires were found within vacuoles at 24 h post infection, suggesting that bacterial replication occurs within a membrane-bound compartment. In contrast, L. biflexa were completely degraded at 24 h post infection. Furthermore, L. interrogans but not L. biflexa, were released to the extracellular milieu. These results suggest that pathogenic leptospires are able to survive, replicate and exit from mouse macrophages, enabling their eventual spread to target organs. © 2011 Blackwell Publishing Ltd.

MASTITE E SÍNDROME DA QUEDA DO LEITE / INFECÇÃO POR Leptospira interrogans EM OVELHAS DA RAÇA SANTA INÊS NO DISTRITO FEDERAL

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Adriana Helena Rosa

2012-06-01

Full Text Available The aim of the present work was to determine the relationship between clinical mastitis and Leptospira interrogans / Milk Drop Syndrome in Santa Inês ewes. One thousand sheep were examined on 12 farms in the Distrito Federal, Brazil, as to their clinical condition, in order to identify animals with clinical mastitis. The animals were divided into two groups: animals which presented clinical mastitis symptoms (G1 and animals which did not have clinical mastitis symptoms (G2. Blood samples were collected from all ewes of the two groups. The microscopic agglutination test was performed in order to identify the seropositive animals to Leptospira spp. Four (4.08% animals of the first group were seropositives to Leptospira spp., of which three were positives to Hardjoprajitno (Norma and Hardjoprajitno (OMS sorovars and one to both Australis and Autumnalis sorovars. In the second group, two (5.26% animals were seropositives to both Hardjoprajitno (Norma and Hardjoprajitno (OMS sorovars. A relationship between Leptospira spp. infection / milk drop syndrome and the presence of clinical mastitis in ewes (p > 0.05 was not observed.

Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

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Lilian Terezinha de Queiroz Leite

1996-10-01

Full Text Available Monoclonal antibodies (MABs ivere produced against an etbylenediaminetetraacetate (EDTA extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b. The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.

Leptospira interrogans en una población canina del Gran Buenos Aires: variables asociadas con la seropositividad Leptospira interrogans in a canine population of Greater Buenos Aires: variables associated with seropositivity

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Diana Rubel

1997-08-01

Full Text Available Se determinó la seroprevalencia de leptospirosis en una población canina suburbana con el objeto de analizar la asociación entre distintas variables individuales y ambientales y la seropositividad a leptospirosis. El estudio, de diseño transversal, se llevó a cabo durante julio de 1992 en un barrio del Gran Buenos Aires en el que viven unos 9 500 habitantes y una población canina de unos 2 000 animales. Se estudió una muestra aleatoria de 223 perros, de cada uno de los cuales se obtuvo una muestra de sangre. La ficha epidemiológica del animal se obtuvo por encuesta al ama de casa. Las determinaciones serológicas se realizaron por microaglutinación frente a 10 serotipos de Leptospira interrogans. Se halló seropositividad en 57% de los 223 perros examinados; 82% de los sueros positivos coaglutinaron con dos o más serotipos. Los serotipos detectados con mayor frecuencia fueron canicola y pyrogenes. La seroprevalencia en hembras fue menor que en machos (P We determined the seroprevalence of leptospirosis in a suburban canine population for the purpose of analyzing the association between different individual and environmental variables and seropositivity for leptospirosis. The study, which was cross-sectional, was performed in July 1992 in a neighborhood of Greater Buenos Aires with approximately 9 500 inhabitants and a canine population of around 2 000 animals. We studied a random sample of 223 dogs and obtained a blood sample from each. Each animal’s epidemiologic history was obtained by interviewing the housewife. Serologic measurements were performed by the microagglutination technique with the use of 10 different serotypes of Leptospira interrogans. Of the 223 dogs that were tested, 57% showed seropositivity; 82% of the positive sera coagglutinated with two or more serotypes. The most frequently detected serotypes were canicola and pyrogenes. Seroprevalence in females was less common than in males (P <0,05 and in puppies less than

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

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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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

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Ram, S.

2012-01-01

Full Text Available Aim: Isolation, dark field detection and microscopic agglutination test (MAT are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR - based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septicemia. Methodology and Results: Two hundred and seven (207 blood samples from patients were diagnosed with septicemia which includes 100 bacterial (other than Leptospira culture positive and 107 bacterial culture negative samples were studied. Primers for Nested PCR targeting LipL32 gene of Leptospira interrogans were designed and the specificity of primers was tested against serum samples positive/negative by either MAT or dark field microscopy. PCR amplified products were further confirmed by DNA sequencing. The standardized nPCR was sensitive and specific to Leptospira interrogans. Twenty-one (21% out of 100 culture positive blood samples, three (2.8% out of 107 culture negative samples showed nPCR positivity and were confirmed as Leptospira interrogans by DNA sequencing (p<0.001. A sensitive nPCR specific to Leptospira interrogans was developed. Conclusion, significance and impact of study: The p value (<0.001 signifies that Leptospira is commonly associated with other bacteria circulating in blood indicating that a decreased immune status is created primarily by a bacterium with enhanced possibility of development of Leptospiral infection probably be of an endogenous origin.

LipL53, a temperature regulated protein from Leptospira interrogans that binds to extracellular matrix molecules.

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Oliveira, Tatiane R; Longhi, Mariana T; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-03-01

The regulation of gene expression by environmental signals, such as temperature and osmolarity, has been correlated with virulence. In this study, we characterize the protein LipL53 from Leptospira interrogans, previously shown to react with serum sample of individual diagnosed with leptospirosis and to be up-regulated by shift to physiological osmolarity. The recombinant protein was expressed in Escherichia coli system, in insoluble form, recovered by urea solubilization and further refolded by decreasing the denaturing agent concentration during the purification procedure. The secondary structure content of the recombinant LipL53, as assessed by circular dichroism, showed a mixture of beta-strands and alpha-helix. The presence of LipL53 transcript at 28 degrees C was only detected within the virulent strains. However, upon shifted of attenuated cultures of pathogenic strains from 28 degrees C to 37 degrees C and to 39 degrees C, this transcript could also be observed. LipL53 binds laminin, collagen IV, cellular and plasma fibronectin in dose-dependent and saturable manner. Animal challenge studies showed that LipL53, although immunogenic, elicited only partial protection in hamsters. LipL53 is probably surface exposed as seen through immunofluorescence confocal microscopy. Our results suggest that LipL53 is a novel temperature regulated adhesin of L. interrogans that may be relevant in the leptospiral pathogenesis. Copyright 2009 Elsevier Masson SAS. All rights reserved.

Proteína LIC10494 de Leptospira interrogans serovar Copenhageni: modelo estructural y regiones funcionales asociadas

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Orlando Emilio Acevedo

2012-04-01

Full Text Available Protein LIC10494 of Leptospira interrogans serovar Copenhageni: structural model and associated functional regions. Objective.Predict by computational means the 3D structure of the antigenic protein LIC10494 and report associated important functional regionsfor its pathogenicity and immunogenicity. Materials and methods. We performed a computational analysis of the primary structure ofLIC10494 using the servers BLAST, PROTPARAM, PROTSCALE, DAS, SOSUI, TOPPRED, TMAP, TMpred, SPLIT4, PHDHTM,TMHMM2, HMMTOP2, GLOBPLOT and PROSITE. The secondary structure was obtained by consensus of the algorithms SOPM,PREDATOR GOR4, DPM and DSC. The approach to the tertiary structure was obtained using the algorithm MUSTER. The energyminimization was done using the AMBER94 force field of the Schrodinger suite of molecular analysis, and the stereochemistry andenergy model validation was performed by the RAMPAGE server. The final model was visualized using PyMol V.0,98. Results. Thisstudy proposes a computational model that describes the 3D structure of the hypothetical lipoprotein LIC10494 and agrees with previousexperimental reports; thus, our study demonstrates the existence of patterns that could play an important role in the pathogenicity andprotection of the bacteria against the host immune system; the presence of a disorganized region between amino acids 80 and 140, andof a transmembrane segment between amino acids 8 and 22. Conclusion. The coincidence between structural and functional segments suggests that our model can be used to predict certain aspects of the biological behaviour of the protein according to the pathogenic andimmunogenic characteristics of the bacteria.

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

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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

Interaction of bovine peripheral blood polymorphonuclear cells and Leptospira species; innate responses in the natural bovine reservoir host.

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Cattle are the reservoir hosts of Leptospira borgpetersenii serovar Hardjo, and also be reservoir hosts of other Leptospira species such as L. kirschneri, and L. interrogans. As a reservoir host, cattle shed Leptospira, infecting other animals, including humans. Previous studies with human and murin...

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

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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...

Detection of reactive canines to Leptospira in Campeche City, Mexico Detección de caninos reaccionantes a Leptospira en la ciudad de Campeche, México

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Selene del C Blum Domínguez

2013-03-01

Full Text Available 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.

[Construction and expression of recombinant Mycobacterium bovis BCG with the ompA-like membrane protein gene Loa22 of Leptospira interrogans serovar].

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Li, Dao-kun; Bao, Lang; Zhang, Ying; Sun, Zhan

2010-03-01

To study the immunity of Loa22 from Leptospira interrogans serovar Lai strain 56601 by expressing its protein in BCG. Amplified the mature peptide of Loa22 gene from the genome of of Leptospira interrogans serovar Lai strain 56601 and constructed recombinant plasmid rpMV36l-1oa22 with the E. coli-BCG integrating shuttle plasmid pMV361 and the Loa22 mature peptide gene. The rpMV36l-1oa22 plasmid was transformed into BCG by electroporation. The rBCG bearing rpMV36l-1oa22 was induced by high temperature of 45 degrees C and expressed protein was identified by SDS-PAGE and Western Blotting. Fifth 6-week-old BALB/c mice were randomly divided into five groups, which were inoculated intraperitoneally two times at 0-day and 21-day with BCG, rBCG-pMV361, rI3CG-1oa22, Loa22 and killed whole-leptospires respectively. All animals were dislocated from cervical vertebra on the 14Ih day after the last immunization. The proliferative reaction of splenic lymphocyte in tuitro were tested by XTT. The rpMV36l-1oa22 plasmid was constructed successfully and transformed into BCG. The rBCG expressed a 19 X io specifical protein identified by SDS-PAGE and Western Blotting. The splenic lymphocyte proliferate activity (SI) in rBCG-ioa22 group in intro was significantly higher than those in BCG group and rBCG-pMV361 group. We explored the expressing feasibility of Loa22 in Mycobacterium bovis BCG. may therefore make further researches on the induction of protective immunity against human and animal leptospirosis.

Effects of temperature on gene expression patterns in Leptospira interrogans serovar Lai as assessed by whole-genome microarrays.

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Lo, Miranda; Bulach, Dieter M; Powell, David R; Haake, David A; Matsunaga, James; Paustian, Michael L; Zuerner, Richard L; Adler, Ben

2006-10-01

Leptospirosis is an important zoonosis of worldwide distribution. Humans become infected via exposure to pathogenic Leptospira spp. from infected animals or contaminated water or soil. The availability of genome sequences for Leptospira interrogans, serovars Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor known to affect leptospiral protein expression. Leptospira spp. can grow in artificial media at a range of temperatures reflecting conditions found in the environment and the mammalian host. Therefore, transcriptional changes were compared between cultures grown at 20 degrees C, 30 degrees C, 37 degrees C, and 39 degrees C to represent ambient temperatures in the environment, growth under laboratory conditions, and temperatures in healthy and febrile hosts. Data from direct pairwise comparisons of the four temperatures were consolidated to examine transcriptional changes at two generalized biological conditions representing mammalian physiological temperatures (37 degrees C and 39 degrees C) versus environmental temperatures (20 degrees C and 30 degrees C). Additionally, cultures grown at 30 degrees C then shifted overnight to 37 degrees C were compared with those grown long-term at 30 degrees C and 37 degrees C to identify genes potentially expressed in the early stages of infection. Comparison of data sets from physiological versus environmental experiments with upshift experiments provided novel insights into possible transcriptional changes at different stages of infection. Changes included differential expression of chemotaxis and motility genes, signal transduction systems, and genes encoding proteins involved in alteration of the outer membrane. These findings indicate that temperature is an important factor regulating expression of proteins that facilitate invasion and establishment of disease.

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

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Eshghi, Azad; Pinne, Marija; Haake, David A; Zuerner, Richard L; Frank, Ami; Cameron, Caroline E

2012-03-01

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 immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.

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

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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

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

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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. Copyright © 2016. Published by Elsevier Ltd.

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

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J Muse Davis

2009-06-01

Full Text Available 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.

Epidemiology of Leptospira Transmitted by Rodents in Southeast Asia

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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

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

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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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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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. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Leptospiral Antigen Lp49 is a Two-Domain Protein with Putative Protein Binding Function

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Oliveira Giuseppe,P.; Oliveira Neves, F.; Nascimento, A.; Gomes Guimaraes, B.

2008-01-01

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Angstroms resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

Protección inducida por nanococleatos derivados de proteoliposomas de Leptospira interrogans serovar Canicola

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Beatriz Tamargo

2012-04-01

Full Text Available Desde los años 20 del pasado siglo, hasta el presente, en el mundo se han desarrollado y empleado vacunas de células enteras contra la leptospirosis que confieren una corta inmunidad; la mayoría no adyuvadas y dirigidas, fundamentalmente, contra los diferentes serogrupos de la especie Leptospira interrogans, contenidos en las preparaciones. Numerosos han sido los intentos realizados para lograr una formulación vacunal más pura, efectiva, de amplio espectro y duración de la protección que las bacterinas de células enteras inactivadas. Sin embargo, hasta el momento no se ha registrado ninguna vacuna con tales características. En el presente trabajo se obtuvieron antígenos de membrana externa a partir de una cepa cubana autóctona (Cepa 87, L. interrogans serovar Canicola, mediante una modificación de la tecnología para la producción de vesículas de membrana, patentada por investigadores del Instituto Finlay. Estos antígenos con estructura nanoproteoliposómica fueron formulados/adyuvados mediante diferentes estrategias, logrando cinco preparaciones con estructura coclear, que constituyen nanopartículas de aproximadamente 100 a 150 nm de largo y entre 15 a 30 nm de diámetro. Los inmunógenos se inocularon en el biomodelo Mesocrisetus aureatus, con dos dosis e intervalo de seis semanas. El reto fue realizado con 100.000 DL 50 . Los resultados demuestran que las nuevas formulaciones vacunales confieren protección frente al reto homólogo y fueron capaces de eliminar el estado de portador, lo que unido a la robustez del método de preparación, el mayor nivel de pureza, en comparación con las bacterinas, y la no necesidad del hidróxido de aluminio, las convierten en una alternativa de interés para continuar su desarrollo.

Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

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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

High-Resolution Melting Curve Analysis of the 16S Ribosomal Gene to Detect and Identify Pathogenic and Saprophytic Leptospira Species in Colombian Isolates.

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Peláez Sánchez, Ronald G; Quintero, Juan Álvaro López; Pereira, Martha María; Agudelo-Flórez, Piedad

2017-05-01

AbstractIt is important to identify the circulating Leptospira agent to enhance the performance of serodiagnostic tests by incorporating specific antigens of native species, develop vaccines that take into account the species/serovars circulating in different regions, and optimize prevention and control strategies. The objectives of this study were to develop a polymerase chain reaction (PCR)-high-resolution melting (HRM) assay for differentiating between species of the genus Leptospira and to verify its usefulness in identifying unknown samples to species level. A set of primers from the initial region of the 16S ribosomal gene was designed to detect and differentiate the 22 species of Leptospira . Eleven reference strains were used as controls to establish the reference species and differential melting curves. Twenty-five Colombian Leptospira isolates were studied to evaluate the usefulness of the PCR-HRM assay in identifying unknown samples to species level. This identification was confirmed by sequencing and phylogenetic analysis of the 16S ribosomal gene. Eleven Leptospira species were successfully identified, except for Leptospira meyeri / Leptospira yanagawae because the sequences were 100% identical. The 25 isolates from humans, animals, and environmental water sources were identified as Leptospira santarosai (twelve), Leptospira interrogans (nine), and L. meyeri / L. yanagawae (four). The species verification was 100% concordant between PCR-HRM and phylogenetic analysis of the 16S ribosomal gene. The PCR-HRM assay designed in this study is a useful tool for identifying Leptospira species from isolates.

Large-scale purification and in vitro characterization of the assembly of MreB from Leptospira interrogans.

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Barkó, Szilvia; Szatmári, Dávid; Bódis, Emőke; Türmer, Katalin; Ujfalusi, Zoltán; Popp, David; Robinson, Robert C; Nyitrai, Miklós

2016-09-01

Weil's syndrome is caused by Leptospira interrogans infections, a Gram negative bacterium with a distinct thin corkscrew cell shape. The molecular basis for this unusual morphology is unknown. In many bacteria, cell wall synthesis is orchestrated by the actin homolog, MreB. Here we have identified the MreB within the L. interrogans genome and expressed the His-tagged protein product of the synthesized gene (Li-MreB) in Escherichia coli. Li-MreB did not purify under standard nucleotide-free conditions used for MreBs from other species, requiring the continual presence of ATP to remain soluble. Covalent modification of Li-MreB free thiols with Alexa488 produced a fluorescent version of Li-MreB. We developed native and denaturing/refolding purification schemes for Li-MreB. The purified product was shown to assemble and disassemble in MgCl2 and KCl dependent manners, as monitored by light scattering and sedimentation studies. The fluorescence spectrum of labeled Li-MreB-Alexa488 showed cation-induced changes in line with an activation process followed by a polymerization phase. The resulting filaments appeared as bundles and sheets under the fluorescence microscope. Finally, since the Li-MreB polymerization was cation dependent, we developed a simple method to measure monovalent cation concentrations within a test case prokaryote, E. coli. We have identified and initially characterized the cation-dependent polymerization properties of a novel MreB from a non-rod shaped bacterium and developed a method to measure cation concentrations within prokaryotes. This initial characterization of Li-MreB will enable future structural determination of the MreB filament from this corkscrew-shaped bacterium. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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Florence Naze

Full Text Available 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.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.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.

Post-translational modification of LipL32 during Leptospira interrogans infection

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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 ...

Prevalence of antibody titers to leptospira spp. in Minnesota white-tailed deer

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Goyal, S.M.; Mech, L.D.; Nelson, M.E.

1992-01-01

Serum samples (n = 204) from 124 white-tailed deer (Odocoileus virginianus) in northeastern Minnesota (USA) were collected from 1984 through 1989 and tested for antibodies to six serovars of Leptospira interrogans (bratislava, canicola, grippotyphosa, hardjo, icterohemorrhagiae, and pomona) using a microtiter agglutination test. Eighty-eight (43%) sera were positive at greater than or equal to 1:100 for antibodies against serovars pomona and/or bratislava; none was positive for any of the other four serovars. None of the 31 sera collected in 1984-85 was positive, whereas all 54 sera collected from 1986 through 1988 had titers of greater than or equal to 1:100. During 1989, only 34 (29%) of 119 sera had titers of greater than or equal to 1:100. Based on these results, we believe there to be wide variability in exposure of Minnesota deer to Leptospira interrogans.

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

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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. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

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

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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

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

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Hélène Pailhoriès

2015-08-01

Full Text Available 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.

Multiple activities of LigB potentiate virulence of Leptospira interrogans: inhibition of alternative and classical pathways of complement.

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Henry A Choy

Full Text Available Microbial pathogens acquire the immediate imperative to avoid or counteract the formidable defense of innate immunity as soon as they overcome the initial physical barriers of the host. Many have adopted the strategy of directly disrupting the complement system through the capture of its components, using proteins on the pathogen's surface. In leptospirosis, pathogenic Leptospira spp. are resistant to complement-mediated killing, in contrast to the highly vulnerable non-pathogenic strains. Pathogenic L. interrogans uses LenA/LfhA and LcpA to respectively sequester and commandeer the function of two regulators, factor H and C4BP, which in turn bind C3b or C4b to interrupt the alternative or classical pathways of complement activation. LigB, another surface-proximal protein originally characterized as an adhesin binding multiple host proteins, has other activities suggesting its importance early in infection, including binding extracellular matrix, plasma, and cutaneous repair proteins and inhibiting hemostasis. In this study, we used a recent model of ectopic expression of LigB in the saprophyte, L. biflexa, to test the hypothesis that LigB also interacts with complement proteins C3b and C4b to promote the virulence of L. interrogans. The surface expression of LigB partially rescued the non-pathogen from killing by 5% normal human serum, showing 1.3- to 48-fold greater survival 4 to 6 d following exposure to complement than cultures of the non-expressing parental strain. Recombinant LigB7'-12 comprising the LigB-specific immunoglobulin repeats binds directly to human complement proteins, C3b and C4b, with respective K(ds of 43±26 nM and 69±18 nM. Repeats 9 to 11, previously shown to contain the binding domain for fibronectin and fibrinogen, are also important in LigB-complement interactions, which interfere with the alternative and classical pathways measured by complement-mediated hemolysis of erythrocytes. Thus, LigB is an adaptable interface

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

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

2016-05-04

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. © The American Society of Tropical Medicine and Hygiene.

Doxycycline Attenuates Leptospira-Induced IL-1β by Suppressing NLRP3 Inflammasome Priming

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Zhang, Wenlong; Xie, Xufeng; Wu, Dianjun; Jin, Xuemin; Liu, Runxia; Hu, Xiaoyu; Fu, Yunhe; Ding, Zhuang; Zhang, Naisheng; Cao, Yongguo

2017-01-01

Doxycycline (Dox), a semisynthetic antibiotic, has been reported to exert multiple immunomodulatory effects. Treatment with Dox has a satisfactory curative effect against leptospirosis. In addition to its antibacterial action, we supposed that Dox also modulated immune response in controlling leptospira infection. Using J774A.1 mouse macrophages, the effects of Dox on protein and mRNA levels of IL-1β and TNF-α were investigated after infection with live or sonicated Leptospira interrogans ser...

Osmotic regulation of expression of two extracellular matrix-binding proteins and a haemolysin of Leptospira interrogans: differential effects on LigA and Sph2 extracellular release.

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Matsunaga, James; Medeiros, Marco A; Sanchez, Yolanda; Werneid, Kristian F; Ko, Albert I

2007-10-01

The life cycle of the pathogen Leptospira interrogans involves stages outside and inside the host. Entry of L. interrogans from moist environments into the host is likely to be accompanied by the induction of genes encoding virulence determinants and the concomitant repression of genes encoding products required for survival outside of the host. The expression of the adhesin LigA, the haemolysin Sph2 (Lk73.5) and the outer-membrane lipoprotein LipL36 of pathogenic Leptospira species have been reported to be regulated by mammalian host signals. A previous study demonstrated that raising the osmolarity of the leptospiral growth medium to physiological levels encountered in the host by addition of various salts enhanced the levels of cell-associated LigA and LigB and extracellular LigA. In this study, we systematically examined the effects of osmotic upshift with ionic and non-ionic solutes on expression of the known mammalian host-regulated leptospiral genes. The levels of cell-associated LigA, LigB and Sph2 increased at physiological osmolarity, whereas LipL36 levels decreased, corresponding to changes in specific transcript levels. These changes in expression occurred irrespective of whether sodium chloride or sucrose was used as the solute. The increase of cellular LigA, LigB and Sph2 protein levels occurred within hours of adding sodium chloride. Extracellular Sph2 levels increased when either sodium chloride or sucrose was added to achieve physiological osmolarity. In contrast, enhanced levels of extracellular LigA were observed only with an increase in ionic strength. These results indicate that the mechanisms for release of LigA and Sph2 differ during host infection. Thus, osmolarity not only affects leptospiral gene expression by affecting transcript levels of putative virulence determinants but also affects the release of such proteins into the surroundings.

Na,K-ATPase: a molecular target for Leptospira interrogans endotoxin

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Younes-Ibrahim M.

1997-01-01

Full Text Available On the basis of our report that a glycolipoprotein fraction (GLP extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1 GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2 Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3 GLP inhibits Na,K-ATPase from intact cells, and 4 GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC50: 120-220 µg protein GLP/ml all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 µg protein GLP/ml for rat and rabbit, respectively, indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 ± SEM; control: 23.8 ± 1.8; GLP, 88 µg protein/ml: 8.2 ± 0.9, demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis

Characterization of the Outer Membrane Proteome of Leptospira interrogans Expressed during Acute Lethal Infection▿

OpenAIRE

Nally, Jarlath E.; Whitelegge, Julian P.; Bassilian, Sara; Blanco, David R.; Lovett, Michael A.

2006-01-01

Pathogenic Leptospira species adapt to a wide range of environmental conditions during disease transmission and infection. While the proteome of in vitro cultivated Leptospira has been characterized in several studies to date, relatively little is known of the proteome as expressed by Leptospira during disease processes. Isolates of Leptospira obtained from patients suffering the severe pulmonary form of leptospirosis cause acute lethal infection in guinea pigs and chronic asymptomatic infect...

In vivo gene expression and immunoreactivity of Leptospira collagenase.

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Janwitthayanan, Weena; Keelawat, Somboon; Payungporn, Sunchai; Lowanitchapat, Alisa; Suwancharoen, Duangjai; Poovorawan, Yong; Chirathaworn, Chintana

2013-06-12

Pulmonary hemorrhage is an increasing cause of death of leptospirosis patients. Bacterial collagenase has been shown to be involved in lung hemorrhage induced by various infectious agents. According to Leptospira whole genome study, colA, a gene suggested to code for bacterial collagenase has been identified. We investigated colA gene expression in lung tissues of Leptospira infected hamsters. Golden Syrian Hamsters were injected intraperitoneally with Leptospira interrogans serovar Pyrogenes. The hamsters were sacrificed on days 3, 5 and 7 post-infection and lung tissues were collected for histological examination and RNA extraction. Lung pathologies including atelectasis and hemorrhage were observed. Expression of colA gene in lung tissues was demonstrated by both RT-PCR and real time PCR. In addition, ColA protein was cloned and the purified protein could react with sera from leptospirosis patients. Leptospira ColA protein may play a role in Leptospira survival or pathogenesis in vivo. Its reaction with leptospirosis sera suggests that this protein is immunogenic and could be another candidate for vaccine development. Copyright © 2012 Elsevier GmbH. All rights reserved.

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

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Hartwig, Daiane D; Bacelo, Kátia L; Oliveira, Thaís L; Schuch, Rodrigo; Seixas, Fabiana K; Collares, Tiago; Rodrigues, Oscar; Hartleben, Cláudia P; Dellagostin, Odir A

2015-02-01

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

A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species

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Boonsilp, Siriphan; Thaipadungpanit, Janjira; 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

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. We modified the existing scheme by replacing one of the

Produção e purificação de imunoglobulinas Y policlonais anti-Leptospira spp.

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Tatiane C.F. Tavares

2013-09-01

Full Text Available Objetivou-se verificar se galinhas imunizadas com uma solução de Leptospira interrogans inativadas e proteínas de membrana externa do sorovar Hardjo, poderiam produzir anticorpos policlonais específicos anti-leptospiras, detectáveis em testes ELISA. Foram imunizados oito galinhas com 25 semanas de idade, da raça White Leghorn, sendo três imunizadas com uma suspensão de leptospiras inativadas, três com uma solução de proteínas de membrana externa extraída do sorovar Hardjo e duas controle. Coletas de sangue foram realizadas quinzenalmente e de ovos diariamente. A IgY foi purificada a partir da gema dos ovos utilizando para a delipidação o método de diluição em água ácida e a precipitação com sulfato de amônio. Nos testes ELISA realizados para verificar a especificidade da IgY, foi demonstrada a produção de anticorpos anti-Leptospira, tanto no soro quanto nas gemas purificadas. O pico de produção de anticorpos específicos ocorreu na 5º semana após a primeira imunização. Ficou demonstrada a possibilidade da indução da produção de anticorpos específicos em galinhas imunizadas com leptospiras do sorovar Hardjo inativadas, bem como, com proteínas de membrana externa (PME extraidas desse sorovar. As galinhas imunizadas com uma suspensão de leptospiras inativadas ou com PME de Leptospira interrogans do sorovar Hardjo produziram anticorpos reativos a PME Hardjo detectáves por teste ELISA.

Infecção experimental em suínos jovens com Leptospira interrogans sorovar wolffi: determinação de parâmetros bioquímicos Experimental infection by Leptospira interrogans serovar wolffi in young pigs: determination of biochemical parameters

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José Carlos Rende

2007-04-01

Full Text Available Um estudo sobre infecção experimental foi realizado em oito suínos, com idade média de 90 dias, machos castrados, da raça Wessex, e distribuídos em dois grupos de quatro suínos cada. Durante 36 dias, foram analisadas as alterações bioquímicas nos soros dos suínos dos dois grupos. O Grupo I foi mantido como testemunho e recebeu 5,0mL de solução fisiológica estéril por via intravenosa (veia cava craniana e, no Grupo II, os suínos foram inoculados pela mesma via com 5,0mL de cultura de Leptospira interrogans sorovar wolffi , amostra L-10 selvagem isolada de tatu (Dasypus novemcinctus, contendo 1,0 x 10(8 leptospiras/mL. A partir do terceiro dia após a inoculação e em intervalos de 72 horas até o décimo oitavo dia, foram feitas coletas de sangue, sem anticoagulante, dos animais inoculados e testemunhas. Os parâmetros bioquímicos analisados foram: bilirrubina total, direta e indireta, ácidos graxos, glicose e proteínas plasmáticas. Foi detectado um aumento da bilirrubina direta no terceiro dia e um aumento no sexto dia da bilirrubina total e indireta após a inoculação. As dosagens de glicose, ácidos graxos e proteínas plasmáticas apresentaram uma diminuição a partir do terceiro dia da inoculação. Com os resultados obtidos, pode-se concluir que o aumento das taxas de bilirrubinas levam a uma definição de um diagnóstico de hemólise aguda, e que a hipoglicemia, a hipolipidemia e a hipoproteinemia podem estar relacionadas com lesões hepáticas e a uma septcemia.Todas as dosagens em todos os animais retornaram aos seus valores normais a partir do décimo quinto dia.Eight, 90 days old pigs, of the Wessex lineage all castrated male were used in experiment, divided into two groups of four animals. Biochemical alterations in the serum of the animals were analyzed in both groups during 36 days. Control (Group I received 5.0mL of a 0.9% sterile sodium chloride solution by intracranial vein injection; Group II animals

Rapid Isolation and Susceptibility Testing of Leptospira spp. Using a New Solid Medium, LVW Agar

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Wuthiekanun, Vanaporn; Amornchai, Premjit; Paris, Daniel H.; Langla, Sayan; Thaipadunpanit, Janjira; Chierakul, Wirongrong; Smythe, Lee D.; White, Nicholas J.; Day, Nicholas P. J.; Peacock, Sharon J.

2013-01-01

Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO2 incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO2 for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC90 values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 μg/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research. PMID:23114772

Detección de proteínas de adhesión a fibronectina y colágeno presentes en Leptospira interrogans serovar Canicola

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Gisele Reyes

2007-12-01

Full Text Available Como parte de los estudios encaminados a la obtención de una formulación vacunal por subunidades contra la leptospirosis humana, se describe la purificación y caracterización de las proteínas de unión a fibronectina y a colágeno en Leptospira interrogans. Las proteínas de la membrana externa fueron extraídas mediante la solubilización con Tritón X-114 y se aplicaron en una columna de afinidad de IgG AntiBSA-Sepharose 2B CL, para eliminar la BSA, contaminante principal del medio de cultivo en que crece el microorganismo. La muestra libre de BSA (no fijado se aplicó a una columna de afinidad de fibronectina Sepharose 4B-CNBr, que permitió la separación y detección de una fracción que contenía una proteína de unión a fibronectina presente en la cepa 87 de Leptospira interrogans serovar Canicola, cuyo peso molecular fue estimado en 40 kDa. La proteína aislada demostró ser antigénica y conservada en los serovares Canicola, Copenhageni y Mozdok, en el ensayo de inmunodetección utilizado en este estudio (Dot blot. Para ello se utilizaron sueros específicos obtenidos en ratas infectadas experimentalmente con cada serovar y una mezcla de sueros de humanos convalecientes de leptospirosis. Las proteínas de membrana externa solubilizadas con Tritón X-114, libres de BSA, fueron aplicadas también a una columna de afinidad colágeno-Sepharosa 4B-CNBr, que permitió la purificación de una proteína de unión a colágeno con un peso molecular de aproximadamente 25 kDa, la cual resultó ser antigénica frente a sueros de humanos convalecientes de la enfermedad. Ambas proteínas seleccionadas (40 kD y 25 kD podrían ser evaluadas como posibles inmunógenos en futuros estudios encaminados a la obtención de nuevos antígenos vacunales.

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

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Daiane D Hartwig

2015-02-01

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

Development of the leptospirosis by experimental infection in hamsters (Mesocricetus auratus with Leptospira interrogans serovar Canicola, strain LO4, by intact and scratched skin exposures

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Carolina de Sousa Américo Batista

2010-10-01

Full Text Available The establishment and evolution of leptospirosis in hamster (Mesocricetus auratus by experimental infection with Leptospira interrogans serovar Canicola, LO4 strain, by intact and scratched skin exposures, having as control the intraperitoneal route, were evaluated. Hundred-twenty female hamsters distributed in two groups according to inoculation route (intact and scratched skin were used. Infectious inoculum was constituted by a pure culture of L. interrogans serovar Canicola (strain LO4, isolated from liver from a slaughtered swine in Londrina, Paraná state and typified by agglutinins adsortion technique with monoclonal antibody kit at the Royal Tropical Institute, Amsterdam, the Netherlands. The animals were observed twice a day during 21 days. Animals that died were necropsied and kidneys, liver, genital tract (uterus and ovaries and brain were aseptically collected. On the 21st post-inoculation day, surviving animals were euthanized. In these animals, serum samples were also collected by cardiac puncture to antileptospires agglutinins research using microscopic agglutination test (MAT. Fresh direct microscopy and microbiological culture were used for the detection of leptospires. Scratched skin route induced larger lethality when compared to intact skin route, with establishment and evolution of leptospirosis. On the other hand, intact skin route induced renal and/or genital carrier state more frequently. LO4 strain presented low immunogenic power, characterized by soroconversion at the MAT in only one inoculated animal.

Leptospira Species in Feral Cats and Black Rats from Western Australia and Christmas Island.

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Dybing, Narelle A; Jacobson, Caroline; Irwin, Peter; Algar, David; Adams, Peter J

2017-05-01

Leptospirosis is a neglected, re-emerging bacterial disease with both zoonotic and conservation implications. Rats and livestock are considered the usual sources of human infection, but all mammalian species are capable of carrying Leptospira spp. and transmitting pathogenic leptospires in their urine, and uncertainty remains about the ecology and transmission dynamics of Leptospira in different regions. In light of a recent case of human leptospirosis on tropical Christmas Island, this study aimed to investigate the role of introduced animals (feral cats and black rats) as carriers of pathogenic Leptospira spp. on Christmas Island and to compare this with two different climatic regions of Western Australia (one island and one mainland). Kidney samples were collected from black rats (n = 68) and feral cats (n = 59) from Christmas Island, as well as feral cats from Dirk Hartog Island (n = 23) and southwest Western Australia (n = 59). Molecular (PCR) screening detected pathogenic leptospires in 42.4% (95% confidence interval 29.6-55.9) of cats and 2.9% (0.4-10.2) of rats from Christmas Island. Sequencing of cat- and rat-positive samples from Christmas Island showed 100% similarity for Leptospira interrogans. Pathogenic leptospires were not detected in cats from Dirk Hartog Island or southwest Western Australia. These findings were consistent with previous reports of higher Leptospira spp. prevalence in tropical regions compared with arid and temperate regions. Despite the abundance of black rats on Christmas Island, feral cats appear to be the more important reservoir species for the persistence of pathogenic L. interrogans on the island. This research highlights the importance of disease surveillance and feral animal management to effectively control potential disease transmission.

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

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Cristina Kraemer Zimpel

Full Text Available Abstract 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.

Eficácia dos tratamentos estabelecidos pelo Manual da IETS, em oócitos, expostos à Leptospira interrogans

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A.C. Goes

2012-02-01

Full Text Available Avaliou-se a eficácia dos tratamentos, definidos pela International Embryo Transfer Society (IETS, de oócitos bovinos, maturados in vitro e expostos experimentalmente à Leptospira interrogans sorovar Grippotyphosa. Os oócitos foram obtidos por meio de punção folicular, selecionados e distribuídos em quatro grupos, expostos ao patógeno e submetidos aos diferentes tipos de tratamentos. Foram expostos à cepa na concentração de 4,7.10(5/µL, virulenta e não adaptada ao meio de manutenção EMJH, e, de 6,3.10(5/µL, avirulenta e adaptada ao meio, por 24 horas. Os grupos tratados com tripsina ou antibióticos apresentaram eficácia de 21,7%, e o grupo lavado sequencialmente 33,4%. Os tratamentos não foram eficazes para os contaminados com a cepa avirulenta. Concluiu-se que as normas de controle de qualidade estabelecidas pela IETS poderiam ser revisadas e, possivelmente, redefinidas, uma vez que a eficácia dos tratamentos, provavelmente, não depende somente da espécie do patógeno, pois há interferência da virulência e de ação dos tratamentos sobre o tipo de patógeno.

Cloning, expression, and homology modeling of GroEL protein from Leptospira interrogans serovar autumnalis strain N2.

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Natarajaseenivasan, Kalimuthusamy; Shanmughapriya, Santhanam; Velineni, Sridhar; Artiushin, Sergey C; Timoney, John F

2011-10-01

Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and sequenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indicated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and validated. The results indicated a Z-score of -8.35, which is in good agreement with the expected value for a protein. The superposition of the Ca traces of cpn60 structure and predicted structure of leptospiral GroEL indicates good agreement of secondary structure elements with an RMSD value of 1.5 Å. Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component. Copyright © 2011 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

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

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Henry A Choy

Full Text Available 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

Characterization of a lipopolysaccharide mutant of Leptospira derived by growth in the presence of an anti-lipopolysaccharide monoclonal antibody

NARCIS (Netherlands)

Zapata, Sonia; Trueba, Gabriel; Bulach, Dieter M.; Boucher, David; Adler, Ben; Hartskeerl, Rudy

2010-01-01

A lipopolysaccharide mutant of Leptospira interrogans (LaiMut) was obtained by growth in the presence of an agglutinating monoclonal antibody (mAb) against lipopolysaccharide. Agglutination reactions with anti-lipopolysaccharide mAbs and polyclonal antibodies showed that LaiMut had lost some

[Eukaryotic expression of Leptospira interrogans lipL32/1-ompL1/1 fusion gene encoding genus-specific protein antigens and the immunoreactivity of expression products].

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Yan, Jie; Zhao, Shou-feng; Mao, Ya-fei; Ruan, Ping; Luo, Yi-hui; Li, Shu-ping; Li, Li-wei

2005-01-01

immunoreactivity, which can be used as a potential antigen for developing a novel vaccine of L.interrogans.

Antibodies to a novel leptospiral protein, LruC, in the eye fluids and sera of horses with Leptospira-associated uveitis.

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Verma, Ashutosh; Matsunaga, James; Artiushin, Sergey; Pinne, Marija; Houwers, Dirk J; Haake, David A; Stevenson, Brian; Timoney, John F

2012-03-01

Screening of an expression library of Leptospira interrogans with eye fluids from uveitic horses resulted in identification of a novel protein, LruC. LruC is located in the inner leaflet of the leptospiral outer membrane, and an lruC gene was detected in all tested pathogenic L. interrogans strains. LruC-specific antibody levels were significantly higher in eye fluids and sera of uveitic horses than healthy horses. These findings suggest that LruC may play a role in equine leptospiral uveitis.

Evidence of Rickettsial and Leptospira Infections in Andean Northern Peru

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2004-01-01

about health in six Latin American countries, 1973- 1992. Rev Panam Salud Publica 1: 23–34. 3. Cowan G, 2000. Rickettsial diseases: the typhus group of...Leonardo Mendoza Instituto Nacional de Salud , Capac Yupanqui No. 1400, Jesús Maria, Lima 11, Peru, Telephone: 51-1-471-9920. Allen L. Richards, Viral and...Colli C, 1997. Leptospira interrogans in a canine population of greater Bue- nos Aires: variables associated with seropositivity. Rev Panam Salud

Expanding the genetic toolbox for Leptospira species by generation of fluorescent bacteria.

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Aviat, Florence; Slamti, Leyla; Cerqueira, Gustavo M; Lourdault, Kristel; Picardeau, Mathieu

2010-12-01

Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.

Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira.

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Christian A Ganoza

2006-08-01

Full Text Available Although previous data indicate that the overall incidence of human leptospirosis in the Peruvian Amazon is similar in urban and rural sites, severe leptospirosis has been observed only in the urban context. As a potential explanation for this epidemiological observation, we tested the hypothesis that concentrations of more virulent Leptospira would be higher in urban than in rural environmental surface waters.A quantitative real-time PCR assay was used to compare levels of Leptospira in urban and rural environmental surface waters in sites in the Peruvian Amazon region of Iquitos. Molecular taxonomic analysis of a 1,200-bp segment of the leptospiral 16S ribosomal RNA gene was used to identify Leptospira to the species level. Pathogenic Leptospira species were found only in urban slum water sources (Fisher's exact test; p = 0.013. The concentration of pathogen-related Leptospira was higher in urban than rural water sources (approximately 10(3 leptospires/ml versus 0.5 x 10(2 leptospires/ml; F = 8.406, p < 0.05. Identical 16S rRNA gene sequences from Leptospira interrogans serovar Icterohaemorrhagiae were found in urban slum market area gutter water and in human isolates, suggesting a specific mode of transmission from rats to humans. In a prospective, population-based study of patients presenting with acute febrile illness, isolation of L. interrogans-related leptospires from humans was significantly associated with urban acquisition (75% of urban isolates; human isolates of other leptospiral species were associated with rural acquisition (78% of rural isolates (chi-square analysis; p < 0.01. This distribution of human leptospiral isolates mirrored the distribution of leptospiral 16S ribosomal gene sequences in urban and rural water sources.Our findings data support the hypothesis that urban severe leptospirosis in the Peruvian Amazon is associated with higher concentrations of more pathogenic leptospires at sites of exposure and transmission

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

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Lo, Miranda; Murray, Gerald L; Khoo, Chen Ai; Haake, David A; Zuerner, Richard L; Adler, Ben

2010-11-01

Leptospirosis is a globally significant zoonosis caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since iron availability is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In many bacteria, expression of iron uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutation in one of these, la1857. We conducted microarray analysis to identify iron-responsive genes and to study the effects of la1857 mutation on gene expression. Under iron-limiting conditions, 43 genes were upregulated and 49 genes were downregulated in the wild type. Genes encoding proteins with predicted involvement in inorganic ion transport and metabolism (including TonB-dependent proteins and outer membrane transport proteins) were overrepresented in the upregulated list, while 54% of differentially expressed genes had no known function. There were 16 upregulated genes of unknown function which are absent from the saprophyte L. biflexa and which therefore may encode virulence-associated factors. Expression of iron-responsive genes was not significantly affected by mutagenesis of la1857, indicating that LA1857 is not a global regulator of iron homeostasis. Upregulation of heme biosynthetic genes and a putative catalase in the mutant suggested that LA1857 is more similar to PerR, a regulator of the oxidative stress response. Indeed, the la1857 mutant was more resistant to peroxide stress than the wild type. Our results provide insights into the role of iron in leptospiral metabolism and regulation of the oxidative stress response, including genes likely to be important for virulence.

Characterization of Leptospira isolates from humans and the environment in Uruguay.

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Meny, Paulina; Menéndez, Clara; Quintero, Jair; Hernández, Elba; Ríos, Cristina; Balassiano, Ilana Teruszkin; Trindade, Camilla Nunes Dos Reis; Vital-Brazil, Juliana Magalhães; Ramos, Tatiane Mendes Varela; Ashfield, Natalia; Feble, Camila; Avila, Esthefani; Schelotto, Felipe; Varela, Gustavo

2017-12-21

Laboratory diagnosis of human leptospirosis usually relies on indirect methods exploring specific immune response. Isolation and identification of the involved strains are cumbersome, but can provide biological resources for pathogenic studies and relevant information for guiding prevention and control measures. The aim of the research we are hereby reporting was the characterization of Leptospira isolates obtained from humans and the environment in Uruguay. Blood cultures were performed from early samples of 302 Uruguayan patients, mainly rural workers, and from 36 water samples taken from their living or working environments. Eight human isolates and seven environmental isolates were obtained and analyzed by end point Polymerase Chain Reaction (PCR), Multilocus Variable Number of Tandem Repeat Analysis (MLVA) and other molecular methods. Human isolates corresponded to several serogroups and serovars of Leptospira interrogans and Leptospira kirschneri species, probably reflecting the infection with similar involved Leptospira species and serovars of an extended animal reservoir in rural settings of the country, mostly dedicated to meat and dairy production. Culture-positive patients were older than usually affected workers, and presented signs and symptoms of severe illness. A high organic and circulating bacterial burden may explain an easier positive result from these workers' samples. Environmental isolates were mainly identified as Leptospira biflexa strains, with a single L. meyeri isolate of uncertain significance.

Preliminary identification of secreted proteins by Leptospira interrogans serovar Kennewicki strain Pomona Fromm

International Nuclear Information System (INIS)

Ricardi, L.M.P.; Portaro, F.C.; Abreu, P.A.E.; Barbosa, A.S.; Morais, Z.M.; Vasconcellos, S.A.

2012-01-01

Full text: This project aimed to identify secreted proteins by pathogenic Leptospira interrogans serovar Kennewicki strain Pomona Fromm (LPF) by proteomic analyses. The strain LPF, whose virulence was maintained by passages in hamsters, were cultured in EMJH medium. The supernatants were centrifuged, dialyzed and subjected to lyophilization. Protein samples were resolved first by IEF at pH 3 to 10, immobilized pH gradient 13-cm strips. Strips were then processed for the second-dimension separation on SDS-polyacrylamide gels. Proteins from gel spots were subjected to reduction, cysteine-alkylation, and in-gel tryptic digestion, and analyzed by LC/MS/MS spectrometry. Liquid chromatography-based separation followed by automated tandem mass spectrometry was also used to identify secreted proteins. In silico analyses were performed using the PSORTbV.3.0 program and SignalP server. One major obstacle to secretome studies is the difficulty to obtain extracts of secreted proteins without citoplasmatic contamination. In addition, the extraction of low concentration proteins from large volumes of culture media, which are rich in salts, BSA and other compounds, frequently interfere with most proteomics techniques. For these reasons, several experimental approaches were used to optimize the protocol applied. In spite of this fact, our analysis resulted in the identification of 200 proteins with high confidence. Only 5 of 63 secreted proteins predicted by in silico analysis were found. Other classes identified included proteins that possess signal peptide but whose cellular localization prediction is unknown or may have multiple localization sites, and proteins that lack signal peptide and are thus thought to be secreted via non conventional mechanisms or resulting from cytoplasmic contamination by cell lysis. Many of these are hypothetical proteins with no putative conserved domains detected. To our knowledge, this is the first study to identify secreted proteins by

Preliminary identification of secreted proteins by Leptospira interrogans serovar Kennewicki strain Pomona Fromm

Energy Technology Data Exchange (ETDEWEB)

Ricardi, L.M.P.; Portaro, F.C.; Abreu, P.A.E.; Barbosa, A.S. [Instituto Butantan, Sao Paulo, SP (Brazil); Morais, Z.M.; Vasconcellos, S.A. [Universidade de Sao Paulo (USP), SP (Brazil)

2012-07-01

Full text: This project aimed to identify secreted proteins by pathogenic Leptospira interrogans serovar Kennewicki strain Pomona Fromm (LPF) by proteomic analyses. The strain LPF, whose virulence was maintained by passages in hamsters, were cultured in EMJH medium. The supernatants were centrifuged, dialyzed and subjected to lyophilization. Protein samples were resolved first by IEF at pH 3 to 10, immobilized pH gradient 13-cm strips. Strips were then processed for the second-dimension separation on SDS-polyacrylamide gels. Proteins from gel spots were subjected to reduction, cysteine-alkylation, and in-gel tryptic digestion, and analyzed by LC/MS/MS spectrometry. Liquid chromatography-based separation followed by automated tandem mass spectrometry was also used to identify secreted proteins. In silico analyses were performed using the PSORTbV.3.0 program and SignalP server. One major obstacle to secretome studies is the difficulty to obtain extracts of secreted proteins without citoplasmatic contamination. In addition, the extraction of low concentration proteins from large volumes of culture media, which are rich in salts, BSA and other compounds, frequently interfere with most proteomics techniques. For these reasons, several experimental approaches were used to optimize the protocol applied. In spite of this fact, our analysis resulted in the identification of 200 proteins with high confidence. Only 5 of 63 secreted proteins predicted by in silico analysis were found. Other classes identified included proteins that possess signal peptide but whose cellular localization prediction is unknown or may have multiple localization sites, and proteins that lack signal peptide and are thus thought to be secreted via non conventional mechanisms or resulting from cytoplasmic contamination by cell lysis. Many of these are hypothetical proteins with no putative conserved domains detected. To our knowledge, this is the first study to identify secreted proteins by

Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira.

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Dhandapani, Gunasekaran; Sikha, Thoduvayil; Rana, Aarti; Brahma, Rahul; Akhter, Yusuf; Gopalakrishnan Madanan, Madathiparambil

2018-04-10

Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti-leptospiral strategies. © 2018 Wiley Periodicals, Inc.

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

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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

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

Science.gov (United States)

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

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

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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.

Household transmission of leptospira infection in urban slum communities.

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Elves A P Maciel

2008-01-01

Full Text Available Leptospirosis, a spirochaetal zoonotic disease, is the cause of epidemics associated with high mortality in urban slum communities. Infection with pathogenic Leptospira occurs during environmental exposures and is traditionally associated with occupational risk activities. However, slum inhabitants reside in close proximity to environmental sources of contamination, suggesting that transmission during urban epidemics occurs in the household environment.A survey was performed to determine whether Leptospira infection clustered within households located in slum communities in the city of Salvador, Brazil. Hospital-based surveillance identified 89 confirmed cases of leptospirosis during an outbreak. Serum samples were obtained from members of 22 households with index cases of leptospirosis and 52 control households located in the same slum communities. The presence of anti-Leptospira agglutinating antibodies was used as a marker for previous infection. In households with index cases, 22 (30% of 74 members had anti-Leptospira antibodies, whereas 16 (8% of 195 members from control households had anti-Leptospira antibodies. Highest titres were directed against L. interrogans serovars of the Icterohaemorrhagiae serogroup in 95% and 100% of the subjects with agglutinating antibodies from case and control households, respectively. Residence in a household with an index case of leptospirosis was associated with increased risk (OR 5.29, 95% CI 2.13-13.12 of having had a Leptospira infection. Increased infection risk was found for all age groups who resided in a household with an index case, including children <15 years of age (P = 0.008.This study identified significant household clustering of Leptospira infection in slum communities where recurrent epidemics of leptospirosis occur. The findings support the hypothesis that the household environment is an important transmission determinant in the urban slum setting. Prevention therefore needs to target

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

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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

Features of two new proteins with OmpA-like domains identified in the genome sequences of Leptospira interrogans.

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Aline F Teixeira

Full Text Available 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.

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

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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.

[Construction and application of prokaryotic expression system of Leptospira interrogans lipL32/1-lipL41/1 fusion gene].

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Luo, Dong-jiao; Yan, Jie; Mao, Ya-fei; Li, Shu-ping; Luo, Yi-hui; Li, Li-wei

2005-01-01

To construct lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system and to determine frequencies of carrying and expression of lipL32 and lipL41 genes in L.interrogans wild strains and specific antibody levels in sera from leptospirosis patients. lipL32/1-lipL41/1 fusion gene was constructed using linking primer PCR method and the prokaryotic expression system of the fusion gene done with routine techniques. SDS-PAGE was used to examine expression of the target recombinant protein rLipL32/1-rLipL41/1. Immunogenicity of rLipL32/1-rLipL41/1 was identified by Western blot. PCR and MAT were performed to detect carrying and expression of lipL32 and lipL41 genes in 97 wild L.interrogans strains. Antibodies against products of lipL32 and lipL41 genes in serum samples from 228 leptospirosis patients were detected by ELISA method. The homogeneity of nucleotide and putative amino acid sequence of lipL32/1-lipL41/1 fusion gene were 99.9 % and 99.8 % in comparison with the reported sequences. Expression output of the target recombinant protein rLipL32/1-rLipL41/1, mainly present in inclusion body, accounted for 10 % of the total bacterial proteins. Both the rabbit antisera against rLipL32/1 and rLipL41/1 could combine to rLipL32/1-rLipL41/1. 97.9 % and 87.6 % of the L.interrogans wild strains had lipL32 and lipL41 genes, respectively. 95.9 % and 84.5 % of the wild strains were positive for MAT with titers of 1:4 - 1:128 using rabbit anti-rLipL32s or anti-rLipL41s sera, respectively. 94.7 % - 97.4 % of the patients'serum samples were positive for rLipL32s antibodies, while 78.5 % - 84.6 % of them were rLipL41s antibodies detectable. lipL32/1-jlipL41/1 fusion gene and its prokaryotic expression system were successfully constructed. The expressed fusion protein had qualified immunogenicity. Both the lipL32 and lipL41 genes are extensively carried and frequently expressed by different serogroups of L.interrogans, and their expression products exhibit cross-antigenicity.

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

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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.

2005-01-01

Summary 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 pseudo-gene, 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

Detection of virulence factors and molecular typing of pathogenic Leptospira from capybara (Hydrochaeris hydrochaeris).

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Jorge, Sérgio; Monte, Leonardo G; Coimbra, Marco Antonio; Albano, Ana Paula; Hartwig, Daiane D; Lucas, Caroline; Seixas, Fabiana K; Dellagostin, Odir A; Hartleben, Cláudia P

2012-10-01

Leptospirosis is a globally prevalent zoonosis caused by pathogenic Leptospira spp.; several serologic variants have reservoirs in synanthropic rodents. The capybara is the largest living rodent in the world, and it has a wide geographical distribution in Central and South America. This rodent is a significant source of Leptospira since the agent is shed via urine into the environment and is a potential public health threat. In this study, we isolated and identified by molecular techniques a pathogenic Leptospira from capybara in southern Brazil. The isolated strain was characterized by partial rpoB gene sequencing and variable-number tandem-repeats analysis as L. interrogans, serogroup Icterohaemorrhagiae. In addition, to confirm the expression of virulence factors, the bacterial immunoglobulin-like proteins A and B expression was detected by indirect immunofluorescence using leptospiral specific monoclonal antibodies. This report identifies capybaras as an important source of infection and provides insight into the epidemiology of leptospirosis.

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

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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

Human leptospirosis in Tanzania: sequencing and phylogenetic analysis confirm that pathogenic Leptospira species circulate among agro-pastoralists living in Katavi-Rukwa ecosystem.

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Muller, Shabani K; Assenga, Justine A; Matemba, Lucas E; Misinzo, Gerald; Kazwala, Rudovick R

2016-06-10

Leptospirosis is a neglected zoonotic disease of worldwide public health importance. The disease affects humans, domestic animals and wildlife. However, leptospirosis is challenging in its diagnosis in humans. Culture technique, which is time consuming, is not recommended for clinical diagnosis. For these reasons, serological and molecular techniques remain the test of choice. The major objective of this study was to explore the genetic characteristic of Leptospira species which are prevalent among agro-pastoralists living in Katavi-Rukwa Ecosystem, Tanzania. A cross-sectional epidemiological study was carried out in the Katavi-Region South-west, Tanzania between August, 2013 and November, 2014. A total of 267 participants were randomly recruited for the study. Microscopic agglutination test (MAT) was used to detect antibody against six Leptospira antigens including local serogroups Icterohaemorrhagiae, Ballum, Grippotyphosa, Sejroe and reference serogroups Hebdomadis, and Australis. Samples with MAT titers ≥ 1:160 were scored as positive, samples with MAT titers ranging from 1:20 to 1:80 were scored as exposed to Leptospira, and absence of agglutination titers was scored as negative. All MAT positive samples, including the low titre samples were subjected to PCR using the respective 16S rRNA primers for the pathogenic and non-pathogenic species. Out of 267 samples tested, 80 (29.9 %) were positive with MAT. The major circulating leptospiral serogroups were Sejroe (15.7 %,), Icterohaemorrhagiae (8.9 %), Grippotyphosa (4.8 %), Hebdomadis (3.37 %), Australis (1.49 %) and Ballum (1.19 %). By using PCR, 33 (15.7 %) out of 210 samples were pathogenic Leptospira while no saprophytic Leptospira spp. was detected. Partial 16S rRNA gene sequences of Leptospira species which were obtained from this study were submitted to GenBank and acquired accession numbers KP313246 and KP313247. Phylogenetic analysis of the nucleotide sequences revealed that species

Diversification of an emerging pathogen in a biodiversity hotspot: Leptospira in endemic small mammals of Madagascar.

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Dietrich, Muriel; Wilkinson, David A; Soarimalala, Voahangy; Goodman, Steven M; Dellagi, Koussay; Tortosa, Pablo

2014-06-01

Biodiversity hotspots and associated endemism are ideal systems for the study of parasite diversity within host communities. Here, we investigated the ecological and evolutionary forces acting on the diversification of an emerging bacterial pathogen, Leptospira spp., in communities of endemic Malagasy small mammals. We determined the infection rate with pathogenic Leptospira in 20 species of sympatric rodents (subfamily Nesomyinae) and tenrecids (family Tenrecidae) at two eastern humid forest localities. A multilocus genotyping analysis allowed the characterization of bacterial diversity within small mammals and gave insights into their genetic relationships with Leptospira infecting endemic Malagasy bats (family Miniopteridae and Vespertilionidae). We report for the first time the presence of pathogenic Leptospira in Malagasy endemic small mammals, with an overall prevalence of 13%. In addition, these hosts harbour species of Leptospira (L. kirschneri, L. borgpetersenii and L. borgpetersenii group B) which are different from those reported in introduced rats (L. interrogans) on Madagascar. The diversification of Leptospira on Madagascar can be traced millions of years into evolutionary history, resulting in the divergence of endemic lineages and strong host specificity. These observations are discussed in relation to the relative roles of endemic vs. introduced mammal species in the evolution and epidemiology of Leptospira on Madagascar, specifically how biodiversity and biogeographical processes can shape community ecology of an emerging pathogen and lead to its diversification within native animal communities. © 2014 John Wiley & Sons Ltd.

Doxycycline Attenuates Leptospira-Induced IL-1β by Suppressing NLRP3 Inflammasome Priming

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Wenlong Zhang

2017-07-01

Full Text Available Doxycycline (Dox, a semisynthetic antibiotic, has been reported to exert multiple immunomodulatory effects. Treatment with Dox has a satisfactory curative effect against leptospirosis. In addition to its antibacterial action, we supposed that Dox also modulated immune response in controlling leptospira infection. Using J774A.1 mouse macrophages, the effects of Dox on protein and mRNA levels of IL-1β and TNF-α were investigated after infection with live or sonicated Leptospira interrogans serovar Lai strain Lai (56601. Specifically, the level of IL-1β but not TNF-α was sharply decreased when treated with Dox in leptospira-infected macrophages. Western blot analysis showed that Dox suppressed the activation of leptospira-induced MAPK and NF-κB signaling pathways. Using NLRP3-deficient and NLRC4-deficient mice, the data showed that the expression of leptospira-induced IL-1β was mainly dependent on the presence of NLRP3 inflammasome in macrophages. Meanwhile, Dox suppressed leptospira-induced NLRP3 inflammasome priming with the upregulation of the Na/K-ATPase Pump β1 subunit. The inhibition effect of Dox on IL-1β was also conspicuous in cells with lipopolysaccharide and ATP stimulation. These results were confirmed in vivo, as peritoneal fluids of mice and organs of hamsters expressed less IL-1β after treatment of leptospiral infection with Dox. Our results indicated that Dox also modulated immune response to attenuate leptospira-induced IL-1β by suppressing p38, JNK, p65, and NLRP3 inflammasome priming.

Doxycycline Attenuates Leptospira-Induced IL-1β by Suppressing NLRP3 Inflammasome Priming

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Zhang, Wenlong; Xie, Xufeng; Wu, Dianjun; Jin, Xuemin; Liu, Runxia; Hu, Xiaoyu; Fu, Yunhe; Ding, Zhuang; Zhang, Naisheng; Cao, Yongguo

2017-01-01

Doxycycline (Dox), a semisynthetic antibiotic, has been reported to exert multiple immunomodulatory effects. Treatment with Dox has a satisfactory curative effect against leptospirosis. In addition to its antibacterial action, we supposed that Dox also modulated immune response in controlling leptospira infection. Using J774A.1 mouse macrophages, the effects of Dox on protein and mRNA levels of IL-1β and TNF-α were investigated after infection with live or sonicated Leptospira interrogans serovar Lai strain Lai (56601). Specifically, the level of IL-1β but not TNF-α was sharply decreased when treated with Dox in leptospira-infected macrophages. Western blot analysis showed that Dox suppressed the activation of leptospira-induced MAPK and NF-κB signaling pathways. Using NLRP3-deficient and NLRC4-deficient mice, the data showed that the expression of leptospira-induced IL-1β was mainly dependent on the presence of NLRP3 inflammasome in macrophages. Meanwhile, Dox suppressed leptospira-induced NLRP3 inflammasome priming with the upregulation of the Na/K-ATPase Pump β1 subunit. The inhibition effect of Dox on IL-1β was also conspicuous in cells with lipopolysaccharide and ATP stimulation. These results were confirmed in vivo, as peritoneal fluids of mice and organs of hamsters expressed less IL-1β after treatment of leptospiral infection with Dox. Our results indicated that Dox also modulated immune response to attenuate leptospira-induced IL-1β by suppressing p38, JNK, p65, and NLRP3 inflammasome priming. PMID:28791016

Leptospira interrogans en una población canina del Gran Buenos Aires: variables asociadas con la seropositividad

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Diana Rubel

1997-08-01

Full Text Available Se determinó la seroprevalencia de leptospirosis en una población canina suburbana con el objeto de analizar la asociación entre distintas variables individuales y ambientales y la seropositividad a leptospirosis. El estudio, de diseño transversal, se llevó a cabo durante julio de 1992 en un barrio del Gran Buenos Aires en el que viven unos 9 500 habitantes y una población canina de unos 2 000 animales. Se estudió una muestra aleatoria de 223 perros, de cada uno de los cuales se obtuvo una muestra de sangre. La ficha epidemiológica del animal se obtuvo por encuesta al ama de casa. Las determinaciones serológicas se realizaron por microaglutinación frente a 10 serotipos de Leptospira interrogans. Se halló seropositividad en 57% de los 223 perros examinados; 82% de los sueros positivos coaglutinaron con dos o más serotipos. Los serotipos detectados con mayor frecuencia fueron canicola y pyrogenes. La seroprevalencia en hembras fue menor que en machos (P <0,05 y entre los cachorros de menos de 1 año de edad, menor que en los animales de mayor edad (P <0,01. El callejeo del perro y la presencia de agua estancada frente a la vivienda del propietario fueron los factores de riesgo más importantes entre los que se estudiaron. Las asociaciones de la seropositividad con el contacto con un basural, con el comportamiento de caza del perro y con la presencia de roedores en la vivienda no fueron estadísticamente significativas. Se discuten distintas medidas de control.

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

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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

Characterization of three novel adhesins of Leptospira interrogans.

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Siqueira, Gabriela H; Atzingen, Marina V; Alves, Ivy J; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2013-12-01

We report cloning, expression, purification, and characterization of three predicted leptospiral membrane proteins (LIC11360, LIC11009, and LIC11975). In silico analysis and proteinase K accessibility data suggest that these proteins might be surface exposed. We show that proteins encoded by LIC11360, LIC11009 and LIC11975 genes interact with laminin in a dose-dependent and saturable manner. The proteins are referred to as leptospiral surface adhesions 23, 26, and 36 (Lsa23, Lsa26, and Lsa36), respectively. These proteins also bind plasminogen and generate active plasmin. Attachment of Lsa23 and Lsa36 to fibronectin occurs through the involvement of the 30-kDa and 70-kDa heparin-binding domains of the ligand. Dose-dependent, specific-binding of Lsa23 to the complement regulator C4BP and to a lesser extent, to factor H, suggests that this protein may interfere with the complement cascade pathways. Leptospira spp. may use these interactions as possible mechanisms during the establishment of infection.

Caracterización molecular de serovariedades de Leptospira spp. aisladas de muestras de animales y agua en Colombia

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Claudia M. Romero-Vivas

2013-08-01

Full Text Available Introducción. La leptospirosis es una infección bacteriana transmitida directa o indirectamente de animales a humanos, la cual puede resultar en una enfermedad hemorrágica grave, hepática o renal y pulmonar. Hay 20 especies de Leptospira conocidas y cientos de serovariedades, algunas de las cuales pertenecen a diferentes especies. Es esencial identificar las serovariedades patógenas y sus reservorios potenciales para enfocar estrategias de control. Objetivo. Caracterizar las serovariedades de Leptospira aisladas de muestras de roedores, perros, cerdos y agua en Colombia. Materiales y métodos. Las cepas de leptospiras aisladas fueron identificadas como patógenas usando la reacción en cadena de la polimerasa (PRC. Sus ADN y el ADN de Salmonella Braenderup H9812 (marcador de peso molecular fueron cortados con NotI y corridos en electroforesis de campo pulsado. Los patrones de la ECP se analizaron con base en los criterios de tipificación para cepas bacterianas y el coeficiente de Dice, cuando se compararon con 200 cepas aisladas en otras partes del mundo. Los perfiles de ADN con un coeficiente de Dice entre 73,7 % y 100 % se consideraron pertenecientes a la misma especie. Resultados. Todos los aislamientos fueron cepas patógenas y cinco se caracterizaron genéticamente. El aislamiento P275 (coeficiente de Dice: 84 % y el P282 (coeficiente de Dice: 95 % de cerdos, se relacionaron con Leptospira interrogans de serovariedad Pomona; el aislamiento de rata (I15 fue indistinguible de Leptospira interrogans de serovariedades Icterohaemorrhagiae o Copenhageni (coeficiente de Dice: 100 %, mientras que los aislamientos de perro (C67 y agua (A42 no se relacionaron (coeficiente de Dice <73,7 % con ninguna de las 200 cepas de referencia; las más cercanas fueron Leptospira noguchii de serovariedades Nicaragua (coeficiente de Dice: 63 % y Orleans (coeficiente de Dice: 60 %. Conclusiones. Esta fue la primera caracterización molecular de serotipos de

Leptospira interrogans activation of peripheral blood monocyte glycolipoprotein demonstrated in whole blood by the release of IL-6

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F. Dorigatti

2005-06-01

Full Text Available Glycolipoprotein (GLP from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control or medium (negative control, and supernatants were collected after 6, 20/24, and 48 h, and kept at -80ºC until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.

Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions.

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Wu, Qingzhong; Prager, Katherine C; Goldstein, Tracey; Alt, David P; Galloway, Renee L; Zuerner, Richard L; Lloyd-Smith, James O; Schwacke, Lori

2014-08-11

Several real-time PCR assays are currently used for detection of pathogenic Leptospira spp.; however, few methods have been described for the successful evaluation of clinical urine samples. This study reports a rapid assay for the detection of pathogenic Leptospira spp. in California sea lions Zalophus californianus using real-time PCR with primers and a probe targeting the lipL32 gene. The PCR assay had high analytic sensitivity-the limit of detection was 3 genome copies per PCR volume using L. interrogans serovar Pomona DNA and 100% analytic specificity; it detected all pathogenic leptospiral serovars tested and none of the non-pathogenic Leptospira species (L. biflexa and L. meyeri serovar Semaranga), the intermediate species L. inadai, or the non-Leptospira pathogens tested. Our assay had an amplification efficiency of 1.00. Comparisons between the real-time PCR assay and culture isolation for detection of pathogenic Leptospira spp. in urine and kidney tissue samples from California sea lions showed that samples were more often positive by real-time PCR than by culture methods. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that our real-time PCR assay has high analytic sensitivity and specificity for the rapid detection of pathogenic Leptospira species in urine and kidney tissue samples.

Structural and functional characterization of an orphan ATP-binding cassette ATPase involved in manganese utilization and tolerance in Leptospira spp.

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Benaroudj, Nadia; Saul, Frederick; Bellalou, Jacques; Miras, Isabelle; Weber, Patrick; Bondet, Vincent; Murray, Gerald L; Adler, Ben; Ristow, Paula; Louvel, Hélène; Haouz, Ahmed; Picardeau, Mathieu

2013-12-01

Pathogenic Leptospira species are the etiological agents of the widespread zoonotic disease leptospirosis. Most organisms, including Leptospira, require divalent cations for proper growth, but because of their high reactivity, these metals are toxic at high concentrations. Therefore, bacteria have acquired strategies to maintain metal homeostasis, such as metal import and efflux. By screening Leptospira biflexa transposon mutants for their ability to use Mn(2+), we have identified a gene encoding a putative orphan ATP-binding cassette (ABC) ATPase of unknown function. Inactivation of this gene in both L. biflexa and L. interrogans strains led to mutants unable to grow in medium in which iron was replaced by Mn(2+), suggesting an involvement of this ABC ATPase in divalent cation uptake. A mutation in this ATPase-coding gene increased susceptibility to Mn(2+) toxicity. Recombinant ABC ATPase of the pathogen L. interrogans exhibited Mg(2+)-dependent ATPase activity involving a P-loop motif. The structure of this ATPase was solved from a crystal containing two monomers in the asymmetric unit. Each monomer adopted a canonical two-subdomain organization of the ABC ATPase fold with an α/β subdomain containing the Walker motifs and an α subdomain containing the ABC signature motif (LSSGE). The two monomers were arranged in a head-to-tail orientation, forming a V-shaped particle with all the conserved ABC motifs at the dimer interface, similar to functional ABC ATPases. These results provide the first structural and functional characterization of a leptospiral ABC ATPase.

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

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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.

Detection of pathogenic Leptospira species associated with phyllostomid bats (Mammalia: Chiroptera) from Veracruz, Mexico.

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Ballados-González, G G; Sánchez-Montes, S; Romero-Salas, D; Colunga Salas, P; Gutiérrez-Molina, R; León-Paniagua, L; Becker, I; Méndez-Ojeda, M L; Barrientos-Salcedo, C; Serna-Lagunes, R; Cruz-Romero, A

2018-06-01

The genus Leptospira encompass 22 species of spirochaetes, with ten pathogenic species that have been recorded in more than 160 mammals worldwide. In the last two decades, the numbers of records of these agents associated with bats have increased exponentially, particularly in America. Although order Chiroptera represents the second most diverse order of mammals in Mexico, and leptospirosis represents a human and veterinary problem in the country, few studies have been conducted to identify potential wildlife reservoirs. The aim of this study was to detect the presence and diversity of Leptospira sp. in communities of bats in an endemic state of leptospirosis in Mexico. During January to September 2016, 81 bats of ten species from three localities of Veracruz, Mexico, were collected with mist nets. Kidney samples were obtained from all specimens. For the detection of Leptospira sp., we amplified several genes using specific primers. Amplicons of the expected size were submitted to sequencing, and sequences recovered were compared with those of reference deposited in GenBank using the BLAST tool. To identify their phylogenetic position, we realized a reconstruction using maximum-likelihood (ML) method. Twenty-five samples from three bat species (Artibeus lituratus, Choeroniscus godmani and Desmodus rotundus) showed the presence of Leptospira DNA. Sequences recovered were close to Leptospira noguchii, Leptospira weilii and Leptospira interrogans. Our results include the first record of Leptospira in bats from Mexico and exhibit a high diversity of these pathogens circulating in the state. Due to the finding of a large number of positive wild animals, it is necessary to implement a surveillance system in populations of the positive bats as well as in related species, in order to understand their role as carriers of this bacterial genus. © 2018 Blackwell Verlag GmbH.

Diagnostic accuracy of an in-house ELISA using the intermediate species Leptospira fainei as antigen for diagnosis of acute leptospirosis in dogs.

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Penna, Bruno; Marassi, Carla D; Libonati, Hugo; Narduche, Lorena; Lilenbaum, Walter; Bourhy, Pascale

2017-02-01

Diagnosis of animal leptospirosis is still challenging. The microscopic agglutination test, is the current method for diagnosing leptospirosis. However, this technique requires specific equipment, highly trained staff and the maintenance of live cultures of several reference strains of Leptospira for use as antigens. Recently, an ELISA (enzyme-linked immunosorbent assay) employing a Leptospira fainei serovar Hurstbridge based antigen for the early diagnostic of human leptospirosis was developed. In this study we estimate the diagnostic sensitivity and specificity of this test in identifying acute canine leptospirosis. A total of 271 serum samples divided into five panels and tested by MAT as a reference test, were used to evaluate the ELISA. Comparing acutely and non-acutely infected dogs, ELISA-Hb showed 95.6% sensitivity and 93% specificity. L. fainei-based ELISA is adequate for diagnosing acute canine leptospirosis, with high sensitivity and specificity and presenting practical advantages when compared to current techniques. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis.

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Turk, Nenad; Milas, Zoran; Mojcec, Vesna; Ruzic-Sabljic, Eva; Staresina, Vilim; Stritof, Zrinka; Habus, Josipa; Postic, Daniele

2009-11-01

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

Proteomic Analysis of Leptospira interrogans Shed in Urine of Chronically Infected Hosts▿

OpenAIRE

Monahan, Avril M.; Callanan, John J.; Nally, Jarlath E.

2008-01-01

Leptospirosis is a global zoonotic disease. The causative agent, pathogenic Leptospira species, survives in the renal tubules of chronically infected hosts, from where leptospires are shed via urine into the environment. Infection of new hosts can present as an array of acute and chronic disease processes reflecting variations in host-pathogen interactions. The present study was designed to reproduce the carrier phase of infection in Rattus norvegicus, thus facilitating shedding of leptospire...

Perfil dos cães sororreagentes para aglutininas anti-Leptospira interrogans em Belo Horizonte, Minas Gerais, 2001/2002 Serological profile of seropositive dogs to anti-Leptospira interrogans agglutinins in Belo Horizonte, Minas Gerais, Brazil, 2001-2002

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D.F. Magalhães

2007-10-01

Full Text Available The serological profile of seropositive dogs according to anti-Leptospira agglutinins was checked in Belo Horizonte including variables such as race, sex, age and whether the dog had an owner or not. The dogs were captured by the Zoonosis Control Center in nine neighborhoods around the city and were separated in two categories - with owners or captured on the streets. The prevalence of anti-Leptospira agglutinins was evaluated in 3,417 blood samples using the microscopic agglutination test (MAT from September 2001 to September 2002. It was found that 13.1% of the dogs had seropositive results with the most reactive serovars being Canicola (7.0%, Ballum (6.1%, Pyrogenes (3.2% and Icterohaemorrhagiae (2.9%. The prevalence of other serovars was less than 1.0%. Greater prevalence was found in male, crossbred dogs, without owners. There were no significant results due to age in 95% (P=0.808 of the cases. According to the results, more research should be done in order to isolate and classify the serovars in positive dogs, especially Ballum and Pyrogenes, which will suggest their inclusion in the commercial vaccines against leptospira used in dogs in this city.

Crystallization and preliminary X-ray analysis of LipL32 from Leptospira interrogans serovar Copenhageni

International Nuclear Information System (INIS)

Hauk, Pricila; Guzzo, Cristiane R.; Ho, Paulo L.; Farah, Chuck S.

2009-01-01

Recombinant selenomethionine-labelled LipL32, the major surface protein of pathogenic Leptospira, has been purified and crystallized. Data sets from two crystals were collected, one of which diffracted to 2.25 Å resolution. LipL32 is a major surface protein that is expressed during infection by pathogenic Leptospira. Here, the crystallization of recombinant LipL32 21–272 , which corresponds to the mature LipL32 protein minus its N-terminal lipid-anchored cysteine residue, is described. Selenomethionine-labelled LipL32 21–272 crystals diffracted to 2.25 Å resolution at a synchrotron source. The space group was P3 1 21 or P3 2 21 and the unit-cell parameters were a = b = 126.7, c = 96.0 Å

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

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

2016-08-01

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. 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. 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.

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

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Meghan R Mason

2016-08-01

Full Text Available 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.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.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.

Association between Opisthorchis viverrini and Leptospira spp. infection in endemic Northeast Thailand.

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Van, Chinh Dang; Doungchawee, Galayanee; Suttiprapa, Sutas; Arimatsu, Yuji; Kaewkes, Sasithorn; Sripa, Banchob

2017-08-01

Opisthorchiasis caused by Opisthorchis viverrini is an important foodborne trematodiasis in Thailand, Laos and Cambodia. Interestingly, the opisthorchiasis endemic region overlaps with an area of leptospirosis emergence. Here we report an association between opisthorchiasis and leptospirosis in Thailand. Of 280 sera collected from villagers living around the Lawa wetland complex in Khon Kaen province, 199 (71%) were seropositive for leptospirosis by immunochromatography. Individuals with O. viverrini infection had a significantly higher rate of leptospirosis than those without (P=0.001). Significant higher leptospirosis prevalence was found in males than females (P=0.002). However, females but not males with O. viverrini infection showed a significantly higher seroprevalence of leptospirosis. Twenty-one of 35 environmental samples from the lake (water, mud and fish skin mucus) were positive for Leptospira spp. DNA sequencing, sequence alignment, and phylogenetic analysis of some positive nested PCR products revealed both pathogenic and intermediate pathogenic strains of Leptospira in the samples. Strikingly, O. viverrini metacercariae from the fish were positive for L. interrogans. These results suggest a close association between opisthorchiasis and leptospirosis. Contact with water, mud or eating raw fish harboring liver fluke metacercariae may be risk factors for Leptospira infection. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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.

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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. Copyright © 2016. Published by Elsevier GmbH.

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

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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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

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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 ...

NCBI nr-aa BLAST: CBRC-CJAC-01-0924 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-CJAC-01-0924 ref|NP_713695.1| Heme exporter protein A [Leptospira interrogans ...serovar Lai str. 56601] gb|AAN50713.1|AE011508_10 Heme exporter protein A [Leptospira interrogans serovar Lai str. 56601] NP_713695.1 3.5 22% ...

Interaction of bovine peripheral blood polymorphonuclear cells and Leptospira species; innate responses in the natural bovine reservoir host.

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Jennifer H Wilson-Welder

2016-07-01

Full Text Available 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 L. 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

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.

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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

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

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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.

ORF Alignment: NC_000919 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available Carbon storage regulator csrA [Leptospira interrogans ... serovar Lai str. 56601] gb|AAN51504.1| Carbo...n storage ... regulator csrA [Leptospira interrogans serovar lai str. ... ...r. ... Fiocruz L1-130] sp|Q8EYB0|CSRA_LEPIN Carbon storage ... regulator homolog sp|Q72MJ6|CSRA_

ORF Alignment: NC_006087 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available Carbon storage regulator csrA [Leptospira interrogans ... serovar Lai str. 56601] gb|AAN51504.1| Carbo...n storage ... regulator csrA [Leptospira interrogans serovar lai str. ... ...r. ... Fiocruz L1-130] sp|Q8EYB0|CSRA_LEPIN Carbon storage ... regulator homolog sp|Q72MJ6|CSRA_

ORF Alignment: NC_006156 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available Carbon storage regulator csrA [Leptospira interrogans ... serovar Lai str. 56601] gb|AAN51504.1| Carbo...n storage ... regulator csrA [Leptospira interrogans serovar lai str. ... ...r. ... Fiocruz L1-130] sp|Q8EYB0|CSRA_LEPIN Carbon storage ... regulator homolog sp|Q72MJ6|CSRA_

ORF Alignment: NC_002570 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available Carbon storage regulator csrA [Leptospira interrogans ... serovar Lai str. 56601] gb|AAN51504.1| Carbo...n storage ... regulator csrA [Leptospira interrogans serovar lai str. ... ...r. ... Fiocruz L1-130] sp|Q8EYB0|CSRA_LEPIN Carbon storage ... regulator homolog sp|Q72MJ6|CSRA_

ORF Alignment: NC_003030 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available Carbon storage regulator csrA [Leptospira interrogans ... serovar Lai str. 56601] gb|AAN51504.1| Carbo...n storage ... regulator csrA [Leptospira interrogans serovar lai str. ... ...r. ... Fiocruz L1-130] sp|Q8EYB0|CSRA_LEPIN Carbon storage ... regulator homolog sp|Q72MJ6|CSRA_

ORF Alignment: NC_004193 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available Carbon storage regulator csrA [Leptospira interrogans ... serovar Lai str. 56601] gb|AAN51504.1| Carbo...n storage ... regulator csrA [Leptospira interrogans serovar lai str. ... ...r. ... Fiocruz L1-130] sp|Q8EYB0|CSRA_LEPIN Carbon storage ... regulator homolog sp|Q72MJ6|CSRA_

ORF Alignment: NC_001318 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available Carbon storage regulator csrA [Leptospira interrogans ... serovar Lai str. 56601] gb|AAN51504.1| Carbo...n storage ... regulator csrA [Leptospira interrogans serovar lai str. ... ...r. ... Fiocruz L1-130] sp|Q8EYB0|CSRA_LEPIN Carbon storage ... regulator homolog sp|Q72MJ6|CSRA_

ORF Alignment: NC_000964 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available Carbon storage regulator csrA [Leptospira interrogans ... serovar Lai str. 56601] gb|AAN51504.1| Carbo...n storage ... regulator csrA [Leptospira interrogans serovar lai str. ... ...r. ... Fiocruz L1-130] sp|Q8EYB0|CSRA_LEPIN Carbon storage ... regulator homolog sp|Q72MJ6|CSRA_

ORF Alignment: NC_005823 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available Carbon storage regulator csrA [Leptospira interrogans ... serovar Lai str. 56601] gb|AAN51504.1| Carbo...n storage ... regulator csrA [Leptospira interrogans serovar lai str. ... ...r. ... Fiocruz L1-130] sp|Q8EYB0|CSRA_LEPIN Carbon storage ... regulator homolog sp|Q72MJ6|CSRA_

ORF Alignment: NC_004342 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available Carbon storage regulator csrA [Leptospira interrogans ... serovar Lai str. 56601] gb|AAN51504.1| Carbo...n storage ... regulator csrA [Leptospira interrogans serovar lai str. ... ...r. ... Fiocruz L1-130] sp|Q8EYB0|CSRA_LEPIN Carbon storage ... regulator homolog sp|Q72MJ6|CSRA_

Genetic diversity of Leptospira in northwestern Colombia: first report of Leptospira santarosai as a recognised leptospirosis agent

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Ronald Guillermo Peláez Sanchez

Full Text Available The region of Antioquia in northeastern Colombia has the highest number of reported leptospirosis cases in the country. It also shows high seroprevalence indexes in the general population and socio-environmental conditions favourable for the transmission of the disease between humans and animals. In this study, 25 Leptospira isolates from Colombia’s Antioquia department were identified to the species level as L. santarosai (12, L. interrogans (9 and L. meyeri (4 using phylogenetic analysis of the Amidohydrolase gene. Typing at the serovar level was performed using multilocus sequence typing (MLST and monoclonal antibodies. The serovars Canalzonae, Babudieri, Alice, Beye, and Copenhageni have been identified as causing human or animal infections in Antioquia, Colombia. The four environmental isolates were not identified to the serovar level. L. santarosai serovar Canalzonae and Alice were identified as new etiologic agents of human leptospirosis in Antioquia, Colombia. This paper reports species and serovars that were previously unknown in the region.

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

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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.

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

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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

Identification of a novel prophage-like gene cluster actively expressed in both virulent and avirulent strains of Leptospira interrogans serovar Lai.

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Qin, Jin-Hong; Zhang, Qing; Zhang, Zhi-Ming; Zhong, Yi; Yang, Yang; Hu, Bao-Yu; Zhao, Guo-Ping; Guo, Xiao-Kui

2008-06-01

DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains.

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

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

2016-02-15

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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Seroepidemiological detection of antibodies against Leptospira spp using microscopic agglutination test in Urmia cows and sheep

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Ramin Ag

2013-01-01

Full Text Available The study was designed to determine the level of incidence, titer and various serovars of leptospira in 203 cows and 166 sheep at Urmia abattoir in 2011. Blood samples were collected during the slaughter of animals and sera were separated to evaluate the serological reaction to Leptospira spp by Microscopic Agglutination Test (MAT using live antigens representing Leptospira interrogans serogroups: pomona, grippotyphosa, canicola, hardjo, icterrohaemoragiae, and ballum. Overall, 36% of cows and 19.3% of sheep including 33.8% of bulls, 40.5% of female cows, 18.3% of rams and 25% of ewes had a positive reaction to at least one of the leptospira serovars. The most prevalent serovars in cows were pomona (22.7%, grippotyphosa (13.8%, and hardjo (8.4%, and in sheep were grippotyphosa (66.7%, pomona (26.2% and canicola (7.1%. Other serovars were not detected in cows and sheep. The most prevalent serological titers of 1:100 and 1:200 in cows was 18.2% and 26.6%, and for sheep were 13.5% and 8%, respectively, and of 1:400 in sheep was 2.3%. Cows with a positive reaction to one, two and three serovars were 28.6%, 5.9%, and 1.5% and sheep positive to one and two serovars were 13.3% and 6%, respectively. Age comparison in seropositive cows and sheep showed a significantly increased infection (p<0.05 from young to adult ruminants, while no differences were seen regarding gender. The main mixed serovars were between grippotyphosa/pomona, grippotyphosa/canicola and canicola/pomona. The gender comparison of the serovars' distribution revealed that the pomona and grippotyphosa were predominant among other leptospiral serovars in cows and sheep, respectively. In conclusion, the rate of leptospirosis in Urmia cows was about 2 fold in sheep. The most current serovars in cows and sheep were pomona and grippotyphosa, respectively. The majority of animals was infected with one serovar, but polyserovars, are also possible. The highest titer (1:200 was observed in cows

3D modelling of the pathogenic Leptospira protein LipL32: A bioinformatics approach.

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Kumaran, Sharmilah Kumari; Bakar, Mohd Faizal Abu; Mohd-Padil, Hirzahida; Mat-Sharani, Shuhaila; Sakinah, S; Poorani, K; Alsaeedy, Hiba; Peli, Amira; Wei, Teh Seoh; Ling, Mok Pooi; Hamat, Rukman Awang; Neela, Vasantha Kumari; Higuchi, Akon; Alarfaj, Abdullah A; Rajan, Mariappan; Benelli, Giovanni; Arulselvan, Palanisamy; Kumar, S Suresh

2017-12-01

Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira species (Leptospiraceae). LipL32 is an abundant lipoprotein from the outer membrane proteins (OMPs) group, highly conserved among pathogenic and intermediate Leptospira species. Several studies used LipL32 as a specific gene to identify the presence of leptospires. This research was aimed to study the characteristics of LipL32 protein gene code, to fill the knowledge gap concerning the most appropriate gene that can be used as antigen to detect the Leptospira. Here, we investigated the features of LipL32 in fourteen Leptospira pathogenic strains based on comparative analyses of their primary, secondary structures and 3D modeling using a bioinformatics approach. Furthermore, the physicochemical properties of LipL32 in different strains were studied, shedding light on the identity of signal peptides, as well as on the secondary and tertiary structure of the LipL32 protein, supported by 3D modelling assays. The results showed that the LipL32 gene was present in all the fourteen pathogenic Leptospira strains used in this study, with limited diversity in terms of sequence conservation, hydrophobic group, hydrophilic group and number of turns (random coil). Overall, these results add basic knowledge to the characteristics of LipL32 protein, contributing to the identification of potential antigen candidates in future research, in order to ensure prompt and reliable detection of pathogenic Leptospira species. Copyright © 2017 Elsevier B.V. All rights reserved.

MAT FOR LEPTOSPIROSIS DIAGNOSIS

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Esti Rahardianingtyas.

2014-06-01

Full Text Available Leptospirosis is a disease caused by bacterial infection leptospira interrogans.Leptospira bacteria is a spiral bacterium with solid strands with two flagella periplasmik.Septicaemic phase patient samples taken from the blood and cerebrospinal fluid, whereassamples taken at phase immune extracted from urine. The diagnosis of leptospirosis occurdirectly or indirectly. Diagnosis is done by directly isolate and identify the causative agents ofthe agent. Diagnosis is done indirectly by detecting specific antibodies from the patient's body.Gold Standard of the diagnosis of leptospirosis is MAT. Mat made by reacting antibodies toleptospira antigen. Positive results seen with clump formed.Key words: Leptospirosis, Leptospirosis Diagnostic, MAT (Microscopic Agglutination Test Leptospirosis merupakan penyakit yang disebabkan karena infeksi bakteri leptospirainterrogans. Bakteri leptospira merupakan bakteri spiral dengan untaian yang padat dengan duaflagella periplasmik. Sampel pasien pada fase septicaemic diambil dari darah dan cairanserebrospinal, sedangkan sampel yang diambil pada fase immune diambil dari urine. Diagnosisleptospirosis dilakukan secara langsung maupun tidak langsung. Diagnosis secara langsungdilakukan dengan cara mengisolasi agen penyebab dan mengidentifikasi agen tersebut. Diagnosissecara tidak langsung dilakukan dengan cara mendeteksi antibodi spesiflk dari dalam tubuhpasien. Gold Standart dari diagnosis leptospirosis adalah MAT. Mat dilakukan dengan caramereaksikan antibodi dengan antigen leptospira. Hasil positif dilihat dengan terbentuk gumpalanagglutinasiKata kunci: Leptospirosis, Leptospira, Leptospirosis Diagnosis.

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

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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

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

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Sylvia Grune Loffler

Full Text Available 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

Crystallization of FcpA from Leptospira, a novel flagellar protein that is essential for pathogenesis.

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San Martin, Fabiana; Mechaly, Ariel E; Larrieux, Nicole; Wunder, Elsio A; Ko, Albert I; Picardeau, Mathieu; Trajtenberg, Felipe; Buschiazzo, Alejandro

2017-03-01

The protein FcpA is a unique component of the flagellar filament of spirochete bacteria belonging to the genus Leptospira. Although it plays an essential role in translational motility and pathogenicity, no structures of FcpA homologues are currently available in the PDB. Its three-dimensional structure will unveil the novel motility mechanisms that render pathogenic Leptospira particularly efficient at invading and disseminating within their hosts, causing leptospirosis in humans and animals. FcpA from L. interrogans was purified and crystallized, but despite laborious attempts no useful X ray diffraction data could be obtained. This challenge was solved by expressing a close orthologue from the related saprophytic species L. biflexa. Three different crystal forms were obtained: a primitive and a centred monoclinic form, as well as a hexagonal variant. All forms diffracted X-rays to suitable resolutions for crystallographic analyses, with the hexagonal type typically reaching the highest limits of 2.0 Å and better. A variation of the quick-soaking procedure resulted in an iodide derivative that was instrumental for single-wavelength anomalous diffraction methods.

Factor analysis of serogroups botanica and aurisina of Leptospira biflexa.

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Cinco, M

1977-11-01

Factor analysis is performed on serovars of Botanica and Aurisina serogroup of Leptospira biflexa. The results show the arrangement of main factors serovar and serogroup specific, as well as the antigens common with serovars of heterologous serogroups.

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

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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. © 2016 John Wiley & Sons Ltd.

Serosurvey of Leptospirainterrogans, Brucellaabortus and Chlamydophilaabortus infection in free-ranging giant anteaters (Myrmecophaga tridactyla from Brazil

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Flávia R. Miranda

2015-05-01

Full Text Available Abstract:A serological survey for antibodies against Leptospira interrogans, Brucella abortus, and Chlamydophila abortus was conducted in 21 clinically healthy, free-ranging giant ant- eaters (Myrmecophaga tridactyla from Parque Nacional das Emas (Goiás State, Brazil; n=6, Parque Nacional da Serra da Canastra (Minas Gerais State, Brazil; n=9, and RPPN SESC Pantanal (Mato Grosso State, Brazil; n=6 between July 2001 and September 2006. Sera were screened for antibodies against 22 serovars of Leptospira interrogans with a microscopic agglutination test. Twelve tested positive for L. interrogansserovars sentot (n=5 in PN Emas, n=2 in PN Serra da Canastra, butembo (n=2 in PN Serra da Canastra, autumnalis, bataviae, and shermani/icterohaemorrhagiae(n=1 each in SESC PantanalOne adult female tested positive for B. abortus with the buffered plate antigen test. All sera were negative for C. abortususing the complement fixation text. This is the first report of pathogens that may interfere with the reproduction and population dynamics of free-ranging giant anteaters.

High Leptospira Diversity in Animals and Humans Complicates the Search for Common Reservoirs of Human Disease in Rural Ecuador.

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Barragan, Veronica; Chiriboga, Jorge; Miller, Erin; Olivas, Sonora; Birdsell, Dawn; Hepp, Crystal; Hornstra, Heidie; Schupp, James M; Morales, Melba; Gonzalez, Manuel; Reyes, Soraya; de la Cruz, Carmen; Keim, Paul; Hartskeerl, Rudy; Trueba, Gabriel; Pearson, Talima

2016-09-01

Leptospirosis is a zoonotic disease responsible for high morbidity around the world, especially in tropical and low income countries. Rats are thought to be the main vector of human leptospirosis in urban settings. However, differences between urban and low-income rural communities provide additional insights into the epidemiology of the disease. Our study was conducted in two low-income rural communities near the coast of Ecuador. We detected and characterized infectious leptospira DNA in a wide variety of samples using new real time quantitative PCR assays and amplicon sequencing. We detected infectious leptospira in a high percentage of febrile patients (14.7%). In contrast to previous studies on leptospirosis risk factors, higher positivity was not found in rats (3.0%) but rather in cows (35.8%) and pigs (21.1%). Six leptospira species were identified (L. borgpetersenii, L kirschnerii, L santarosai, L. interrogans, L noguchii, and an intermediate species within the L. licerasiae and L. wolffii clade) and no significant differences in the species of leptospira present in each animal species was detected (χ2 = 9.89, adj.p-value = 0.27). A large portion of the world's human population lives in low-income, rural communities, however, there is limited information about leptospirosis transmission dynamics in these settings. In these areas, exposure to peridomestic livestock is particularly common and high prevalence of infectious leptospira in cows and pigs suggest that they may be the most important reservoir for human transmission. Genotyping clinical samples show that multiple species of leptospira are involved in human disease. As these genotypes were also detected in samples from a variety of animals, genotype data must be used in conjunction with epidemiological data to provide evidence of transmission and the importance of different potential leptospirosis reservoirs.

InvA protein is a Nudix hydrolase required for infection by pathogenic Leptospira in cell lines and animals.

Science.gov (United States)

Luo, Yihui; Liu, Yan; Sun, Dexter; Ojcius, David M; Zhao, Jinfang; Lin, Xuai; Wu, Dong; Zhang, Rongguang; Chen, Ming; Li, Lanjuan; Yan, Jie

2011-10-21

Leptospirosis caused by pathogenic species of the genus Leptospira is a re-emerging zoonotic disease, which affects a wide variety of host species and is transmitted by contaminated water. The genomes of several pathogenic Leptospira species contain a gene named invA, which contains a Nudix domain. However, the function of this gene has never been characterized. Here, we demonstrated that the invA gene was highly conserved in protein sequence and present in all tested pathogenic Leptospira species. The recombinant InvA protein of pathogenic L. interrogans strain Lai hydrolyzed several specific dinucleoside oligophosphate substrates, reflecting the enzymatic activity of Nudix in Leptospira species. Pathogenic leptospires did not express this protein in media but temporarily expressed it at early stages (within 60 min) of infection of macrophages and nephric epithelial cells. Comparing with the wild type, the invA-deficient mutant displayed much lower infectivity and a significantly reduced survival rate in macrophages and nephric epithelial cells. Moreover, the invA-deficient leptospires presented an attenuated virulence in hamsters, caused mild histopathological damage, and were transmitted in lower numbers in the urine, compared with the wild-type strain. The invA revertant, made by complementing the invA-deficient mutant with the invA gene, reacquired virulence similar to the wild type in vitro and in vivo. The LD(50) in hamsters was 1000-fold higher for the invA-deficient mutant than for the invA revertant and wild type. These results demonstrate that the InvA protein is a Nudix hydrolase, and the invA gene is essential for virulence in pathogenic Leptospira species.

Diminuição da fragilidade osmótica eritrocitária na leptospirose canina

OpenAIRE

Santoro, Marcelo L.; Kogika, Marcia M.; Hagiwara, Mitika K.; Mirandola, Regina M. S.; Castelar, Izaura L. C. G.

1994-01-01

Erythrocyte osmotic fragility (EOF) was carried out in nineteen dogs naturally infected by Leptospira interrogans serovar icterohaemorrhagiae/copenhagi. A decreased EOF was observed, suggesting a modification of erythrocyte components secondary to disturbances that occur during canine leptospirosis, such as renal damage and hepatic disease.A fragilidade osmótica eritrocitária foi estudada em dezenove cães infectados naturalmente pela Leptospira interrogans serovar icterohaemorrhagiae/copenhag...

DETECTION OF Leptospira spp. AND Brucella abortus ANTIBODIES IN FREE-LIVING JAGUARS (Panthera onca IN TWO PROTECTED AREAS OF NORTHERN PANTANAL, BRAZIL

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Selma Samiko Miyazaki ONUMA

2015-04-01

Full Text Available 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.

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

Science.gov (United States)

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.

Exposure to Rats and Rat-Associated Leptospira and Bartonella Species Among People Who Use Drugs in an Impoverished, Inner-City Neighborhood of Vancouver, Canada.

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McVea, David A; Himsworth, Chelsea G; Patrick, David M; Lindsay, L Robbin; Kosoy, Michael; Kerr, Thomas

2018-02-01

Rat infestations are common, particularly in impoverished, inner-city neighborhoods. However, there has been little research into the nature and consequences of rat exposure in these neighborhoods, particularly in Canada. In this study, we sought to characterize exposure to rats and rat-associated Leptospira interrogans and Bartonella tribocorum, as well as risk factors associated with exposure, in residents (n = 202) of the Downtown Eastside (DTES) neighborhood of Vancouver, Canada. There was no evidence of exposure to rat-associated L. interrogans but 6/202 (3.0%) of participants were exposed to B. tribocorum, which is known to be circulating among DTES rats. We also found that frequent and close rat exposure was common among DTES residents, and that this exposure was particularly associated with injection drug use and outdoor income-generating activities (e.g., drug dealing). These risk factors may be good targets for interventions geared toward effectively reducing rat exposure.

Isolation and clinical sample typing of human leptospirosis cases in Argentina.

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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. Copyright © 2015 Elsevier B.V. All rights reserved.

Letter to the Editor

Science.gov (United States)

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...

An Extended Multilocus Sequence Typing (MLST Scheme for Rapid Direct Typing of Leptospira from Clinical Samples.

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Sabrina Weiss

2016-09-01

Full Text Available Rapid typing of Leptospira is currently impaired by requiring time consuming culture of leptospires. The objective of this study was to develop an assay that provides multilocus sequence typing (MLST data direct from patient specimens while minimising costs for subsequent sequencing.An existing PCR based MLST scheme was modified by designing nested primers including anchors for facilitated subsequent sequencing. The assay was applied to various specimen types from patients diagnosed with leptospirosis between 2014 and 2015 in the United Kingdom (UK and the Lao Peoples Democratic Republic (Lao PDR. Of 44 clinical samples (23 serum, 6 whole blood, 3 buffy coat, 12 urine PCR positive for pathogenic Leptospira spp. at least one allele was amplified in 22 samples (50% and used for phylogenetic inference. Full allelic profiles were obtained from ten specimens, representing all sample types (23%. No nonspecific amplicons were observed in any of the samples. Of twelve PCR positive urine specimens three gave full allelic profiles (25% and two a partial profile. Phylogenetic analysis allowed for species assignment. The predominant species detected was L. interrogans (10/14 and 7/8 from UK and Lao PDR, respectively. All other species were detected in samples from only one country (Lao PDR: L. borgpetersenii [1/8]; UK: L. kirschneri [1/14], L. santarosai [1/14], L. weilii [2/14].Typing information of pathogenic Leptospira spp. was obtained directly from a variety of clinical samples using a modified MLST assay. This assay negates the need for time-consuming culture of Leptospira prior to typing and will be of use both in surveillance, as single alleles enable species determination, and outbreaks for the rapid identification of clusters.

The OmpL37 surface-exposed protein is expressed by pathogenic Leptospira during infection and binds skin and vascular elastin.

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Pinne, Marija; Choy, Henry A; Haake, David A

2010-09-07

Pathogenic Leptospira spp. shed in the urine of reservoir hosts into freshwater can be transmitted to a susceptible host through skin abrasions or mucous membranes causing leptospirosis. The infection process involves the ability of leptospires to adhere to cell surface and extracellular matrix components, a crucial step for dissemination and colonization of host tissues. Therefore, the elucidation of novel mediators of host-pathogen interaction is important in the discovery of virulence factors involved in the pathogenesis of leptospirosis. In this study, we assess the functional roles of transmembrane outer membrane proteins OmpL36 (LIC13166), OmpL37 (LIC12263), and OmpL47 (LIC13050), which we recently identified on the leptospiral surface. We determine the capacity of these proteins to bind to host tissue components by enzyme-linked immunosorbent assay. OmpL37 binds elastin preferentially, exhibiting dose-dependent, saturating binding to human skin (K(d), 104±19 nM) and aortic elastin (K(d), 152±27 nM). It also binds fibrinogen (K(d), 244±15 nM), fibrinogen fragment D (K(d), 132±30 nM), plasma fibronectin (K(d), 359±68 nM), and murine laminin (K(d), 410±81 nM). The binding to human skin elastin by both recombinant OmpL37 and live Leptospira interrogans is specifically enhanced by rabbit antiserum for OmpL37, suggesting the involvement of OmpL37 in leptospiral binding to elastin and also the possibility that host-generated antibodies may promote rather than inhibit the adherence of leptospires to elastin-rich tissues. Further, we demonstrate that OmpL37 is recognized by acute and convalescent leptospirosis patient sera and also by Leptospira-infected hamster sera. Finally, OmpL37 protein is detected in pathogenic Leptospira serovars and not in saprophytic Leptospira. Thus, OmpL37 is a novel elastin-binding protein of pathogenic Leptospira that may be promoting attachment of Leptospira to host tissues.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp.

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Souza, Natalie M; Vieira, Monica L; Alves, Ivy J; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2012-09-01

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coli BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K(D)) of 292 ± 24 nm and 157 ± 35 nm, respectively. Moreover, the Lsa30 is a plasminogen (PLG) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

Duration of immunity of a multivalent (DHPPi/L4R) canine vaccine against four Leptospira serovars.

Science.gov (United States)

Wilson, Stephen; Stirling, Catrina; Thomas, Anne; King, Vickie; Plevová, Edita; Chromá, Ludmila; Siedek, Elisabeth; Illambas, Joanna; Salt, Jeremy; Sture, Gordon

2013-06-28

Despite effective vaccines against common Leptospira serovars, the development of new products with long duration of immunity is still important to protect dogs against leptospirosis. The results from four challenge studies performed one year after vaccination of dogs with a multivalent vaccine containing four Leptospira antigens are reported. Six week old dogs received two vaccinations, three weeks apart, and were challenged 367 days later. Clinical observations were recorded, while blood (culture, biochemistry and haematology), urine (culture) and liver and kidney (culture) samples were collected throughout the study or at necropsy. All control dogs remained seronegative until challenge, when they seroconverted. Antibody titres to Leptospira antigens were seen in vaccinated dogs 21 days after first vaccination and peaked three to six weeks after the second vaccination. Titres decreased in all studies over the following 12 months, until challenge when anamnestic responses were observed. In all studies control dogs demonstrated various abnormal clinical signs, while no vaccinated dogs were affected; differences between groups were only significant following L. bratislava challenge. Analysis of blood cultures showed all control and five of the 24 vaccinated dogs were Leptospira positive after challenge; all studies showed significant differences between treatment groups in mean number of days with positive cultures. Significant differences between vaccinated and control groups in mean number of days with positive urine cultures were also observed, with all non-vaccinated and one vaccinated dog Leptospira positive. The urine culture positive vaccinated dog also gave positive culture from kidney and liver samples. All except one control dog also showed positive Leptospira isolation from kidney or liver, with significant differences between vaccinated and control groups observed. The results demonstrate that administration of a new vaccine to six week old puppies

Classification of Leptospira genomospecies 1, 3, 4 and 5 as Leptospira alstonii sp. nov., Leptospira vanthielii sp. nov., Leptospira terpstrae sp. nov. and Leptospira yanagawae sp. nov., respectively

NARCIS (Netherlands)

Smythe, L.; Adler, B.; Hartskeerl, R. A.; Galloway, R. L.; Turenne, C. Y.; Levett, P. N.

2013-01-01

The genus Leptospira currently comprises 16 named species. In addition, four unnamed hybridization groups were designated Leptospira genomospecies 1, 3, 4 and 5. These groups represent valid species-level taxa, but were not assigned names in the original description by Brenner et al. [Int J Syst

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

Science.gov (United States)

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. © The American Society of Tropical Medicine and Hygiene.

LipL32 Is a Subsurface Lipoprotein of Leptospira interrogans: presentation of new data and reevaluation of previous studies.

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Marija Pinne

Full Text Available The agents of leptospirosis, a zoonosis with worldwide distribution, are pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via fresh water and colonization of the renal tubules of their reservoir hosts. Infection of accidental hosts, including humans, may result in life-threatening sequelae. Bacterial outer membrane proteins (OMPs, particularly those with surface-exposed regions, play crucial roles in pathogen virulence mechanisms and adaptation to environmental conditions, including those found in the mammalian host. Therefore, elucidation and characterization of the surface-exposed OMPs of Leptospira spp. is of great interest in the leptospirosis field. A thorough, multi-pronged approach for assessing surface exposure of leptospiral OMPs is essential. Herein, we present evidence for a sub-surface location for most or all of the major leptospiral lipoprotein, LipL32, based on surface immunofluorescence utilizing three different types of antibodies and four different permeabilization methods, as well as surface proteolysis of intact and lysed leptospires. We reevaluate prior evidence presented in support of LipL32 surface-exposure and present a novel perspective on a protein whose location has been misleading researchers, due in large part to its extraordinary abundance in leptospiral cells.

Isolation and characterization of new Leptospira genotypes from patients in Mayotte (Indian Ocean.

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Pascale Bourhy

Full Text Available 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.

A study on the relationship dose-effect in case of gamma irradiation on leptospira

International Nuclear Information System (INIS)

Vasilevska, M.; Yankov, N.; Grigorov, L.

1977-01-01

The influence of gamma rays in doses 400, 2000, 10000 and 100000 rads upon two serotypes of leptospira has been examined. The development of the leptospira after the irradiation was traced upon a dark field of vision and by counting in Petroff-chamber. The morphological and functional changes (size, agility, reproduction and degree of lysis) have been determined and proved to be dependent on the dose. Differences in the indices have been observed in the pathogenic (L.Pomona (local strain Tsalapitsa)) and sapropyte (L.Thraciae (local strain Bulgaria 4)) serotypes, the saprophytic one being more resistant. The age of the culture was significant for the degree of the observed changes. Regarding the eventual practical use of the present study for obtaining killed leptospira antigens doses of 10000 and 100000 rad which are dependent of the age and the pathogenicity of the culture might be taken into consideration. (author)

Decreased erytrocyte osmotic fragility during canine leptospirosis Diminuição da fragilidade osmótica eritrocitária na leptospirose canina

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Marcelo L. Santoro

1994-02-01

Full Text Available Erythrocyte osmotic fragility (EOF was carried out in nineteen dogs naturally infected by Leptospira interrogans serovar icterohaemorrhagiae/copenhagi. A decreased EOF was observed, suggesting a modification of erythrocyte components secondary to disturbances that occur during canine leptospirosis, such as renal damage and hepatic disease.A fragilidade osmótica eritrocitária foi estudada em dezenove cães infectados naturalmente pela Leptospira interrogans serovar icterohaemorrhagiae/copenhagi. Observou-se uma redução da fragilidade osmótica eritrocitária, sem a presença de anemia, possivelmente relacionada aos distúrbios hepato-renais que ocorrem nesta patologia.

Spatial characterization of Leptospira spp. infection in equids from the Brejo Paraibano micro-region in Brazil

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Ruy Brayner Oliveira Filho

2014-05-01

Full Text Available The present study, the first to spatially characterize Leptospira spp. infection among equids in the Brejo Paraibano micro-region of the Paraiba state in the northeast of Brazil, investigated 257 animals in 26 farms properties. Serum samples from 204 horses, 46 mules and seven donkeys were serologically diagnosed using the microscopic agglutination test (MAT. The distribution of Leptospira spp. was studied by employing specific antigens from 24 different Leptospira serovars. All farms were georeferenced and their distribution visualised on a map of the Brejo Paraibano micro-region. In addition, rainfall data were obtained from the same year, in which the sampling was performed. Among the 20 farms found to harbour animals with leptospirosis, 14 (70% exhibited low prevalence, five (25% medium prevalence and one (5%, high prevalence. Certain areas had a higher density of infected farms and required intervention to control the infection. Many serovars were widely distributed, while others were more common in particular areas. There was no significant association between the prevalence of Leptospira spp. infection and rainfall.

Leptospira spp. vaccinal antibodies do not react with Borrelia burgdorferi peptides used in the AccuPlex 4.

Science.gov (United States)

Caress, Amber L; Moroff, Scott; Lappin, Michael R

2017-11-01

We attempted to determine if Leptospira spp. antibodies induced by vaccination would cross-react with Borrelia burgdorferi antigens used in a commercial automated immunofluorescent assay (AccuPlex 4 BioCD; Antech). Staff- and student-owned dogs ( n = 31) were recruited at a veterinary teaching hospital in a B. burgdorferi nonendemic area. The dogs were randomized and administered 1 of 4 commercial Leptospira spp. vaccines that contained serovars Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona, then booster vaccinated 3 wk later. Blood was collected on weeks 0, 3, 4, 8, and 12. After confirming that maximal Leptospira spp. titers occurred on week 4, aliquots of sera from week 4 were shipped frozen for analysis of B. burgdorferi antibodies against OspA, OspC, OspF, P39, and SLP with the AccuPlex system. Week 4 sera from all 31 dogs had a titer of 1:100 for at least 1 Leptospira spp. serovar. Titers of 1:800 or greater were detected against multiple serovars in 27 dogs. None of the samples contained antibodies against the B. burgdorferi OspA, OspC, OspF, P39, and SLP peptides used in the commercial assay. The B. burgdorferi peptides used in the AccuPlex system do not recognize naturally occurring Leptospira spp. antibodies or those induced by the commercial Leptospira spp. vaccines administered in our study.

9 CFR 113.102 - Leptospira Icterohaemorrhagiae Bacterin.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Leptospira Icterohaemorrhagiae... REQUIREMENTS Inactivated Bacterial Products § 113.102 Leptospira Icterohaemorrhagiae Bacterin. Leptospira Icterohaemorrhagiae Bacterin shall be produced from a culture of Leptospira icterohaemorrhagiae which has been...

9 CFR 113.104 - Leptospira Grippotyphosa Bacterin.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Leptospira Grippotyphosa Bacterin. 113... REQUIREMENTS Inactivated Bacterial Products § 113.104 Leptospira Grippotyphosa Bacterin. Leptospira Grippotyphosa Bacterin shall be produced from a culture of Leptospira grippotyphosa which has been inactivated...

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin.

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Figueira, Cláudio Pereira; Croda, Julio; Choy, Henry A; Haake, David A; Reis, Mitermayer G; Ko, Albert I; Picardeau, Mathieu

2011-06-09

In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.

Newly recognized Leptospira species ("Leptospira inadai" serovar lyme) isolated from human skin.

OpenAIRE

Schmid, G P; Steere, A C; Kornblatt, A N; Kaufmann, A F; Moss, C W; Johnson, R C; Hovind-Hougen, K; Brenner, D J

1986-01-01

Leptospira strain 10, which represents a new Leptospira species, was isolated from a skin biopsy of a patient with Lyme disease. Although pathogenic for laboratory animals, the organism was not considered to have a significant role in the patient's illness.

9 CFR 113.101 - Leptospira Pomona Bacterin.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Leptospira Pomona Bacterin. 113.101... Inactivated Bacterial Products § 113.101 Leptospira Pomona Bacterin. Leptospira Pomona Bacterin shall be produced from a culture of Leptospira pomona which has been inactivated and is nontoxic. Each serial of...

9 CFR 113.103 - Leptospira Canicola Bacterin.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Leptospira Canicola Bacterin. 113.103... Inactivated Bacterial Products § 113.103 Leptospira Canicola Bacterin. Leptospira Canicola Bacterin shall be produced from a culture of Leptospira canicola which has been inactivated and is nontoxic. Each serial of...

9 CFR 113.105 - Leptospira Hardjo Bacterin.

Science.gov (United States)

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 of...

Glicolipoproteína de Leptospira interrogans sorogrupo icterohaemorrhagiae: distribuição em fígado e rim de cobaias experimentalmente infectadas

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R.T. Macêdo Santos

1989-08-01

Full Text Available Acredita-se que as lesões teciduais na leptospirose possam decorrer da ação direta das leptospiras, de toxinas sintetizadas ou liberadas durante sua lise. O presente estudo visou a extração quimica da glicolipoproteína (GLP da leptospira, a produção de anti-soro anti-GLP e a avaliação de sua distribuição em cortes de fígado e rim de cobaias inoculadas e sacrificadas em estudo seqüencial diário até o 6°dia de infecção, correspondente ao pico da doença. Procurou-se também correlacionar a expressão tecidual da GLP com o grau de lesões locais, em busca de novos subsídios para a compreensão da patogenia da leptospirose. A QLP foi detectada em fígado e rim de 2 dentre 6 cobaias no 5°dia e em todas as 6 no 6° dia de infecção, sob a forma de grânulos no citoplasma de macrófagos, livres no interstício ou acolados à membrana de células endoteliais e parenquimatosas, especialmente nas regiões mais lesadas. A cronologia do aparecimento da GLP e sua distribuição sugerem tratar-se de produto dc lise de leptospiras fagocitadas por macrófagos e que esta substância, conquanto não comprovada como iniciadora das lesões, associa-se a seu agravamento nas etapas mais avançadas da leptospirose.

A novel approach for differentiating pathogenic and non-pathogenic Leptospira based on molecular fingerprinting.

Science.gov (United States)

Xiao, Di; Zhang, Cuicai; Zhang, Huifang; Li, Xiuwen; Jiang, Xiugao; Zhang, Jianzhong

2015-04-24

disease that has become an important public health problem. Traditional serological methods are the gold standard for the detection of pathogenic strains of Leptospira. However, serological procedures are cumbersome, require more complex experimental techniques, and are based on a large number of international and domestic reference strains. Additionally, these experiments involve the immunization of animals with antigens from different serotypes to produce immune serum, and improper techniques may result in a rapid decrease in antibody titer, which would affect the final results. It is difficult to perform cumbersome detection procedures in a basic laboratory. Therefore, the use of conventional serological methods is limited, which significantly impacts daily leptospirosis epidemic surveillance, prevention, and control. Molecular biology methods, such as 16S rRNA and PCR-based methods, can be used to identify the pathogenic Leptospira. However, DNA extraction and gene sequencing methods are laborious and time consuming. Therefore, more rapid and reliable high-throughput identification methods are urgently needed for the clinical diagnosis of leptospirosis to improve epidemic control. Here, molecular fingerprinting technique was use to identify the pathogenicity. We constructed the reference spectra database and the super reference spectra of pathogenic and non-pathogenic Leptospira, which can rapidly identified Leptospira at the species level and the pathogenicity of these isolates can be simultaneously confirmed. Furthermore, the protein components of Leptospira pathogenicity were revealed. These findings thus provide a new way for Leptospira pathogenicity identification. Copyright © 2014. Published by Elsevier B.V.

A Western-blot assay for the detection of antibodies against pathogenic Leptospira serogroups with recombinant outer membrane protein LipL32

OpenAIRE

Hong-yuan DUAN; Zhi-guo LIU; Shao-fu QIU; Bin HE; Hai ZHAO; Li-hua SONG; Hong ZHU; Qing DUAN

2011-01-01

Objective To provide a possible antigen for rapid serodiagnosis of leptospirosis,the present study focused on the activity of immune-reaction and cross-reaction between outer membrane protein LipL32 and multi-serogroup anti-pathogenic Leptospira antibodies.Methods Based on the given sequence of LipL32 gene of Leptospira icterohaemorrhagiae strain 56601,the primer pair was designed and the DNA fragment was amplified by PCR.The amplified product was inserted into vector pET-28a-(c) to construct...

A study of the effect of fractional gamma irradiation of leptospirae

International Nuclear Information System (INIS)

Vasilevska, M.; Khristova, M.; Yankova, N.; Pandev, I.

1977-01-01

In view of the theoretical and practical interest in obtaining antigens from killed leptospirae for immune or diagnostic serums, a two hour leptospiral culture from the pathogenic strain Pomona (Calapica) was fractionally gamma-irradiated with a total dose of 2 Mrad. Changes were observed in the size, motility and survival duration of the leptospiral strains. The results are interpreted and compared with those of single administration of the same dose, studied in a previous work. (author)

Temperature and Oxidative Stress as Triggers for Virulence Gene Expression in Pathogenic Leptospira spp.

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Tricia Fraser

2017-05-01

Full Text Available Leptospirosis is a zooanthroponosis aetiologically caused by pathogenic bacteria belonging to the genus, Leptospira. Environmental signals such as increases in temperatures or oxidative stress can trigger response regulatory modes of virulence genes during infection. This study sought to determine the effect of temperature and oxidative stress on virulence associated genes in highly passaged Leptospira borgpeterseneii Jules and L. interrogans Portlandvere. Bacteria were grown in EMJH at 30°C, 37°C, or at 30°C before being transferred to 37°C. A total of 14 virulence-associated genes (fliY, invA, lenA, ligB, lipL32, lipL36, lipL41, lipL45, loa22, lsa21, mce, ompL1, sph2, and tlyC were assessed using endpoint PCR. Transcriptional analyses of lenA, lipL32, lipL41, loa22, sph2 were assessed by quantitative real-time RT-PCR at the temperature conditions. To assess oxidative stress, bacteria were exposed to H2O2 for 30 and 60 min with or without the temperature stress. All genes except ligB (for Portlandvere and ligB and mce (for Jules were detectable in the strains. Quantitatively, temperature stress resulted in significant changes in gene expression within species or between species. Temperature changes were more influential in gene expression for Jules, particularly at 30°C and upshift conditions; at 37°C, expression levels were higher for Portlandvere. However, compared to Jules, where temperature was influential in two of five genes, temperature was an essential element in four of five genes in Portlandvere exposed to oxidative stress. At both low and high oxidative stress levels, the interplay between genetic predisposition (larger genome size and temperature was biased towards Portlandvere particularly at 30°C and upshift conditions. While it is clear that expression of many virulence genes in highly passaged strains of Leptospira are attenuated or lost, genetic predisposition, changes in growth temperature and/or oxidative intensity and

Leptospira Exposure and Gardeners: A Case-Control Seroprevalence Study

Science.gov (United States)

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

21 CFR 866.3350 - Leptospira spp. serological reagents.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leptospira spp. serological reagents. 866.3350... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3350 Leptospira spp. serological reagents. (a) Identification. Leptospira spp. serological reagents are devices that...

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African Journals Online (AJOL)

... Comparison of the effects of UV-A radiation on Leptospira interrogan serovar ... Comparison of three methods for determination of protein concentration in lactic .... differential gene expression from the xerophyte Ammopiptanthus mongolicus ...

A quantitative PCR (TaqMan assay for pathogenic Leptospira spp

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Symonds Meegan L

2002-07-01

Full Text Available Abstract Background Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA and slide agglutination test (SAT, can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. Methods The polymerase chain reaction (PCR has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. Results and Conclusions The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.

Leptospira mayottensis sp. nov., a pathogenic species of the genus Leptospira isolated from humans.

Science.gov (United States)

Bourhy, Pascale; Collet, Louis; Brisse, Sylvain; Picardeau, Mathieu

2014-12-01

A group of strains representing species of the genus Leptospira, isolated from patients with leptospirosis in Mayotte (Indian Ocean), were previously found to be considerably divergent from other known species of the genus Leptospira. This was inferred from sequence analysis of rrs (16S rRNA) and other genetic loci and suggests that they belong to a novel species. Two strains from each serogroup currently identified within this novel species were studied. Spirochaete, aerobic, motile, helix-shaped strains grew well at 30-37 °C, but not at 13 °C or in the presence of 8-azaguanine. Draft genomes of the strains were also analysed to study the DNA relatedness with other species of the genus Leptospira. The new isolates formed a distinct clade, which was most closely related to Leptospira borgpetersenii, in multilocus sequence analysis using concatenated sequences of the genes rpoB, recA, fusA, gyrB, leuS and sucA. Analysis of average nucleotide identity and genome-to-genome distances, which have recently been proposed as reliable substitutes for classical DNA-DNA hybridization, further confirmed that these isolates should be classified as representatives of a novel species. The G+C content of the genomic DNA was 39.5 mol%. These isolates are considered to represent a novel species, for which the name Leptospira mayottensis sp. nov. is proposed, with 200901116(T) ( = CIP 110703(T) = DSM 28999(T)) as the type strain. © 2014 IUMS.

SEROPREVALENCIA Y SEROTIPIFICACIÓN DE LEPTOSPIROSIS CANINA EN EL MUNICIPIO DE BUENAVENTURA (VALLE DEL CAUCA)

OpenAIRE

Romero Peñuela, Marlyn Hellen; Astudillo Hernández, Myriam; Quintero Martínez, Mauricio Elías

2009-01-01

El presente estudio tuvo como objetivo verificar la infección con serovares de Leptospira spp. en caninos con propietario en Buenaventura, Valle del Cauca, Colombia. Las muestras de suero fueron tomadas de 100 caninos y fueron probadas con los serogrupos de Leptospira interrogans: Bataviae, Mini, Autumnalis, Canicola, Shermani, Icterohaemorrhagiae, Cynopteri, Australis, Celledonis, Grippotyphosa, Hebdomadis, Javanica, Manhao, Pomona, Pyrogenes, Sejroe, Tarassovi y Ballum, usando la técnica de...

Structure and calcium binding activity of LipL32, the major surface antigen of pathogenic Leptospira sp

Energy Technology Data Exchange (ETDEWEB)

Hauk, Pricila; Roman-Ramos, Henrique; Ho, Paulo Lee [Instituto Butantan, Sao Paulo, SP (Brazil). Centro de Biotecnologia; Guzzo, Cristiane R.; Farah, Chuck S. [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Quimica. Dept. de Bioquimica

2009-07-01

Leptospirosis, caused by the spirochaete Leptospira is an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospira. It is highly immunogenic and has been shown to bind to host extracellular matrix components, including collagens, fibronectin and laminin. In this work we crystallized recombinant LipL32 protein and determined its structure to 2.25 A resolution. Initial phases were determined using the multi-wavelength anomalous dispersion technique with data collected from selenomethionine-containing crystals at the MX2 beamline at the LNLS. The LipL32 monomer is made of a jelly-roll fold core from which protrude several peripheral secondary structures. Some structural features suggested that LipL32 could bind Ca{sup 2+} ions and indeed, spectroscopic data (circular (dichroism. intrinsic tryptophan fluorescence and extrinsic 1-amino-2-anaphthol-4-sulfonic acid fluorescence) confirmed the calcium binding properties of LipL32. (author)

Structure and calcium binding activity of LipL32, the major surface antigen of pathogenic Leptospira sp

International Nuclear Information System (INIS)

Hauk, Pricila; Roman-Ramos, Henrique; Ho, Paulo Lee; Guzzo, Cristiane R.; Farah, Chuck S.

2009-01-01

Leptospirosis, caused by the spirochaete Leptospira is an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospira. It is highly immunogenic and has been shown to bind to host extracellular matrix components, including collagens, fibronectin and laminin. In this work we crystallized recombinant LipL32 protein and determined its structure to 2.25 A resolution. Initial phases were determined using the multi-wavelength anomalous dispersion technique with data collected from selenomethionine-containing crystals at the MX2 beamline at the LNLS. The LipL32 monomer is made of a jelly-roll fold core from which protrude several peripheral secondary structures. Some structural features suggested that LipL32 could bind Ca 2+ ions and indeed, spectroscopic data (circular (dichroism. intrinsic tryptophan fluorescence and extrinsic 1-amino-2-anaphthol-4-sulfonic acid fluorescence) confirmed the calcium binding properties of LipL32. (author)

[Genotyping and evaluation of infection dynamics in a Colombian isolate of Leptospira santarosai in hamster as an experimental model].

Science.gov (United States)

Agudelo-Flórez, Piedad; Durango, Harold; Aranzazu, Diego; Rodas, Juan David; Travi, Bruno

2014-01-01

Is necessary to develop models for the study of leptospirosis. To genotype a Colombian strain of Leptospira isolated from a human with Weil´s syndrome and to evaluate its infection dynamics in the hamster experimental model. Genotyping was performed by amplification and sequence analysis of the rrs 16S and lipL32 genes. The median lethal dose was determined in intraperitoneally inoculated hamsters. The patterns of clinical chemistry, the duration of leptospiremia, leptospiruria and pathological findings were studied and compared in the same animal model infected with L. interrogans (Fiocruz L1-130). Molecular typing revealed that the isolate corresponded to the pathogenic species L. santarosai, which was recovered from hamsters´ kidneys and lungs and detected by lipL32 PCR from day 3 post-infection in these organs. There was a marked increase of C-reactive protein in animals at day 5 post-infection (3.25 mg/dl; normal value: 0.3 mg/dl) with decreases by day 18 (2.60 mg/dl: normal value: 0.8 mg/dl). Biomarkers of urea showed changes consistent with possible renal acute failure (day 5 post-infection: 49.01 mg/dl and day 18 post-infection: 53.71 mg/dl). Histopathological changes included interstitial pneumonia with varying degrees of hemorrhage and interstitial nephritis. The pathogenic species L. santarosai was identified in Colombia. Its pathogenicity as determined by tropism to lung and kidney was comparable to that of L. interrogans Fiocruz L1-130, well known for its virulence and pulmonar tropism. The biological aspects studied here had never before been evaluated in an autochthonous isolate.

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

Science.gov (United States)

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.

Complement Evasion by Pathogenic Leptospira.

Science.gov (United States)

Fraga, Tatiana Rodrigues; Isaac, Lourdes; Barbosa, Angela Silva

2016-01-01

Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira . Pathogenic microorganisms, notably those which reach the blood circulation such as Leptospira , have evolved multiple strategies to escape the host complement system, which is important for innate and acquired immunity. Leptospira avoid complement-mediated killing through: (i) recruitment of host complement regulators; (ii) acquisition of host proteases that cleave complement proteins on the bacterial surface; and, (iii) secretion of proteases that inactivate complement proteins in the Leptospira surroundings. The recruitment of host soluble complement regulatory proteins includes the acquisition of Factor H (FH) and FH-like-1 (alternative pathway), C4b-binding protein (C4BP) (classical and lectin pathways), and vitronectin (Vn) (terminal pathway). Once bound to the leptospiral surface, FH and C4BP retain cofactor activity of Factor I in the cleavage of C3b and C4b, respectively. Vn acquisition by leptospires may result in terminal pathway inhibition by blocking C9 polymerization. The second evasion mechanism lies in plasminogen (PLG) binding to the leptospiral surface. In the presence of host activators, PLG is converted to enzymatically active plasmin, which is able to degrade C3b, C4b, and C5 at the surface of the pathogen. A third strategy used by leptospires to escape from complement system is the active secretion of proteases. Pathogenic, but not saprophytic leptospires, are able to secrete metalloproteases that cleave C3 (central complement molecule), Factor B (alternative pathway), and C4 and C2 (classical and lectin pathways). The purpose of this review is to fully explore these complement evasion mechanisms, which act together to favor Leptospira survival and multiplication in the host.

Pathogenic Leptospira spp. in bats: Molecular investigation in Southern Brazil.

Science.gov (United States)

Mayer, Fabiana Quoos; Dos Reis, Emily Marques; Bezerra, André Vinícius Andrade; Cerva, Cristine; Rosa, Júlio; Cibulski, Samuel Paulo; Lima, Francisco Esmaile Sales; Pacheco, Susi Missel; Rodrigues, Rogério Oliveira

2017-06-01

The present study aimed to investigate the frequency of pathogenic Leptospira spp. in Brazilian bats and to determine possible risk factors associated to it. Ninety two bats of 12 species were evaluated. Whole genomic DNA from kidneys was extracted and real-time PCR specific to pathogenic Leptospira spp. was applied. Association between the frequency of specimens positive for Leptospira spp. and sex, age, bat species or family, season of collection, geographic localization and feeding habits was evaluated. The results showed that 39.13% of analyzed bats were found positive for Leptospira spp. Nine bat species had at least one positive result. There was no association among the evaluated variables and frequency of pathogenic Leptospira spp. Although the limitations due to lack of Leptospira spp. isolation, leptospiral carriage was demonstrated in bats of different species from southern Brazil, which reinforces the need for surveillance of infectious agents in wild animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

X-ray analysis in lung leptospira disease

International Nuclear Information System (INIS)

Deng Shiyong; Peng Shi; He Guoman

2006-01-01

Objective: To analysis the X-ray signs and subtype of the lung leptospira disease, and improve the undersdand, reduce the error diagnosis of this diseases. Methods: 40 cases of lung leptospira disease were evaluated about the check X-ray sings and clinical data, the check X-ray sings were dynamic observated and typed, and 40 cases had a diagnostic treatment. Results: There were various X-ray changes of lung leptospira disease. in 40 cases, 12 cases (30%) pulmonary marking, 21 cases (52%) little lesions, and 7 cases(18%) lager lesions, respectively. The patients who were correctly diagnosed made a recovery after effective treatment, the patients who were error diagnosed died because of multiple system organ damage. Conclusion: The check X-ray signs in lung leptospira disease have some characteristics. It may play an important role in improving this disease' diagnosis combining the dynamic observation of check X-ray sings with clinical data. (authors)

Signaling via ITGB1/FAK and microfilament rearrangement mediates the internalization of Leptospira interrogans in mouse J774A.1 macrophages

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Zhao Xin

2015-01-01

Full Text Available Leptospirosis caused by pathogenic Leptospira species is a worldwide zoonotic 2 infectious disease, but the mechanisms of leptospiral internalization remain poorly understood. Here, we report that mouse J774A.1 macrophages expressed integrin-subfamily proteins (ITGB1, ITGB2 and ITGB3. Antibody blockage and siRNA-based knockdown of ITGB1 decreased the internalization of leptospires into mouse J774A.1 macrophage cells. The internalization required focal adhesion kinase (FAK activation in J774A.1 cells rather than phosphoinositide-3-kinase (PI3K, and microfilament rather than microtubule aggregation during infection. The data indicated that the ITGB1/FAK/microfilament signaling pathway is responsible for leptospiral internalization in mouse macrophages.

The OmpA-like protein Loa22 is essential for leptospiral virulence.

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Paula Ristow

2007-07-01

Full Text Available Pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetic manipulations of pathogenic species. In this study, we characterized a mutant obtained by insertion of the transposon Himar1 into a gene encoding a putative lipoprotein, Loa22, which has a predicted OmpA domain based on sequence identity. The resulting mutant did not express Loa22 and was attenuated in virulence in the guinea pig and hamster models of leptospirosis, whereas the genetically complemented strain was restored in Loa22 expression and virulence. Our results show that Loa22 was expressed during host infection and exposed on the cell surface. Loa22 is therefore necessary for virulence of L. interrogans in the animal model and represents, to our knowledge, the first genetically defined virulence factor in Leptospira species.

PATHOGENIC LEPTOSPIRA CONTAMINATION IN HOUSEHOLD WATER IN SETTLEMENT AREA OF DEMAK REGENCY

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Dyah Widiastuti

2016-01-01

Full Text Available Leptospirosis merupakan penyakit zoonosis penting di dunia termasuk Indonesia. Penularan penyakit ini dapat terjadi melalui kontak dengan air yang terkontaminasi bakteri Leptospira patogen. Tujuan penelitian ini untuk mendeteksi Leptospira patogen pada air konsumsi di pemukiman Kabupaten Demak. Penelitian observasional ini dilaksanakan pada bulan Juli 2014. Sebanyak 15 sampel air konsumsi dikumpulkan dari area pemukiman di sekitar kasus leptospirosis terbaru di Kabupaten Demak. Sampel diperiksa menggunakan Polymerase Chain Reaction (PCR untuk mendeteksi kontaminasi Leptospira patogenik dengan gen target LipL32. Lima sampel (46,7% menunjukkan kontaminasi positif dari spesies patogenik dari Leptospira berdasarkan PCR. Leptospira patogenik dapat dideteksi dalam lingkungan Demak dan ini berpotensi menyebabkan terjadinya penularan leptospirosis. Kata kunci: Leptospira, air konsumsi, Demak

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

Science.gov (United States)

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

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

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Derrick E Fouts

2016-02-01

Full Text Available 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

Hematologic changes in dogs naturally infected Leptospira spp., Brucella abortus and Brucella canis

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Jacqueline Ribeiro de Castro

2014-01-01

Full Text Available ABSTRACT. Castro J.R., Silva C.B., Souza M.A., Salaberry S.R.S., Guimarães E.C., Mundim A.V. & Lima-Ribeiro A.M.C. [Hematologic changes in dogs naturally infected Leptospira spp., Brucella abortus and Brucella canis.] Altera- ções hematológicas em cães naturalmente infectados por Leptospira spp., Brucella abortus e Brucella canis. Revista Brasileira de Medicina Veterinária, 36(1:49-54, 2014. Laboratório de Doenças Infectocontagiosas, Faculdade de Medicina Veterinária, Universidade Federal de Uberlândia, Av. Ceará s/n, Bloco 2D, Sala 33, Campus Umuarama, Uberlândia, MG 38400-902, Brasil. E-mail: jack_ufu@yahoo.com.br The investigations of leptospirosis and brucellosis canine act as sanitary control in public health and zoonoses because they were established by close contact between dog and human. The aim was to determine the main hematological reagents in asymptomatic dogs against Leptospira spp. Brucella abortus and Brucella canis naturally infected, living in urban areas in the city of Uberlandia, Minas Gerais. We examined 140 blood samples from clinically healthy dogs, males and females and different ages. Leptospirosis was diagnosed by microscopic agglutination test (MAT, with a collection of twelve serovars, whereas, brucellosis was identified through the tests of Agar Gel Immunodiffusion (AGID for B. canis and buffered acidified antigen (TAA confirmed 2-Mercaptoethanol (2-ME for B. abortus. The results were analyzed using descriptive statistics with the calculation of simple percentages, mean and standard deviation. He applied and short sample t test for two independent samples to assess whether there were significant differences (p<0.05 between hematological parameters obtained. Dogs evaluated, 15% (21/140 and 2.85% (4/140 were reactive to Leptospira spp. and B. abortus, respectively. There was no sample reagent against B. canis. It was concluded that although no specific thrombocytopenia may be a significant finding in dogs

Investigation on the presence of leptospires in ovaries of hamsters experimentally infected whith Leptospiras interrogans serovar pomona

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Claudio Roberto de Almeida Camargo

1993-12-01

Full Text Available After inoculating L. interrogans serovar pomona in 75 primiparous hamsters (Mesocricetus auratus, the invasiveness of leptospires into lhe ovaries and lhe ability in causing ovary morphologic alterations were investigated by means of microscopic examination and bacterial isolation. For this purpose, 75 hamsters were inoculated with 0.5 ml of virulent strain containing 30-40 leptospires by the microscopic field and the other 15 hamsters were held as the uninfected controls. Signs and symptoms (prostration, tachypnea, rufled hair, jaundice, and nasal, bucal and perineal hemorrage were detected in all inoculated animals. The animals were killed in the agonic state of the illness, which were done through 4th and 7th day post inoculation. The ovaries were taken asseptically during the necropsies, thoroughly washed using the sterile phosphate buffered saline, in order to eliminate the possible external contamination. The fresh ovary samples were submitted to the dark field direct microscopic examination. After the formalin fixation, the specimens were stained by means of histopathologic techniques using the Levaditi and Hematoxylin Eosin stains. The ovary smears were also examined by the direct fluorescent antibody technique andlhe bacterial isolation was carried out in the Fletcher’s medium. The dark field direct microscopic examination was found tobe less sensitive in demonstrating the presence of leptospiresin the ovaries. In those specimens stained by the Lcvadititechnique, leptospires were visualized in different ovaryinternal structures, involving the interspace, pellucid zone andin the inner ovules. Through the histopathologic examination,typical morphologic alterations resembling acute infiamatoryprocess were found in 57% of ovaries examined.

Diversity of Leptospira spp. in Rats and Environment from Urban Areas of Sarawak, Malaysia

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Pui, Chai Fung; Apun, Kasing; Su'ut, Lela

2017-01-01

Various prevalence studies on Leptospira in animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophytic Leptospira in selected animals and environments. This study was therefore conducted to detect Leptospira spp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR) method. A total of 107 rats, 292 soil samples, and 324 water samples were collected from April 2014 to February 2015. Pathogenic Leptospira was present in 5.6% (6/107) of rats, 11.6% (34/292) of soil samples, and 1.9% (6/324) of water samples. Intermediate Leptospira was present in 2.7% (8/292) of soil samples and 1.9% (6/324) of water samples. Saprophytic Leptospira was present in 10.3% (11/107) of rats, 1.4% (4/292) of soil samples, and 0.3% (1/324) of water samples. From this study, 76 Leptospira spp. were isolated. Based on DNA sequencing, the dominant Leptospira spp. circulating in urban areas of Sarawak are pathogenic Leptospira noguchii, intermediate Leptospira wolffii serovar Khorat, and saprophytic Leptospira meyeri, respectively. Overall, this study provided important surveillance data on the prevalence of Leptospira spp. from rats and the environment, with dominant local serovars in urban areas of Sarawak. PMID:28348601

Diversity of Leptospira spp. in Rats and Environment from Urban Areas of Sarawak, Malaysia.

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Pui, Chai Fung; Bilung, Lesley Maurice; Apun, Kasing; Su'ut, Lela

2017-01-01

Various prevalence studies on Leptospira in animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophytic Leptospira in selected animals and environments. This study was therefore conducted to detect Leptospira spp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR) method. A total of 107 rats, 292 soil samples, and 324 water samples were collected from April 2014 to February 2015. Pathogenic Leptospira was present in 5.6% (6/107) of rats, 11.6% (34/292) of soil samples, and 1.9% (6/324) of water samples. Intermediate Leptospira was present in 2.7% (8/292) of soil samples and 1.9% (6/324) of water samples. Saprophytic Leptospira was present in 10.3% (11/107) of rats, 1.4% (4/292) of soil samples, and 0.3% (1/324) of water samples. From this study, 76 Leptospira spp. were isolated. Based on DNA sequencing, the dominant Leptospira spp. circulating in urban areas of Sarawak are pathogenic Leptospira noguchii , intermediate Leptospira wolffii serovar Khorat, and saprophytic Leptospira meyeri , respectively. Overall, this study provided important surveillance data on the prevalence of Leptospira spp. from rats and the environment, with dominant local serovars in urban areas of Sarawak.

Diversity of Leptospira spp. in Rats and Environment from Urban Areas of Sarawak, Malaysia

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Chai Fung Pui

2017-01-01

Full Text Available Various prevalence studies on Leptospira in animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophytic Leptospira in selected animals and environments. This study was therefore conducted to detect Leptospira spp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR method. A total of 107 rats, 292 soil samples, and 324 water samples were collected from April 2014 to February 2015. Pathogenic Leptospira was present in 5.6% (6/107 of rats, 11.6% (34/292 of soil samples, and 1.9% (6/324 of water samples. Intermediate Leptospira was present in 2.7% (8/292 of soil samples and 1.9% (6/324 of water samples. Saprophytic Leptospira was present in 10.3% (11/107 of rats, 1.4% (4/292 of soil samples, and 0.3% (1/324 of water samples. From this study, 76 Leptospira spp. were isolated. Based on DNA sequencing, the dominant Leptospira spp. circulating in urban areas of Sarawak are pathogenic Leptospira noguchii, intermediate Leptospira wolffii serovar Khorat, and saprophytic Leptospira meyeri, respectively. Overall, this study provided important surveillance data on the prevalence of Leptospira spp. from rats and the environment, with dominant local serovars in urban areas of Sarawak.

[TYPING OF LEPTOSPIRA SPP. STRAINS BASED ON 16S rRNA].

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Ostankova, Yu V; Semenov, A V; Stoyanova, N A; Tokarevich, N K; Lyubimova, N E; Petrova, O A; Ananina, Yu V; Petrov, E M

2016-01-01

Comparative typing of Leptospira spp. strain collection based on analysis of 16S RNA fragment. 2 pairs of primers were used for PCR, that jointly flank 1423b.p. sized fragment. Sequences of Leptospira spp. strain 16S rRNA, presented in the international database, were used for phylogenetic analysis. A high similarity, including interspecies, of the 16S fragment in Leptospira spp. strains was shown independently of the source, serovar and serogroup. Heterogeneity of the primary matrix, spontaneous mutations of hotspots and erroneous nucleotide couplings, characteristic for 16S sequence of pathogenic Leptospira spp. strains, are discussed. Molecular-genetic characteristic of certain reference Leptospira spp. strains by 16S sequence is obtained. Results of the studies give evidence on expedience of introduction into clinical practice of identification of Leptospira spp. by 16S sequence directly from the clinical material, that would allow to significantly reduce identification time, dismiss complex type-specific sera and other labor-intensive methods.

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

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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. © The American Society of Tropical Medicine and Hygiene.

H+ and Na+ are involved in flagellar rotation of the spirochete Leptospira

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Islam, Md. Shafiqul; Morimoto, Yusuke V.; Kudo, Seishi; Nakamura, Shuichi

2015-01-01

Leptospira is a spirochete possessing intracellular flagella. Each Leptospira flagellar filament is linked with a flagellar motor composed of a rotor and a dozen stators. For many bacterial species, it is known that the stator functions as an ion channel and that the ion flux through the stator is coupled with flagellar rotation. The coupling ion varies depending on the species; for example, H + is used in Escherichia coli, and Na + is used in Vibrio spp. to drive a polar flagellum. Although genetic and structural studies illustrated that the Leptospira flagellar motor also contains a stator, the coupling ion for flagellar rotation remains unknown. In the present study, we analyzed the motility of Leptospira under various pH values and salt concentrations. Leptospira cells displayed motility in acidic to alkaline pH. In the presence of a protonophore, the cells completely lost motility in acidic to neutral pH but displayed extremely slow movement under alkaline conditions. This result suggests that H + is a major coupling ion for flagellar rotation over a wide pH range; however, we also observed that the motility of Leptospira was significantly enhanced by the addition of Na + , though it vigorously moved even under Na + -free conditions. These results suggest that H + is preferentially used and that Na + is secondarily involved in flagellar rotation in Leptospira. The flexible ion selectivity in the flagellar system could be advantageous for Leptospira to survive in a wide range of environment. - Highlights: • This is a study on input energy for motility in the spirochete Leptospira. • Leptospira biflexa exhibited active motility in acidic to alkaline pH. • Both H + and Na + are involved in flagellar rotation in Leptospira. • H + is a primary energy source, but Na + can secondarily enhance motility.

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

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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

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

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

2015-08-01

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. 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. 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. 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.

Quantification of pathogenic Leptospira in the soils of a Brazilian urban slum.

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Andrew G Schneider

2018-04-01

Full Text Available Leptospirosis is an important zoonotic disease that causes considerable morbidity and mortality globally, primarily in residents of urban slums. While contact with contaminated water plays a critical role in the transmission of leptospirosis, little is known about the distribution and abundance of pathogenic Leptospira spp. in soil and the potential contribution of this source to human infection.We collected soil samples (n = 70 from three sites within an urban slum community endemic for leptospirosis in Salvador, Brazil. Using qPCR of Leptospira genes lipl32 and 16S rRNA, we quantified the pathogenic Leptospira load in each soil sample. lipl32 qPCR detected pathogenic Leptospira in 22 (31% of 70 samples, though the median concentration among positive samples was low (median = 6 GEq/g; range: 4-4.31×102 GEq/g. We also observed heterogeneity in the distribution of pathogenic Leptospira at the fine spatial scale. However, when using 16S rRNA qPCR, we detected a higher proportion of Leptospira-positive samples (86% and higher bacterial concentrations (median: 4.16×102 GEq/g; range: 4-2.58×104 GEq/g. Sequencing of the qPCR amplicons and qPCR analysis with all type Leptospira species revealed that the 16S rRNA qPCR detected not only pathogenic Leptospira but also intermediate species, although both methods excluded saprophytic Leptospira. No significant associations were identified between the presence of pathogenic Leptospira DNA and environmental characteristics (vegetation, rat activity, distance to an open sewer or a house, or soil clay content, though samples with higher soil moisture content showed higher prevalences.This is the first study to successfully quantify the burden of pathogenic Leptospira in soil from an endemic region. Our results support the hypothesis that soil may be an under-recognized environmental reservoir contributing to transmission of pathogenic Leptospira in urban slums. Consequently, the role of soil should be

ETIOLOGY OF YELLOW FEVER : X. COMPARATIVE IMMUNOLOGICAL STUDIES ON LEPTOSPIRA ICTEROIDES AND LEPTOSPIRA ICTEROHAEMOORRHAGIAE.

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Noguchi, H

1920-01-31

It has been previously reported that a filterable microorganism belonging to the genus Leptospira has been recovered from the blood or organs of human beings suffering from the disease known as yellow fever in Guayaquil, and that the organism, which has been termed Leptospira icteroides, induces in certain experimental animals the characteristic symptoms and lesions observed in the patients from whom it was isolated. It has also been previously shown that the serum from patients recovering from an attack of yellow fever in Guayaquil had the power to agglutinate and dissolve the organism when introduced into the peritoneal cavity of a normal guinea pig (Pfeiffer phenomenon). Moreover, the guinea pigs which had once been inoculated with the blood of yellow fever patients without succumbing to the infection, notwithstanding the fact that they had shown a definite febrile reaction after 4 to 5 days, were found to be refractory to a subsequent inoculation of a culture of Leptospira icteroides All these observations pointed to the possible relation of this organism to the disease known as yellow fever in Guayaquil. The demonstration of the filterability of the organism and the transmission of the infection with the same organism by Stegomyia calopus have further strengthened the probable etiological significance of the organism in yellow fever. It was by no means a simple problem to determine the relation existing between Leptospira icteroides and Leptospira icterohaemorrhagiae. An experiment reported in a previous paper seemed to justify the view that the two leptospiras are closely related but not identical, yet it was necessary to exhaust various other modes of differentiation before the distinction between them was firmly established. The present paper continues this phase of the inquiry in further detail. There have been taken up here the phenomena of agglutination, the reaction of Pfeiffer, complement fixation, the protective properties of various monovalent and

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

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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. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A new multivalent (DHPPi/L4R) canine combination vaccine prevents infection, shedding and clinical signs following experimental challenge with four Leptospira serovars.

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Wilson, Stephen; Stirling, Catrina; Thomas, Anne; King, Vickie; Plevová, Edita; Chromá, Ludmila; Siedek, Elisabeth; Illambas, Joanna; Salt, Jeremy; Sture, Gordon

2013-06-28

Although effective vaccines have been developed against the common Leptospira serovars, they are still reported in clinical cases, while others are increasingly prevalent. The results from four challenge studies following vaccination of dogs with a new combination vaccine (DHPPi/L4R) containing inactivated L. serovars, L. canicola, L. icterohaemorrhagiae, L. bratislava and L. grippotyphosa conducted to satisfy the requirements of the European Pharmacopoeia monograph (01/2008:0447), are reported. Six week old dogs received two vaccinations, three weeks apart, and were challenged 25 days later with different isolates of the L. serovars. Clinical observations were recorded, and blood, urine and tissue samples were collected for analysis. Following challenge, non-vaccinated dogs demonstrated various clinical signs, while no vaccinated dogs were affected; significant differences in mean clinical scores were observed. Measurable antibody titres to each Leptospira antigen were seen in vaccinated dogs 21 days following the first vaccination, with further increases in antibody titres observed following challenge with the respective Leptospira strain. Non-vaccinated dogs remained seronegative until challenge. Leptospira were re-isolated from the blood, urine, kidney and liver of all non-vaccinated dogs following challenge. In contrast no vaccinated dogs had Leptospira re-isolated from the same tissues. Significant differences were seen in number of days with positive isolation (blood and urine) and in number of dogs with positive samples (kidney and liver). In conclusion, vaccination of dogs with the new vaccine induces protective immunity 25 days after second vaccination with protection against infection, renal infection and clinical signs following challenge. Copyright © 2013 Elsevier Ltd. All rights reserved.

Leptospira Exposure and Patients with Liver Diseases: A Case-Control Seroprevalence Study

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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

Brote de leptospirosis en terneros en recría en la provincia de Corrientes, Argentina Leptospirosis outbreak in calves from Corrientes Province, Argentina

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María G. Draghi

2011-03-01

Full Text Available La leptospirosis es una enfermedad infecciosa que produce importantes pérdidas económicas en la producción ganadera. Los signos característicos de la enfermedad son aborto, muerte embrionaria, muerte de terneros de pocos días de vida y mastitis. Se describe un brote de leptospirosis en terneros en actividad de recría. Se realizaron estudios histopatológicos, de hemoparásitos, inmunofluorescencia y cultivos bacterianos. Se aisló Leptospira interrogans serovar Pomona a partir de muestras de los terneros muertos.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.

Evolution of the RNase P RNA structural domain in Leptospira spp.

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Ravishankar, Vigneshwaran; Ahmed, Ahmed; Sivagnanam, Ulaganathan; Muthuraman, Krishnaraja; Karthikaichamy, Anbarasu; Wilson, Herald A; Devendran, Ajay; Hartskeerl, Rudy A; Raj, Stephen M L

2014-12-01

We have employed the RNase P RNA (RPR) gene, which is present as single copy in chromosome I of Leptospira spp. to investigate the phylogeny of structural domains present in the RNA subunit of the tRNA processing enzyme, RNase P. RPR gene sequences of 150 strains derived from NCBI database along with sequences determined from 8 reference strains were examined to fathom strain specific structural differences present in leptospiral RPR. Sequence variations in the RPR gene impacted on the configuration of loops, stems and bulges found in the RPR highlighting species and strain specific structural motifs. In vitro transcribed leptospiral RPR ribozymes are demonstrated to process pre-tRNA into mature tRNA in consonance with the positioning of Leptospira in the taxonomic domain of bacteria. RPR sequence datasets used to construct a phylogenetic tree exemplified the segregation of strains into their respective lineages with a (re)speciation of strain SH 9 to Leptospira borgpetersenii, strains Fiocruz LV 3954 and Fiocruz LV 4135 to Leptospira santarosai, strain CBC 613 to Leptospira kirschneri and strain HAI 1536 to Leptospira noguchii. Furthermore, it allowed characterization of an isolate P2653, presumptively characterized as either serovar Hebdomadis, Kremastos or Longnan to Leptospira weilii, serovar Longnan. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Leptospira species in floodwater during the 2011 floods in the Bangkok Metropolitan Region, Thailand.

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Thaipadungpanit, Janjira; Wuthiekanun, Vanaporn; Chantratita, Narisara; Yimsamran, Surapon; Amornchai, Premjit; Boonsilp, Siriphan; Maneeboonyang, Wanchai; Tharnpoophasiam, Prapin; Saiprom, Natnaree; Mahakunkijcharoen, Yuvadee; Day, Nicholas P J; Singhasivanon, Pratap; Peacock, Sharon J; Limmathurotsakul, Direk

2013-10-01

Floodwater samples (N = 110) collected during the 2011 Bangkok floods were tested for Leptospira using culture and polymerase chain reaction (PCR); 65 samples were PCR-positive for putatively non-pathogenic Leptospira species, 1 sample contained a putatively pathogenic Leptospira, and 6 samples contained Leptospira clustering phylogenetically with the intermediate group. The low prevalence of pathogenic and intermediate Leptospira in floodwater was consistent with the low number of human leptospirosis cases reported to the Bureau of Epidemiology in Thailand. This study provides baseline information on environmental Leptospira in Bangkok together with a set of laboratory tests that could be readily deployed in the event of future flooding.

Genus I. Leptospira

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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...

In Vitro Antimicrobial Susceptibility of Pathogenic Leptospira Biofilm.

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Vinod Kumar, Kirubakaran; Lall, Chandan; Raj, Ratchagadasse Vimal; Vedhagiri, Kumaresan; Sunish, Ittoop Pulikkottil; Vijayachari, Paluru

2016-10-01

Pathogenic Leptospira spp. are the causative agent of leptospirosis. Biofilm formation in leptospires is a new area of study, and its role in pathogenesis is not fully explored. As in other biofilm-forming bacteria, Leptospira biofilm may play a significant role in antibiotic resistance. In this study, the antimicrobial susceptibility of Leptospira biofilm was investigated by 96-well plate assay using Alamar Blue. Leptospira biofilm showed five to sixfold increase in resistance in all the strains used. The range of minimal bactericidal concentrations for penicillin G, ampicillin, tetracycline, and doxycycline was 1,600 U/ml, 800-1,600 μg/ml, 800-1,600 μg/ml, and 800-1,600 μg/ml, respectively. In agar substrate, the biofilm showed six- to sevenfold increase in resistance to antibiotics compared to planktonic cell. The present study emphasizes the importance of biofilm formation and its antibiotic susceptibility patterns. This could pave the way for devising appropriate strategy to prevent the occurrence of potential chronic leptospirosis in endemic areas and also during an outbreak situation.

Cloning and sequence analysis of a partial CDS of leptospiral ligA gene in pET-32a - Escherichia coli DH5α system

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Manju Soman

2018-04-01

Full Text Available Aim: This study aims at cloning, sequencing, and phylogenetic analysis of a partial CDS of ligA gene in pET-32a - Escherichia coli DH5α system, with the objective of identifying the conserved nature of the ligA gene in the genus Leptospira. Materials and Methods: A partial CDS (nucleotide 1873 to nucleotide 3363 of the ligA gene was amplified from genomic DNA of Leptospira interrogans serovar Canicola by polymerase chain reaction (PCR. The PCR-amplified DNA was cloned into pET-32a vector and transformed into competent E. coli DH5α bacterial cells. The partial ligA gene insert was sequenced and the nucleotide sequences obtained were aligned with the published ligA gene sequences of other Leptospira serovars, using nucleotide BLAST, NCBI. Phylogenetic analysis of the gene sequence was done by maximum likelihood method using Mega 6.06 software. Results: The PCR could amplify the 1491 nucleotide sequence spanning from nucleotide 1873 to nucleotide 3363 of the ligA gene and the partial ligA gene could be successfully cloned in E. coli DH5α cells. The nucleotide sequence when analyzed for homology with the reported gene sequences of other Leptospira serovars was found to have 100% homology to the 1910 bp to 3320 bp sequence of ligA gene of L. interrogans strain Kito serogroup Canicola. The predicted protein consisted of 470 aminoacids. Phylogenetic analysis revealed that the ligA gene was conserved in L. interrogans species. Conclusion: The partial ligA gene could be successfully cloned and sequenced from E. coli DH5α cells. The sequence showed 100% homology to the published ligA gene sequences. The phylogenetic analysis revealed the conserved nature of the ligA gene. Further studies on the expression and immunogenicity of the partial LigA protein need to be carried out to determine its competence as a subunit vaccine candidate.

The role of Leptospira spp. in horses affected with recurrent uveitis in the UK.

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Malalana, F; Blundell, R J; Pinchbeck, G L; Mcgowan, C M

2017-11-01

Equine recurrent uveitis (ERU) is a common cause of ocular pain and blindness in horses. Leptospira spp. have been commonly implicated in the pathophysiology of ERU in mainland Europe and the USA. No recent studies have been carried out in the UK, but Leptospira is reported not to be a major factor in the aetiology of ERU in the UK. To establish the prevalence of Leptospira-associated ERU in the UK and to identify the serovars involved in these cases; to compare serum vs. aqueous humour antibody levels in cases and controls in order to confirm the diagnosis of Leptospira-associated ERU, and to assess the usefulness of serology alone as a confirmatory test for Leptospira-associated ERU in the UK. Case-control study. Eyes enucleated for clinical reasons in ERU-affected horses were collected. Blood and aqueous humour were obtained to determine antibody levels against a variety of Leptospira serovars and C-values (aqueous humour value/serum value) were calculated. In addition, eyes, blood and aqueous humour were obtained from control cases for comparison. Histopathology was performed in all eyes to confirm uveitis in each case. Differences in seroprevalences between ERU and control cases and between Leptospira- and non-Leptospira-associated ERU cases were calculated. A total of 30 ERU and 43 control eyes were analysed. Of the ERU eyes, only two had a C-value of >4 (prevalence of Leptospira-associated uveitis: 6.7%). Serovars hardjo and javanica were detected. There was no difference in seroprevalence between horses with uveitis and control cases (65.5% and 41.9%, respectively; P = 0.11) or between Leptospira- and non-Leptospira-associated uveitis cases (100% and 63.0%, respectively; P = 0.52). The study was limited by low case numbers. Eyes were presented at different stages of disease. The only test used to detect Leptospira was the microscopic agglutination test. Leptospira-associated ERU is uncommon in the UK. Serology alone may not help to definitively diagnose

Kejadian Leptospirosis pada Anjing di Daerah Istimewa Yogyakarta (CASE OF CANINE LEPTOSPIROSIS IN THE CITY OF YOGYAKARTA

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Guntari Titik Mulyani

2017-09-01

Full Text Available Leptospirosis is a zoonotic disease, which is caused by Leptospira interrogans. The incidence of leptospirosis in dogs varies according to region and season, and is considered as emerging infectious diseases in humans. Clinical symptoms of leptospirosis in dogs vary greatly, some dogs are asymptomatic, with mild symptoms, and others progress to severe illness until death. The study aims to determine cases of canine leptospirosis in Yogyakarta, and identify the serovar that infect them. A total of 20 dogs without symptoms, with mild symptoms, to severe symptoms of leptospirosis were collected their serum and further tested using Microscopic Agglutination Test (MAT conducted at the Center for Veterinary Research (BBLitvet Bogor. History of vaccination is recorded as a consideration in interpreting the MAT results. The results showed that three out of the 20 samples were positive leptospirosis. Of the three positive samples, one was identified as serovar bataviae, another one as serovar bataviae and tarrasovi, and the last as serovar bataviae, tarrasovi, ichterohaemorrhagiae, canicola, celledoni, pyrogenes, cynopteri, and rachmati, respectively. The three dogs with leptospirosis showed similar clinical symptoms i.e. Anorexia, lethargy, and fever. It can be concluded that there are cases of canine leptospirosis in Yogyakarta which is predominantly caused by Leptospira interrogans serovar bataviae. ABSTRAK Leptospirosis adalah penyakit zoonosis, yang disebabkan oleh Leptospira interrogans. Kejadian leptospirosis pada anjing bervariasi menurut wilayah dan musim, dan dianggap sebagai penyakit menular yang muncul pada manusia. Gejala klinis leptospirosis pada anjing sangat bervariasi, beberapa anjing tanpa menunjukkan gejala, dengan gejala ringan, dan yang lain melanjut menjadi penyakit yang parah sampai kematian. Penelitian ini bertujuan untuk mengetahui kasus leptospirosis pada anjing di Propinsi Daerah Istimewa Yogyakarta, dan mengidentifikasi

Multiple Co-infections of Rodents with Hantaviruses, Leptospira, and Babesia in Croatia

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Turk, Nenad; Korva, Miša; Margaletić, Josip; Beck, Relja; Vucelja, Marko; Habuš, Josipa; Svoboda, Petra; Županc, Tatjana Avšič; Henttonen, Heikki; Markotić, Alemka

2012-01-01

Abstract Hantaviruses, Leptospira spp., and Babesia spp. are rodent-borne pathogens present worldwide. We studied multiple co-infections of small rodents in Croatia with all three pathogens. Twenty-eight Apodemus flavicollis and 16 Myodes glareolus were tested for the presence of hantavirus RNA by real-time RT-PCR, Leptospira strains by renoculture method and Babesia DNA by PCR. Anti-hantavirus antibodies and anti-Leptospira antibodies were detected by serological methods. Very high infection rates with each pathogen were found in A. flavicollis: 20 of 28 rodents (71%) were infected with Dobrava virus, 13 rodents (46%) were infected with Leptospira, and 5 rodents (18%) were infected with Babesia. Multiple co-infections with all three pathogens were found in 3 of 28 (11%) A. flavicollis animals, suggesting that the same rodent host can be infected with several pathogens at the same time. Dual infections with both hantaviruses and Leptospira were found in 7 of 44 rodents (16%), with hantaviruses and Babesia in 2 rodents (5%), and double infection with both Leptospira and Babesia were found in 1 rodent (2%). Since hantaviruses, Leptospira, and Babesia have similar geographical distributions, it is to be expected that in other parts of the world multiple co-infections, representing a serious threat to public health, can be found. PMID:22217170

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

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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. © 2014 Blackwell Verlag

Leptospira seropositivity as a risk factor for Mesoamerican Nephropathy.

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Riefkohl, Alejandro; Ramírez-Rubio, Oriana; Laws, Rebecca L; McClean, Michael D; Weiner, Daniel E; Kaufman, James S; Galloway, Renee L; Shadomy, Sean V; Guerra, Marta; Amador, Juan José; Sánchez, José Marcel; López-Pilarte, Damaris; Parikh, Chirag R; Leibler, Jessica H; Brooks, Daniel R

2017-01-01

Leptospirosis is postulated as a possible cause of Mesoamerican Nephropathy (MeN) in Central American workers. Investigate job-specific Leptospira seroprevalence and its association with kidney disease biomarkers. In 282 sugarcane workers, 47 sugarcane applicants and 160 workers in other industries, we measured anti-leptospiral antibodies, serum creatinine, and urinary injury biomarkers, including neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), and N-acetyl-D-glucosaminidase (NAG). Leptospira seroprevalence differed among job categories and was highest among sugarcane cutters (59%). Seropositive sugarcane workers had higher NGAL concentrations (relative mean: 1.28; 95% CI: 0.94-1.75) compared to those who were seronegative, with similar findings among field and non-field workers. Leptospira seroprevalence varied by job category. There was some indication that seropositivity was associated with elevated biomarker levels, but results were inconsistent. Additional studies may help establish whether Leptospira infection plays any role in MeN among Central American workers.

Monitoring Leptospira strain collections: the need for quality control

NARCIS (Netherlands)

Cerqueira, Gustavo M.; McBride, Alan J. A.; Queiroz, Adriano; Pinto, Luciano S.; Silva, Everton F.; Hartskeerl, Rudy A.; Reis, Mitermayer G.; Ko, Albert I.; Dellagostin, Odir A.

2010-01-01

The purpose of this study was to perform a 16S sequence-based quality control of two Leptospira strain collections. 16S rRNA gene sequencing was used to verify two Leptospira reference collections provided by the World Health Organization and maintained at a reference laboratory for leptospirosis in

H(+) and Na(+) are involved in flagellar rotation of the spirochete Leptospira.

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Islam, Md Shafiqul; Morimoto, Yusuke V; Kudo, Seishi; Nakamura, Shuichi

2015-10-16

Leptospira is a spirochete possessing intracellular flagella. Each Leptospira flagellar filament is linked with a flagellar motor composed of a rotor and a dozen stators. For many bacterial species, it is known that the stator functions as an ion channel and that the ion flux through the stator is coupled with flagellar rotation. The coupling ion varies depending on the species; for example, H(+) is used in Escherichia coli, and Na(+) is used in Vibrio spp. to drive a polar flagellum. Although genetic and structural studies illustrated that the Leptospira flagellar motor also contains a stator, the coupling ion for flagellar rotation remains unknown. In the present study, we analyzed the motility of Leptospira under various pH values and salt concentrations. Leptospira cells displayed motility in acidic to alkaline pH. In the presence of a protonophore, the cells completely lost motility in acidic to neutral pH but displayed extremely slow movement under alkaline conditions. This result suggests that H(+) is a major coupling ion for flagellar rotation over a wide pH range; however, we also observed that the motility of Leptospira was significantly enhanced by the addition of Na(+), though it vigorously moved even under Na(+)-free conditions. These results suggest that H(+) is preferentially used and that Na(+) is secondarily involved in flagellar rotation in Leptospira. The flexible ion selectivity in the flagellar system could be advantageous for Leptospira to survive in a wide range of environment. Copyright © 2015 Elsevier Inc. All rights reserved.

H{sup +} and Na{sup +} are involved in flagellar rotation of the spirochete Leptospira

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Islam, Md. Shafiqul [Department of Applied Physics, Graduate School of Engineering, Tohoku University, 6-6-05 Aoba, Aoba-ku, Sendai, Miyagi 980-8579 (Japan); Morimoto, Yusuke V. [Quantitative Biology Center, RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874 (Japan); Graduate School of Frontier BioSciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kudo, Seishi [Department of Applied Physics, Graduate School of Engineering, Tohoku University, 6-6-05 Aoba, Aoba-ku, Sendai, Miyagi 980-8579 (Japan); Nakamura, Shuichi, E-mail: naka@bp.apph.tohoku.ac.jp [Department of Applied Physics, Graduate School of Engineering, Tohoku University, 6-6-05 Aoba, Aoba-ku, Sendai, Miyagi 980-8579 (Japan); Graduate School of Frontier BioSciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871 (Japan)

2015-10-16

Leptospira is a spirochete possessing intracellular flagella. Each Leptospira flagellar filament is linked with a flagellar motor composed of a rotor and a dozen stators. For many bacterial species, it is known that the stator functions as an ion channel and that the ion flux through the stator is coupled with flagellar rotation. The coupling ion varies depending on the species; for example, H{sup +} is used in Escherichia coli, and Na{sup +} is used in Vibrio spp. to drive a polar flagellum. Although genetic and structural studies illustrated that the Leptospira flagellar motor also contains a stator, the coupling ion for flagellar rotation remains unknown. In the present study, we analyzed the motility of Leptospira under various pH values and salt concentrations. Leptospira cells displayed motility in acidic to alkaline pH. In the presence of a protonophore, the cells completely lost motility in acidic to neutral pH but displayed extremely slow movement under alkaline conditions. This result suggests that H{sup +} is a major coupling ion for flagellar rotation over a wide pH range; however, we also observed that the motility of Leptospira was significantly enhanced by the addition of Na{sup +}, though it vigorously moved even under Na{sup +}-free conditions. These results suggest that H{sup +} is preferentially used and that Na{sup +} is secondarily involved in flagellar rotation in Leptospira. The flexible ion selectivity in the flagellar system could be advantageous for Leptospira to survive in a wide range of environment. - Highlights: • This is a study on input energy for motility in the spirochete Leptospira. • Leptospira biflexa exhibited active motility in acidic to alkaline pH. • Both H{sup +} and Na{sup +} are involved in flagellar rotation in Leptospira. • H{sup +} is a primary energy source, but Na{sup +} can secondarily enhance motility.

Seroprevalence of Leptospira antibodies among populations at risk

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Abdul-Baki Abdullah Al-Robasi

2015-03-01

Full Text Available Objective: This study was performed to assess the Leptospira IgG antibodies seroprevalence among populations at risk in Hodeida Governorate, Yemen. Methods: A total of 200 subjects (136 males and 64 females participated in this study during June and December 2012.They represented 10 sewage workers, 22 butchers, 16 construction workers, 108 agriculture workers, 20 hospital sanitary workers and 24 blood donors. Predesigned questionnaires and consent were taken from each individual. Blood samples were collected from subjects, and the sera were tested by ELISA to detect the presence of leptospira IgG antibodies. The possible related factors for seropositivity were evaluated. Results: Leptospira IgG antibodies were found positive in 42% of the participants. The highest seroprevalence level was detected in sewage workers (80%, followed by hospital sanitary workers (60%, construction workers (37.5% and farmers (37%. The lowest of antibodies was in butchers (36.4%. Seroprevalence among blood donors was 25% which was comparatively less than of the populations at risk. Seropositivity of Leptospira IgG antibodies was found higher among males than females (42.6% vs. 34.4%. The highest Leptospira antibodies seropositivity was among elderly participants (81.8%. The seropositivity of antibodies in population live in rural and urban areas was not significant differences. As for closely contacting with animals, the highest antibodies were discovered in people who had goats (80% and sheep (60.9%. Conclusion: Individuals engaged in risk activities are often exposed to leptospiral infection. Therefore, control and prevention policy toward these people are necessary. J Microbiol Infect Dis 2015;5(1: 1-4

Leptospira infections in freshwater fish in Morogoro Tanzania: a ...

African Journals Online (AJOL)

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 ...

Molecular epidemiology of pathogenic Leptospira spp. among large ruminants in the Philippines.

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Villanueva, Marvin A; Mingala, Claro N; Balbin, Michelle M; Nakajima, Chie; Isoda, Norikazu; Suzuki, Yasuhiko; Koizumi, Nobuo

2016-12-01

The extent of Leptospira infection in large ruminants resulting to economic problems in livestock industry in a leptospirosis-endemic country like the Philippines has not been extensively explored. Therefore, we determined the prevalence and carrier status of leptospirosis in large ruminants using molecular techniques and assessed the risk factors of acquiring leptospirosis in these animals. Water buffalo and cattle urine samples (n=831) collected from 21 farms during 2013-2015 were subjected to flaB-nested PCR to detect pathogenic Leptospira spp. Leptospiral flaB was detected in both species with a detection rate of 16.1%. Leptospiral DNA was detected only in samples from animals managed in communal farms. Sequence analysis of Leptospira flaB in large ruminants revealed the formation of three major clusters with L. borgpetersenii or L. kirschneri. One farm contained Leptospira flaB sequences from all clusters identified in this study, suggesting this farm was the main source of leptospires for other farms. This study suggested that these large ruminants are infected with various pathogenic Leptospira species causing possible major economic loss in the livestock industry as well as potential Leptospira reservoirs that can transmit infection to humans and other animals in the Philippines.

Long-term preservation of Leptospira spp.: challenges and prospects.

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Philip, Noraini; Garba, Bashiru; Neela, Vasantha Kumari

2018-05-08

Preservation of leptospiral cultures is tantamount to success in leptospiral diagnostics, research, and development of preventive strategies. Each Leptospira isolate has imperative value not only in disease diagnosis but also in epidemiology, virulence, pathogenesis, and drug development studies. As the number of circulating leptospires is continuously increasing and congruent with the importance to retain their original characteristics and properties, an efficient long-term preservation is critically needed to be well-established. However, the preservation of Leptospira is currently characterized by difficulties and conflicting results mainly due to the biological nature of this organism. Hence, this review seeks to describe the efforts in developing efficient preservation methods, to discover the challenges in preserving this organism and to identify the factors that can contribute to an effective long-term preservation of Leptospira. Through the enlightenment of the previous studies, a potentially effective method has been suggested. The article also attempts to evaluate novel strategies used in other industrial and biotechnological preservation efforts and consider their potential application to the conservation of Leptospira spp.

Bid-Induced Release of AIF/EndoG from Mitochondria Causes Apoptosis of Macrophages during Infection with Leptospira interrogans

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Wei-Lin Hu

2017-11-01

Full Text Available Leptospirosis is a global zoonotic infectious disease caused by pathogenic Leptospira species. Leptospire-induced macrophage apoptosis through the Fas/FasL-caspase-8/3 pathway plays an important role in the survival and proliferation of the pathogen in hosts. Although, the release of mitochondrial apoptosis-inducing factor (AIF and endonuclease G (EndoG in leptospire-infected macrophages has been described, the mechanisms linking caspase and mitochondrion-related host-cell apoptosis has not been determined. Here, we demonstrated that leptospire-infection induced apoptosis through mitochondrial damages in macrophages. Apoptosis was caused by the mitochondrial release and nuclear translocation of AIF and/or EndoG, leading to nuclear DNA fragmentation. However, the mitochondrion-related CytC-caspase-9/3 pathway was not activated. Next, we found that the release and translocation of AIF and/or EndoG was preceded by the activation of the BH3-interacting domain death agonist (Bid. Furthermore, our data demonstrated that caspase-8 was activated during the infection and caused the activation of Bid. Meanwhile, high reactive oxygen species (ROS trigged by the infection caused the dephosphorylation of Akt, which also activated Bid. In conclusion, Bid-mediated mitochondrial release of AIF and/or EndoG followed by nuclear translocation is a major mechanism of leptospire- induced apoptosis in macrophages, and this process is modulated by both caspase-8 and ROS-Akt signal pathways.

Serosurvey of Leptospira spp. and Toxoplasma gondii in rats captured from two zoos in Southern Brazil.

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Pellizzaro, Maysa; Conrado, Francisco de Oliveira; Martins, Camila Marinelli; Joaquim, Sâmea Fernandes; Ferreira, Fernando; Langoni, Helio; Biondo, Alexander Welker

2017-01-01

Norway rats (Rattus norvegicus) are zoonotic reservoirs for Leptospira spp. and Toxoplasma gondii, and influence diseases in urban areas. Free-ranging and laboratory-raised rats from two zoos in southern Brazil were tested for Leptospira spp. and T. gondii using microscopic agglutination and modified agglutination tests, respectively. Overall, 25.6% and 4.6% free-ranging rats tested positive for Leptospira spp. and T. gondii, respectively, with co-seropositivity occurring in two animals. For laboratory-raised rats, 20% tested positive for Leptospira spp. Also, Leptospira biflexa serovar Patoc and Leptospira noguchii serovar Panama were found. Serosurveys can show the environmental prevalence of zoonotic pathogens.

Impact of environment and social gradient on Leptospira infection in urban slums.

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Renato B Reis

2008-04-01

Full Text Available 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.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.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 factors that produce unequal health outcomes among

High Seroprevalence of Leptospira Exposure in Meat Workers in Northern Mexico: A Case-Control Study

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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

Opportunities and strategies to further reduce animal use for Leptospira vaccine potency testing.

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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. Published by Elsevier Ltd.

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

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María F Ferrer

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

The multifaceted roles of Leptospira enolase.

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Salazar, Natália; Souza, Matilde Costa Lima de; Biasioli, Amanda Gameiro; Silva, Ludmila Bezerra da; Barbosa, Angela Silva

A previous study had demonstrated that Leptospira enolase is secreted extracellularly by a yet unknown mechanism and reassociates with the bacterial membrane. Surface-anchored leptospiral enolase displays plasminogen binding activity. In this work, we explored the consequences of this interaction and also assessed whether Leptospira enolase might display additional moonlighting functions by interacting with other host effector proteins. We first demonstrated that enolase-bound plasminogen is converted to its active form, plasmin. The protease plasmin targets human fibrinogen and vitronectin, but not the complement proteins C3b and C5. Leptospira enolase also acts as an immune evasion protein by interacting with the negative complement regulators C4b binding protein and factor H. Once bound to enolase, both regulators remain functional as cofactors of factor I, mediating cleavage of C4b and C3b. In conclusion, enolase may facilitate leptospiral survival and dissemination, thus contributing to bacterial virulence. The identification and characterization of moonlighting proteins is a growing field of bacterial pathogenesis, as these multifaceted proteins may represent potential future therapeutic targets to fight bacterial infections. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Detection of Leptospira spp. in wild Phrynops geoffroanus (Geoffroy's side-necked turtle) in urban environment.

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Oliveira, J P; Kawanami, A E; Silva, A S L; Chung, D G; Werther, K

2016-12-01

Leptospira spp., a zoonotic agent relevant for public health, occurs frequently in tropical regions. The aquatic environment represents a viable survival and transmission pathway. This study aimed to investigate the presence of anti-Leptospira spp. antibodies in Phrynops geoffroanus (Geoffroy's side-necked turtle) serum samples using the microagglutination test (MAT), and Leptospira spp. in gastric and cloacal lavage samples using the polymerase chain reaction (PCR) technique. Antibodies against nine different Leptospira spp. serovars were detected in 45.45% (30/66) of the serum samples. Specific amplification of Leptospira spp. genomic material (331bp) was observed in 16.67% (11/66) of the samples. In conclusion, these freshwater testudines host Leptospira spp. and eliminate them. This situation may represent a risk to public health, especially to people who use urban streams for fishing and recreational activities. Additionally, we described some Leptospira spp. serovars, not yet reported in testudines, detected here in P. geoffroanus. Copyright © 2016 Elsevier B.V. All rights reserved.

Isolation of saprophytic Leptospira spp. from a selected environmental water source of Argentina.

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Scialfa, Exequiel; Grune, Sylvia; Brihuega, Bibiana; Aguirre, Pablo; Rivero, Marina

2017-11-29

Ten Leptospira spp. strains were isolated from water samples from Nievas stream, Olavarría, Buenos Aires province (Argentina). The isolates showed the typical motility and morphology of the genus Leptospira under dark field microscopy, developing in liquid EMJH medium after eight days of incubation at 13°C and 30°C. All isolates were negative by the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Molecular identification by 16S rRNA gene sequencing identified all isolates as nonpathogenic leptospires. Four isolates showed a genetic profile identical to that of the reference strain Leptospira biflexa serovar Patoc, and six isolates revealed sequence similarities within the 97-98% range, closely related to Leptospira yanagawae and Leptospira meyeri, respectively. Strains ScialfaASA42, ScialfaASA45, ScialfaASA44, ScialfaASA47, ScialfaASA49, ScialfaASA50 and ScialfaASA51 possibly represent a novel species of the genus Leptospira. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

A duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains.

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Benacer, Douadi; Zain, Siti Nursheena Mohd; Lewis, John W; Khalid, Mohd Khairul Nizam Mohd; Thong, Kwai Lin

2017-01-01

This study aimed to develop a duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains. Primers were designed to target the rrs (LG1/LG2) and ligB (LP1/LP2) genes to confirm the presence of the Leptospira genus and the pathogenic species, respectively. The assay showed 100% specificity against 17 Leptospira strains with a limit of detection of 23.1pg/µl of leptospiral DNA and sensitivity of 103 leptospires/ml in both spiked urine and water. Our duplex endpoint PCR assay is suitable for rapid early detection of Leptospira with high sensitivity and specificity.

Induction of TNF-alfa and CXCL-2 mRNAs in different organs of mice infected with pathogenic Leptospira.

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da Silva, Josefa B; Carvalho, Enéas; Covarrubias, Ambart E; Ching, Ana Tung C; Mattaraia, Vania G M; Paiva, Delhi; de Franco, Marcelo; Fávaro, Regiane Degan; Pereira, Martha M; Vasconcellos, Silvio; Zorn, Telma T M; Ho, Paulo Lee; Martins, Elizabeth A L

2012-04-01

The role of innate immune response in protection against leptospirosis is poorly understood. We examined the expression of the chemokine CXCL2/MIP-2 and the cytokine TNF-α in experimental resistant and susceptible mice models, C3H/HeJ, C3H/HePas and BALB/c strains, using a virulent strain of Leptospira interrogans serovar Copenhageni. Animals were infected intraperitoneally with 10(7) cells and the development of the disease was followed. Mortality of C3H/HeJ mice was observed whereas C3H/HePas presented jaundice and BALB/c mice remained asymptomatic. The infection was confirmed by the presence of leptospiral DNA in the organs of the animals, demonstrated by PCR. Sections of the organs were analyzed, after H&E stain. The relative expression of mRNA of chemokine CXCL2/MIP-2 and cytokine TNF-α was measured in lung, kidney and liver of the mice by qPCR. The concentrations of these proteins were measured in extracts of tissues and in serum of the animals, by ELISA. Increasing levels of transcripts and protein CXCL2/MIP-2 were detected since the first day of infection. The highest expression was observed at third day of infection in kidney, liver and lung of BALB/c mice. In C3H/HeJ the expression of CXCL2/MIP-2 was delayed, showing highest protein concentration in lung and kidney at the 5th day. Increasing in TNF-α transcripts were detected after infection, in kidney and liver of animals from the three mice strains. The expression of TNF-α protein in C3H/HeJ was also delayed, being detected in kidney and lung. Our data demonstrated that Leptospira infection stimulates early expression of CXCL2/MIP-2 and TNF-α in the resistant strain of mice. Histological analysis suggests that the expression of those molecules may be related to the influx of distinct immune cells and plays a role in the naturally acquired protective immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mannose-binding lectin impairs Leptospira activity through the inhibitory effect on the motility of cell.

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Xu, Jun; Guo, Yijie; Nakamura, Shuichi; Islam, Md Shafiqul; Tomioka, Rintaro; Yoneyama, Hiroshi; Isogai, Emiko

2015-02-01

Mannose-binding lectin (MBL) plays key role in lectin pathway of innate immunity, and shows the ability of triggering opsonization intermediately. Substantial increase in the serum level of MBL has been confirmed during leptospirosis, which caused by a pathogenic spirochete, Leptospira. Leptospira has a fascinating locomotion pattern, which simultaneously gyrating and swimming forward, such motility enables that Leptospira is difficult to be captured by immune cells if without any assistance. In this study, the effect of mannose-binding lectin to Leptospira was quantitatively investigated by measuring some kinematic parameters, to discover the mechanism behind MBL-mediated immune responses during leptospiral infection. The results showed that mannose-binding lectin is capable of inhibiting the motility of Leptospira by transforming free swimming cells to tumbled rotating cells, resulted in the increase number of rotating cells. Otherwise, decrease in rotation rate of rotating cell has been observed. However, the swimming speed of swimming Leptospira cells showed no observable change under the effect of MBL. The inhibitory effect were only valid in a relatively short period, Leptospira cells regained their original motility after 2 h. This raises an interesting topic that Leptospira is somehow able to escape from the inhibitory effect of MBL by dragging such unfavorable molecules toward to the cell end and eventually throwing it out. The inhibitory effect of MBL on the motility of Leptospira is expected to provide a new insight into lectin pathway. Copyright © 2015 Elsevier GmbH. All rights reserved.

PEMERIKSAAN BAKTERI LEPTOSPIRA PADA SAMPEL DARAH MANUSIA SUSPECT LEPTOSPIROSIS MENGGUNAKAN METODE PCR (POLYMERASE CHAIN REACTION

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Sefrita Tri Utami

2014-01-01

Full Text Available ABSTRACTLeptospirosis is a zoonotic disease, which is caused by leptospira. Leptospirosis cases often show no specificclinical symptoms and is difficult to diagnose without testing samples in the laboratory. Testing using PCR(Polymerase Chain Reaction is considered more accurate than the other methods. Components required in theexamination Leptospira bacteria in human blood samples using PCR method is DNA template, DNA polymeraseenzyme, forward primer (PU1 and SU1 and reverse primer (Lep R1, nuclease free water, Mg 2 +, and dNTPs.Examination of Leptospira bacteria in human blood samples include sampling, DNA isolation, examination byPCR, and electrophoresis running.Key words: leptospirosis, Leptospira, PCR methodsABSTRAKLeptospirosis adalah penyakit zoonosis yang disebabkan oleh bakteri Leptospira. Kasus leptospirosis seringtidak menunjukkan gejala klinis yang spesifik dan sulit didiagnosis tanpa pengujian sampel di laboratorium.Pengujian dengan menggunakan metode PCR (Polymerase Chain Reaction dinilai lebih akurat dibandingkandengan metode yang lain. Komponen-komponen yang dibutuhkan dalam pemeriksaan bakteri Leptospira padasampel darah manusia menggunakan metode PCR adalah DNA template, enzim polymerase, Primer PU 1 danPrimer SU 1, Primer Lep R1, air, Mg2+ , dan dNTP. Pemeriksaan bakteri Leptospira pada sampel darah manusiameliputi pengambilan sampel, isolasi DNA, pemeriksaan dengan metode PCR, dan running elektroforesis.Kata kunci: leptospirosis, Leptospira, metode PCR

New Leptospira serovar Sokoine of serogroup Icterohaemorrhagiae from cattle in Tanzania

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Mgode, G. F.; Machang'u, R. S.; Goris, M. G.; Engelbert, M.; Sondij, S.; Hartskeerl, R. A.

2006-01-01

The prevalence of leptospirosis is generally high in domestic animals and rodents in Tanzania. Identification of Leptospira isolates from cattle was carried out to establish prevalent Leptospira serovars. Serological typing was done based on monoclonal antibodies and the standard cross-agglutination

Whole genome sequencing revealed host adaptation-focused genomic plasticity of pathogenic Leptospira

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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

Borrelia burgdorferi antibodies in dogs from Cotia county, São Paulo State, Brazil

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JOPPERT Adriana Marques

2001-01-01

Full Text Available Dogs sera samples collected from Cotia County, São Paulo were tested using indirect immunoenzymatic test (ELISA in order to study Lyme disease serology in dogs. ELISA method was standardized and G39/40 North American strain of Borrelia burgdorferi was used as antigen. Positive results were confirmed employing the Western blotting technique. Because of the possibility of cross-reactions, sera were also tested for different serological strains of Leptospira interrogans and L. biflexa using microscopic sera agglutination test. Twenty-three of 237 (9.7% serum samples were positive in the ELISA; 20 of them (86.9% were confirmed by the Western blotting, what suggests that Cotia may be a risk area for Lyme disease. Although 4 samples (1.7% were positive for Lyme disease and leptospirosis, no correlation was found between the results (X² = 0.725; p = 0.394 what suggests absence of serological cross reactivity.

Contribution of Leptospira, Neospora caninum and bovine viral diarrhea virus to fetal loss of beef cattle in New Zealand.

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Sanhueza, J M; Heuer, C; West, D

2013-10-01

The profitability of beef breeding farms in New Zealand depends principally on optimal reproductive performance. The aim of this study was to estimate the impact of four major pathogens, bovine viral diarrhea virus (BVDV), Neospora caninum (N. caninum), Leptospira borgpetersenii serovar Hardjo (Hardjo), and Leptospira interrogans serovar Pomona (Pomona), on rates of fetal loss in commercial beef breeding herds. Farms reporting fetal loss were recruited, and a blood sample from aborting cows (cases) was collected. Controls were normally calving cows from the same farm. At least four controls were selected from each farm contributing cases. Samples were tested using ELISA for detection of antibodies against BVDV and N. caninum, and microscopic agglutination test (MAT) for detection of antibody against Hardjo and Pomona. A selection of titer cut-offs was conducted to evaluate the relationship between fetal loss and seropositivity to each pathogen using conditional logistic regression. The cut-off titer with the strongest association with fetal loss was included in the multivariate model. A significant increased risk of fetal loss was found for animals seropositive to N. caninum (odds ratio (OR)=3.36; 95% confidence interval (95% CI)=1.27-8.89), Hardjo (OR=1.84; 95% CI=1.01-3.33), and Pomona in non-vaccinated cows (OR=14.91, 95% CI=1.73-128.84) at the ELISA titer ≥ 30, and MAT titers of ≥ 1:384 and ≥ 1:768 for a positive sample, respectively. A marginally non-significant increased risk of fetal loss was found for animals exposed to BVDV (OR=2.01; 95% CI=0.99-4.11) at the ELISA titer of ≤ 1. Vaccination did not affect ORs for Hardjo or BVDV and no herd vaccinated against N. caninum. Approximately 14.0% of all fetal loss in the beef breeding cattle population in New Zealand may be attributable to BVDV (3.5%), N. caninum (3.0%), Hardjo (4.7%), and Pomona (3.6%). Copyright © 2013 Elsevier B.V. All rights reserved.

GLOBAL PATTERNS OF LEPTOSPIRA PREVALENCE IN VERTEBRATE RESERVOIR HOSTS.

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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.

DETERMINASI SEROVAR BAKTERI LEPTOSPIRA PADA RESERVOIR DI KABUPATEN BANYUMAS

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Tri Ramadhani

2016-05-01

Full Text Available Leptospirosis is an infectious disease caused by pathogenic Leptospira. Leptospirosis transmitted to human through direct contact with body fluids of infected animals or indirectly through contaminated puddles . The prevalence of leptospirosis in Banyumas tends to increase for 3 years. The purpose of this study was to determine the leptospira serovar in reservoir to prove of a current infection. Surveys was conducted using single live traps for three consecutive days, determination of leptospira serovar was conducted using Microscopic Aglutination Test (MAT. Data analysis was performed by univariate and presented in tables and graphs. The results showed that the trapped animals consisted of Rattus tanezumi (70.6% and Suncus murinus (29.4% with 6.5% succsess trap. Rattus tanezumi were dominantly caught inside the house (51% than outside the house (49%. Female rats were dominantly caught (66.7% than male rats (33.3%. Suncus murinus and Rattus tanezumi shown a titer of 1/100 to be infected with L.icterohaemorrhagiae , L.javanica and L.cynopteri which are pathogenic Leptospira in humans. Efforts are needed to improve community participation in preventing tranmission of leptospirosis by avoiding contact with contaminated water and soil. For people who are risk of exposure to infected animal should wear protective clothes or footwear.

Seroprevalence and factors associated with Leptospira infection in an urban district of Cali, Colombia.

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Escandón-Vargas, Kevin; Osorio, Lyda; Astudillo-Hernández, Miryam

2017-06-12

Few studies have addressed Leptospira seroprevalence and risk factors in urban populations in Colombia. This study aimed to determine seroprevalence and factors associated with Leptospira infection in inhabitants of an urban district of Cali, Colombia. We collected sociodemographic and environmental data, as well as blood samples, from 353 subjects selected through a multistage cluster sampling design. We performed microagglutination test for the eight main Leptospira serogroups circulating in the region, considering a cut-off titer of ≥ 1:100. Most participants were female (226, 64.8%), with mean age 41.4 years, and 89 (32.6%) lived in low-low socioeconomic stratum (SES-1). Overall seroprevalence was 12.2% (95%CI: 10.3%-14.4%). Factors associated with Leptospira infection were SES 1, older age, single marital status, ethnic groups (Afro-Colombian and white/mestizo), school students, absence of toilet, barefoot walking, travel outside Cali in the previous month, and absence of skin and mucous-membrane lesions in the previous month. Our study suggests domestic and peridomiciliary transmission of Leptospira likely related to activities of daily living and inadequate environmental conditions. SES-1 is a major factor associated with Leptospira infection (adjusted OR = 4.08; 95%CI: 2.54-6.53; p Leptospira infection in the study area. Epidemiological surveillance, improvement of environmental and sanitary conditions in various SES-1 areas, and community educational campaigns are recommended.

Comparative Analysis of Leptospira Strains Isolated from Environmental Soil and Water in the Philippines and Japan

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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

Leptospira seroprevalence in animals in the Caribbean region: A systematic review.

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Pratt, Nicola; Rajeev, Sreekumari

2018-06-01

This systematic review summarises the data published on the Leptospira seroprevalence, serovar diversity and distribution among animal species in the Caribbean region. Following Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, and checklist, relevant articles were identified and data were extracted and recorded. The review provided Leptospira seroprevalence data from 16 Caribbean islands (Barbados, Trinidad, Grenada, Puerto Rico, Saint Croix, St. Kitts and Nevis, Jamaica, Antigua, Carriacou, Dominica, Guadalupe, Martinique, Monserrat, St. Lucia, St. Maarten, and St. Vincent) in a variety of animal species. Reviewing the literature highlighted the limited amount of data available from limited number of islands. Many of the studies conducted have recorded seroprevalences based on variable and small samples sizes. Besides, serovar panels used for MAT were not consistent between studies. The review indicates that the Leptospira exposure in a given geographic location may change with time and climatic and environmental conditions, and highlights the need to conduct continual surveillance in tropical countries where the climate supports the survival of Leptospira in the environment. Specific attention must be given to standardization of MAT panels and protocols and providing training across laboratories involved in testing. Further, animal and environment testing to isolate and identify circulating Leptospira spp. in a geographic region must actively be pursued. This knowledge is important to implement geographically specific control programs, as risk factors of Leptospira transmission is favoured by various factors such as change in climatic conditions, urbanization, encroachment of wildlife inhabitation, import/export of animals, increase in adventure travel, and water related recreational activities. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

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

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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

Imported Leptospira licerasiae Infection in Traveler Returning to Japan from Brazil.

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Tsuboi, Motoyuki; Koizumi, Nobuo; Hayakawa, Kayoko; Kanagawa, Shuzo; Ohmagari, Norio; Kato, Yasuyuki

2017-03-01

We describe a case of intermediate leptospirosis resulting from Leptospira licerasiae infection in a traveler returning to Japan from Brazil. Intermediate leptospirosis should be included in the differential diagnosis for travelers with fever returning from South America. This case highlights the need for strategies that detect pathogenic and intermediate Leptospira species.

Seroprevalencia de Leptospira sp., Rickettsia sp. Ehrlichia sp. en trabajadores rurales del departamento de Sucre, Colombia Seroprevalence of Leptospira sp., Rickettsia sp. and Ehrlichia sp. in rural workers of Sucre, Colombia

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Rodrigo Ríos

2008-06-01

Full Text Available Objective. Determinar la seroprevalencia de Leptospira sp., Rickettsia sp. y Ehrlichia sp. en trabajadores de áreas rurales del departamento de Sucre. Material y métodos. Se realizó un estudio escriptivo, prospectivo, de corte transversal, que pretendió determinar la seroprevalencia e Leptospira sp., Rickettsia sp. y Ehrlichia sp. en 90 trabajadores de áreas rurales del departamento de Sucre. Se estableció la presencia de anticuerpos séricos anti-IgM específicos anti-Leptospira por la técnica de ELISA indirecta. Para la determinación de Rickettsia sp. y Ehrlichia sp. se uso la técnica de inmunofluorescencia indirecta. Resultados. La población evaluada estaba compuesta por 27 (30% ordeñadores, 21 (23% jornaleros, 18 (20% profesionales del campo y 24 (27% que realizaban otras actividades. Ventidós (24% muestras resultaron positivas en alguna de las pruebas. De éstas, 12 (13,3% fueron positivas para Leptospira sp., 7 (7,8% para Rickettsia sp. y 3 (3,3% ara Ehrlichia sp. Conclusión. Este fue el primer estudio que se llevó a cabo en el departamento de Sucre y permitió demostrar que existe una prevalencia importante de Leptospira p.,Rickettsia sp. y Ehrlichia sp.. Los factores de riesgo ocupacional fueron factores determinantes en la seropositividad.Objective. To determine the seroprevalence of Leptospira sp., Rickettsia sp. and Ehrlichia sp. in agricultural workers of Sucre. Methods. A descriptive prospective cross-sectional study was conducted in ninety rural workers of Sucre. Presence of serum antibodies anti-IgM specific anti-Leptospira by indirect ELISA was established. For the determination of Rickettsia and Ehrlichia indirect inmunoflorescence was used. Results.The population was composed by 27 (30% milkers, 21 (23% day workers, 18 farm professionals (20% and 24 (26% workers in others activities. A total of 22 (24% samples were positive to some test. Twelve (13.3% were positive to Leptospira sp., seven (7.8% to Rickettsia sp

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

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P. Hauk

2011-04-01

Full Text Available Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272 was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272 per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272 produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira.

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Hauk, P; Carvalho, E; Ho, P L

2011-04-01

Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Lvr, a Signaling System That Controls Global Gene Regulation and Virulence in Pathogenic Leptospira

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Adhikarla, Haritha; Wunder, Elsio A.; Mechaly, Ariel E.; Mehta, Sameet; Wang, Zheng; Santos, Luciane; Bisht, Vimla; Diggle, Peter; Murray, Gerald; Adler, Ben; Lopez, Francesc; Townsend, Jeffrey P.; Groisman, Eduardo; Picardeau, Mathieu; Buschiazzo, Alejandro; Ko, Albert I.

2018-01-01

Leptospirosis is an emerging zoonotic disease with more than 1 million cases annually. Currently there is lack of evidence for signaling pathways involved during the infection process of Leptospira. In our comprehensive genomic analysis of 20 Leptospira spp. we identified seven pathogen-specific Two-Component System (TCS) proteins. Disruption of two these TCS genes in pathogenic Leptospira strain resulted in loss-of-virulence in a hamster model of leptospirosis. Corresponding genes lvrA and lvrB (leptospira virulence regulator) are juxtaposed in an operon and are predicted to encode a hybrid histidine kinase and a hybrid response regulator, respectively. Transcriptome analysis of lvr mutant strains with disruption of one (lvrB) or both genes (lvrA/B) revealed global transcriptional regulation of 850 differentially expressed genes. Phosphotransfer assays demonstrated that LvrA phosphorylates LvrB and predicted further signaling downstream to one or more DNA-binding response regulators, suggesting that it is a branched pathway. Phylogenetic analyses indicated that lvrA and lvrB evolved independently within different ecological lineages in Leptospira via gene duplication. This study uncovers a novel-signaling pathway that regulates virulence in pathogenic Leptospira (Lvr), providing a framework to understand the molecular bases of regulation in this life-threatening bacterium. PMID:29600195

Continuous Excretion of Leptospira borgpetersenii Ballum in Mice Assessed by Viability Quantitative Polymerase Chain Reaction.

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Soupé-Gilbert, Marie-Estelle; Bierque, Emilie; Geroult, Sophie; Teurlai, Magali; Goarant, Cyrille

2017-10-01

Rodents are the main reservoir animals of leptospirosis. In this study, we characterized and quantified the urinary excretion dynamics of Leptospira by Mus musculus infected with 2 × 10 8 virulent Leptospira borgpetersenii serogroup Ballum. Each micturition was collected separately in metabolic cages, at 12 time points from 7 to 117 days post-infection (dpi). We detected Leptospira in all urine samples collected (up to 8 per time point per mouse) proving that Leptospira excretion is continuous with ca. 90% live L. borgpetersenii Ballum, revealed by viability quantitative polymerase chain reaction. Microscopic visualization by Live/Dead fluorescence confirmed this high proportion of live bacteria and demonstrated that L. borgpetersenii Ballum are excreted, at least partly, as bacterial aggregates. We observed two distinct phases in the excretion dynamics, first an increase in Leptospira concentration shed in the urine between 7 and 63 dpi followed by a plateau phase from 63 dpi onward, with up to 3 × 10 7 Leptospira per mL of urine. These two phases seem to correspond to progressive colonization of renal tubules first, then to stable cell survival and maintenance in kidneys. Therefore, chronically infected adult mice are able to contaminate the environment via urine at each micturition event throughout their lifetime. Because Leptospira excretion reached its maximum 2 months after infection, older rodents have a greater risk of contaminating their surrounding environment.

[Research on the relevance between the virulent genes differential expression and pathogenecity of Leptospira with microarray].

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Yu, De-li; Bao, Lang

2015-01-01

To find the change of virulent gene expression and to analyze the relevance between the virulent change and the gene expression. Grouped guinea pigs were inoculated with 1 mL Leptospira cultured in vivo, Leptospira cultured in vitro and the Leptospira culture medium through abdominal subcutaneous respectively. The survival rate, body mass and temperature change of guinea pigs in different groups were measured within 15 d after the inoculation, then the survived guinea pigs were scarified, and the organ coefficient was also measured to know the virulence of Leptospira cultured in different environment. The amplified gene segments from Leptospira were used as probes and wrote the microarray. The total RNA was extracted from Leptospira standard strain cultured in culture medium and guinea pigs. After reverse transcription to cDNA, they were labeled with Cy3 and Cy5 respectively. Labeled cDNA was mixed and hybridized with the microarray. The hybridized mircroarray was scanned and analysed. The survival rate of inoculated guinea pig was different from group to group (in vivo group: 0%; in vitro group: 88.9%; culture medium group: 100%). The guinea pigs in vivo group had a higher temperature (PLeptospira: LA1027, LA1029, LA4004, LA3050, LA3540, LA0327, LA0378, LA1650, LA3937, LA2089, LA2144, LA3576, LA0011 and gene of Loa22 were up regulation after continuously cultured in guinea pigs. The pathogenic ability of Leptospira cultured in different environment is different and the gene expression of Leptospira is different between in vivo and in vitro as well. The understanding of the meaning of this change might help to know the pathogenecity of Leptospira.

Diversity of Leptospira spp. in Rats and Environment from Urban Areas of Sarawak, Malaysia

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Pui, Chai Fung; Bilung, Lesley Maurice; Apun, Kasing; Su’ut, Lela

2017-01-01

Various prevalence studies on Leptospira in animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophytic Leptospira in selected animals and environments. This study was therefore conducted to detect Leptospira spp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR) method. A total of 107 rats, 292 soil...

Leptospira surface adhesin (Lsa21) induces Toll like receptor 2 and 4 mediated inflammatory responses in macrophages

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Syed M. Faisal; Vivek P. Varma; M. Subathra; Sarwar Azam; Anil K. Sunkara; Mohd Akif; Mirza. S. Baig; Yung-Fu Chang

2016-01-01

Leptospirosis is zoonotic and emerging infectious disease of global importance. Little is understood about Leptospira pathogenesis and host immune response. In the present work we have investigated how Leptospira modulates the host innate immune response mediated by Toll-like receptors (TLRs) via surface exposed proteins. We screened Leptospira outer membrane/surface proteins for their ability to activate/inhibit TLR2/4 signaling in HEK293 cell lines. Of these the 21?kDa Leptospira surface ad...

Molecular detection of Leptospira spp. from canine kidney tissues and its association with renal lesions

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Biswajit R. Dash

2018-04-01

Full Text Available Aim: The study aimed to detect the prevalence of Leptospira spp. in kidney tissues collected during necropsy and to establish its association with renal lesions in dogs of Mumbai region. Materials and Methods: Kidney tissues from 40 dogs were collected during necropsy after gross examination and then fixed in neutral buffered formalin and Bouin's fluid for histopathology and histochemistry, respectively. Kidney tissues were also collected for the detection of Leptospira spp. by polymerase chain reaction (PCR in a sterile container and stored at -80°C until further processing. Results: Of 40 cases studied, 13 (32.5% cases showed lesions of nephritis of varying histotype and severity. Glomerulonephritis was reported as the most common type of nephritis in 9 (69.23% cases, and interstitial nephritis was recorded in 4 (30.76% cases. Chronic and acute interstitial nephritis was observed in two cases each. Renal failure as a cause of death was found in 7 (17.5% dogs. Of a total of 40 cases, 9 were found positive for pathogenic Leptospira spp. genome by PCR. However, of nine PCR-positive cases, only four cases showed lesions in kidneys as glomerulonephritis and interstitial nephritis in two cases each. The rest five cases positive for Leptospira spp. by PCR did not show any appreciable lesions in the kidneys. Conclusion: Leptospiral DNA was detected in 9 (22.5% cases by PCR. Of these nine cases, only four cases showed renal lesions. Other five cases which were positive for Leptospira spp. by PCR did not show any appreciable gross and microscopic lesions in the kidneys which might be carriers for Leptospira spp. Considering variable reports on types of nephritis in Leptospira spp. infection and also the prevalence of non-pathogenic Leptospira spp., it is important to conduct an extensive study on the prevalence of Leptospira spp. and its association with renal lesions involving batteries of tests.

Serologic Survey for Selected Viral and Bacterial Swine Pathogens in Colombian Collared Peccaries ( Pecari tajacu) and Feral Pigs ( Sus scrofa).

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Montenegro, Olga L; Roncancio, Nestor; Soler-Tovar, Diego; Cortés-Duque, Jimena; Contreras-Herrera, Jorge; Sabogal, Sandra; Acevedo, Luz Dary; Navas-Suárez, Pedro Enrique

2018-06-14

In South America, wild populations of peccaries coexist with domestic and feral pigs, with poorly understood consequences. We captured 58 collared peccaries ( Pecari tajacu) and 15 feral pigs ( Sus scrofa) in locations of Colombia where coexistence of these species is known. Blood samples were tested for antibodies against four viral agents, classical swine fever virus (CSFV), Aujeszky's disease virus (ADV), porcine circovirus (PCV-2), and vesicular stomatitis virus (New Jersey and Indiana subtypes) and two bacterial agents, Brucella spp. and six serovars of Leptospira interrogans. The prevalence of CSFV was 5% (3/58) in collared peccaries and 7% (1/15) in feral pigs. The prevalence of PCV-2 was 7% (1/15) in collared peccaries and 67% (2/3) in feral pigs. Vesicular stomatitis prevalence was 33% (8/24) in collared peccaries and 67% (4/6) in feral pigs. Leptospira prevalence was 78% (39/50) in collared peccary and 100% (8/8) in feral pigs; bratislava, grippotyphosa, icterohaemorrhagiae, and pomona were the most frequent serovars. Also, the only white-lipped peccary ( Tayassu pecari) sampled was positive for L. interrogans serovar bratislava and for vesicular stomatitis virus, New Jersey strain. No samples were positive for ADV or Brucella. The seroprevalence of antibodies against L. interrogans was similar to that observed in other studies. Icterohaemorrhagiae appears to be a common serovar among in situ and ex situ peccary populations. Positive antibodies against PVC-2 represent a novel report of exposure to this pathogen in Colombian peccaries. Our results indicate the possible transmission of various pathogens, important for pig farms, in the studied pig and peccaries.

Environmental Factors Influencing White-Tailed Deer (Odocoileus virginianus Exposure to Livestock Pathogens in Wisconsin.

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Shelli Dubay

Full Text Available White-tailed deer (Odocoileus virginianus are commonly exposed to disease agents that affect livestock but environmental factors that predispose deer to exposure are unknown for many pathogens. We trapped deer during winter months on two study areas (Northern Forest and Eastern Farmland in Wisconsin from 2010 to 2013. Deer were tested for exposure to six serovars of Leptospira interrogans (grippotyphosa, icterohaemorrhagiae, canicola, bratislava, pomona, and hardjo, bovine viral diarrhea virus (BVDV-1 and BVDV-2, infectious bovine rhinotracheitis virus (IBR, and parainfluenza 3 virus (PI3. We used logistic regression to model potential intrinsic (e.g., age, sex and extrinsic (e.g., land type, study site, year, exposure to multiple pathogens variables we considered biologically meaningful to exposure of deer to livestock pathogens. Deer sampled in 2010-2011 did not demonstrate exposure to BVDV, so we did not test for BVDV in subsequent years. Deer had evidence of exposure to PI3 (24.7%, IBR (7.9%, Leptospira interrogans serovar pomona (11.7%, L. i. bratislava (1.0%, L. i. grippotyphosa (2.5% and L. i. hardjo (0.3%. Deer did not demonstrate exposure to L. interrogans serovars canicola and icterohaemorrhagiae. For PI3, we found that capture site and year influenced exposure. Fawns (n = 119 were not exposed to L. i. pomona, but land type was an important predictor of exposure to L. i. pomona for older deer. Our results serve as baseline exposure levels of Wisconsin white-tailed deer to livestock pathogens, and helped to identify important factors that explain deer exposure to livestock pathogens.

Leptospira spp. in Small Mammals from Areas with Low and High Human Hantavirus Incidences in South-West Germany.

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Obiegala, Anna; Albrecht, Christoph; Dafalla, Maysaa; Drewes, Stephan; Oltersdorf, Carolin; Turni, Hendrik; Imholt, Christian; Jacob, Jens; Wagner-Wiening, Christiane; Ulrich, Rainer G; Pfeffer, Martin

2017-05-01

Leptospirosis is caused by Leptospira spp. and is considered the most widespread zoonotic disease worldwide. It mimics nephropathia epidemica in humans, a disease mainly caused by Puumala hantavirus (PUUV). Small mammals are reservoirs for Leptospira spp. and PUUV. Seewis virus (SWSV) is a shrew-borne hantavirus with unknown pathogenicity. The objective of this study was to estimate the prevalence for Leptospira spp. and the frequency of Leptospira-hantavirus co-infections in small mammals collected at locations with high and low incidences in humans. In 2012 and 2013, 736 small mammals belonging to seven species (Apodemus flavicollis, Microtus agrestis, Microtus arvalis, Myodes glareolus, Sorex araneus, S. coronatus, and S. minutus) were collected at four high incidence sites (H1-H4) and four low (L1-L4) incidence sites for PUUV infection in humans. Kidney-derived DNA samples were tested for Leptospira spp. by real-time PCR targeting the lipl 32 gene and further analyzed by duplex PCR targeting the flaB and the secY genes. For the detection of Seewis virus, lung-derived DNA was tested via RT-PCR targeting the nucleocapsid gene. Altogether, 42 of the 736 small mammals including 27 of 660 bank voles and 11 of 66 shrews, were positive for Leptospira spp., while Sorex spp. (14.7%) showed significantly higher prevalences compared to bank voles (4.1%). Detected Leptospira spp. were pathogenic species other than L. kirschneri. Significantly more Leptospira-positive bank voles were found at H sites than at L sites. Altogether 22.2% of positive bank voles were infected with PUUV. Double infection of PUUV and Leptospira spp. occurrence in bank voles is 1.86 times (OR = 1.86; 95% CI: 0.72-4.73) more likely than infections with each pathogen separately. Leptospira- positive bank voles are focally positively associated with PUUV infection in bank voles and with human hantavirus cases. It should be considered that shrews may serve as Leptospira spp. reservoirs.

Characterization of Leptospira santarosai Serogroup Grippotyphosa Serovar Bananal Isolated from Capybara ( Hydrochaeris hydrochaeris ) in Brazil.

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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.

Prevalence of antibodies to Leptospira in wild mammals trapped on livestock farms in Ontario, Canada.

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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.

Malagasy bats shelter a considerable genetic diversity of pathogenic Leptospira suggesting notable host-specificity patterns.

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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. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Leptospira Contamination in Household and Environmental Water in Rural Communities in Southern Chile

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Claudia Muñoz-Zanzi

2014-06-01

Full Text Available 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.

Six new leptospiral serovars isolated from wild animals in Peru.

OpenAIRE

Liceras de Hidalgo, J L; Sulzer, K R

1984-01-01

Six new serovars of Leptospira interrogans were isolated from opossums (Didelphis marsupialis and Philander opossum) trapped in the Peruvian jungle. The proposed names, type strain designation, and serogroup of the serovars, respectively, were: huallaga, strain M-7, Djasiman serogroup; luis, strain M-6, Tarassovi serogroup; machiguenga, strain MMD-3, Icterohaemorrhagiae serogroup; rioja, strain MR-12, Bataviae serogroup; rupa rupa, strain M-3, Sejroe serogroup; and tingomaria, strain M-13, Cy...

A cross sectional study of leptospirosis and fetal death in Yucatan, Mexico

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Cárdenas-Marrufo, MaríaFidelia; 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 a...

The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.

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Yang, Chih-Wei; Wu, Mai-Szu; Pan, Ming-Jeng; Hsieh, Wang-Ju; Vandewalle, Alain; Huang, Chiu-Ching

2002-08-01

Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 microg/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-alpha mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-kappaB (NF-kappaB) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

Reassessment of MLST schemes for Leptospira spp. typing worldwide.

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Varni, Vanina; Ruybal, Paula; Lauthier, Juan José; Tomasini, Nicolás; Brihuega, Bibiana; Koval, Ariel; Caimi, Karina

2014-03-01

Leptospirosis is a neglected zoonosis of global importance. Several multilocus sequence typing (MLST) methods have been developed for Leptospira spp., the causative agent of leptospirosis. In this study we reassessed the most commonly used MLST schemes in a set of worldwide isolates, in order to select the loci that achieve the maximum power of discrimination for typing Leptospira spp. Global eBURST algorithm was used to detect clonal complexes among STs and phylogenetic relationships among concatenated and individual sequences were inferred through maximum likelihood (ML) analysis. The evaluation of 12 loci combined to type a subset of strains rendered 57 different STs. Seven of these loci were selected into a final scheme upon studying the number of alleles and polymorphisms, the typing efficiency, the discriminatory power and the ratio dN/dS per nucleotide site for each locus. This new 7-locus scheme was applied to a wider collection of worldwide strains. The ML tree constructed from concatenated sequences of the 7 loci identified 6 major clusters corresponding to 6 Leptospira species. Global eBURST established 8 CCs, which showed that genotypes were clearly related by geographic origin and host. ST52 and ST47, represented mostly by Argentinian isolates, grouped the higher number of isolates. These isolates were serotyped as serogroups Pomona and Icterohaemorrhagiae, showing a unidirectional correlation in which the isolates with the same ST belong to the same serogroup. In summary, this scheme combines the best loci from the most widely used MLST schemes for Leptospira spp. and supports worldwide strains classification. The Argentinian isolates exhibited congruence between allelic profile and serogroup, providing an alternative to serological methods. Published by Elsevier B.V.

Direct measurement of helical cell motion of the spirochete leptospira.

Science.gov (United States)

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

2014-01-07

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. Copyright © 2014 Biophysical Society. Published by Elsevier Inc

Detection of asymptomatic renal Leptospira infection in abattoir slaughtered cattle in southeastern Georgia, United States

Directory of Open Access Journals (Sweden)

Sreekumari Rajeev

2014-07-01

Full Text Available Objectives: Leptospirosis is one of the most widespread zoonotic infectious diseases affecting humans and animals. Several animal species, including cattle, can act as potential asymptomatic carriers facilitating zoonotic transmission of Leptospira. This study was conducted to assess the occurrence of asymptomatic renal Leptospira carriers among cattle slaughtered in southeastern Georgia, United States. Methods: A battery of diagnostic tests, including dark field microscopy, direct fluorescent antibody staining, polymerase chain reaction, and culture, were performed on a set of bovine kidneys (n = 37 collected from an abattoir in southeastern Georgia, United States. Virulence of a field isolate obtained from this study was tested in a hamster experimental model. Results: Motile spirochete-like structures were observed by dark field microscopy in 23 (59% out of 37 kidney samples tested. In all, 29 samples (78% were positive by direct fluorescent antibody staining. Only 11 (29.7% samples by polymerase chain reaction and 3 (8.1% by culture were positive for Leptospira sp. The isolates obtained by culture were confirmed as Leptospira borgpetersenii. Hamsters experimentally infected with one of the Leptospira field isolates obtained from this study did not show clinical signs but developed renal infection with interstitial nephritis and tubular necrosis. Conclusions: This study confirms that asymptomatic Leptospira renal infection is present among cattle in the region. Our findings underscore the need for future studies to assess the potential environmental contamination and transmission to humans in contact with infected cattle.

Detection of asymptomatic renal Leptospira infection in abattoir slaughtered cattle in southeastern Georgia, United States

Science.gov (United States)

Ilha, Marcia; Woldemeskel, Moges; Berghaus, Roy D; Pence, Mel E

2014-01-01

Objectives: Leptospirosis is one of the most widespread zoonotic infectious diseases affecting humans and animals. Several animal species, including cattle, can act as potential asymptomatic carriers facilitating zoonotic transmission of Leptospira. This study was conducted to assess the occurrence of asymptomatic renal Leptospira carriers among cattle slaughtered in southeastern Georgia, United States. Methods: A battery of diagnostic tests, including dark field microscopy, direct fluorescent antibody staining, polymerase chain reaction, and culture, were performed on a set of bovine kidneys (n = 37) collected from an abattoir in southeastern Georgia, United States. Virulence of a field isolate obtained from this study was tested in a hamster experimental model. Results: Motile spirochete-like structures were observed by dark field microscopy in 23 (59%) out of 37 kidney samples tested. In all, 29 samples (78%) were positive by direct fluorescent antibody staining. Only 11 (29.7%) samples by polymerase chain reaction and 3 (8.1%) by culture were positive for Leptospira sp. The isolates obtained by culture were confirmed as Leptospira borgpetersenii. Hamsters experimentally infected with one of the Leptospira field isolates obtained from this study did not show clinical signs but developed renal infection with interstitial nephritis and tubular necrosis. Conclusions: This study confirms that asymptomatic Leptospira renal infection is present among cattle in the region. Our findings underscore the need for future studies to assess the potential environmental contamination and transmission to humans in contact with infected cattle. PMID:26770734

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

Science.gov (United States)

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. Copyright © 2016 European Federation of Immunological Societies. All rights reserved.

Pathogenic Leptospira: Advances in understanding the molecular pathogenesis and virulence

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Ghazaei, Ciamak

2018-01-01

Leptospirosis is a common zoonotic disease has emerged as a major public health problem, with developing countries bearing disproportionate burdens. Although the diverse range of clinical manifestations of the leptospirosis in humans is widely documented, the mechanisms through which the pathogen causes disease remain undetermined. In addition, leptospirosis is a much-neglected life-threatening disease although it is one of the most important zoonoses occurring in a diverse range of epidemiological distribution. Recent advances in molecular profiling of pathogenic species of the genus Leptospira have improved our understanding of the evolutionary factors that determine virulence and mechanisms that the bacteria employ to survive. However, a major impediment to the formulation of intervention strategies has been the limited understanding of the disease determinants. Consequently, the association of the biological mechanisms to the pathogenesis of Leptospira, as well as the functions of numerous essential virulence factors still remain implicit. This review examines recent advances in genetic screening technologies, the underlying microbiological processes, the virulence factors and associated molecular mechanisms driving pathogenesis of Leptospira species. PMID:29445617

Pathogenic and Saprophytic Leptospira Species in Water and Soils from Selected Urban Sites in Peninsular Malaysia

Science.gov (United States)

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

Development of Chronic and Acute Golden Syrian Hamster Infection Models with Leptospira borgpetersenii serovar Hardjo

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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...

High prevalence of Leptospira spp. in sewer rats (Rattus norvegicus)

DEFF Research Database (Denmark)

Krøjgaard, L H; Villumsen, S; Markussen, M D K

2009-01-01

Earlier studies on the ecology of leptospirosis in temperate regions focused mainly on free-ranging rats in rural areas. Here we report on the occurrence of Leptospira spp. in Rattus norvegicus living in sewers in a suburban area in Copenhagen, Denmark. In 2006-2007, about 30 rats were captured...... in sewers at each of six different locations. Rat kidneys were screened by PCR for pathogenic Leptospira spp. In one location no infected rats were found, whereas the prevalence in the remaining five locations ranged between 48% and 89%. Micro-agglutination tests showed that serogroup Pomona, Sejroe...

Assessment of exposure to Leptospira serovars in veterinary staff and dog owners in contact with infected dogs.

Science.gov (United States)

Barmettler, Reto; Schweighauser, Ariane; Bigler, Susanne; Grooters, Amy M; Francey, Thierry

2011-01-15

To assess patterns of seroreactivity to Leptospira serovars in veterinary professional staff and dog owners exposed to dogs with acute leptospirosis and to contrast these patterns in people with those observed in dogs. Cross-sectional study. Human subjects consisted of 91 people (50 veterinarians, 19 technical staff, 9 administrative personnel, and 13 dog owners) exposed to dogs with leptospirosis. Canine subjects consisted of 52 dogs with naturally occurring leptospirosis admitted to the University of Bern Vetsuisse Faculty Small Animal Clinic in 2007 and 2008. People were tested for seroreactivity to regionally prevalent Leptospira serovars by use of a complement fixation test. A questionnaire designed to identify risk factors associated with seropositivity was used to collect demographic information from each study participant. Dogs were tested for seroreactivity to Leptospira serovars by use of a microscopic agglutination test. On the basis of microscopic agglutination test results, infected dogs were seropositive for antibodies against Leptospira serovars as follows (in descending order): Bratislava (43/52 [83%]), Australis (43/52 [83%]), Grippotyphosa (18/52 [35%]), Pomona (12/52 [23%]), Autumnalis (6/52 [12%]), Icterohemorrhagiae (4/52 [8%]), Tarassovi (2/52 [4%]), and Canicola (1/52 [2%]). All 91 people were seronegative for antibodies against Leptospira serovars. Therefore, statistical evaluation of risk factors and comparison of patterns of seroreactivity to Leptospira serovars between human and canine subjects were limited to theoretical risks. Seroreactivity to Leptospira serovars among veterinary staff adhering to standard hygiene protocols and pet owners exposed to dogs with acute leptospirosis was uncommon.

The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis

OpenAIRE

Silva, Éverton F.; Medeiros, Marco A.; McBride, Alan J. A.; Matsunaga, Jim; Esteves, Gabriela S.; Ramos, João G. R.; Santos, Cleiton S.; Croda, Júlio; Homma, Akira; Dellagostin, Odir A.; Haake, David A.; Reis, Mitermayer G.; Ko, Albert I.

2007-01-01

Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar...

Testing and Validation of High Density Resequencing Microarray for Broad Range Biothreat Agents Detection

Science.gov (United States)

2009-08-11

concentration of the virus was expressed in plaque forming units (pfu). For synthetic DNA templates, the DNA concentration was used to calculate the...cells ATCC Escherichia coli O157:H7 ATCC 43985 (CDC EDL933) Nucleic acid ATCC Francisella tularensis SHU4 Nucleic acid AFIP Leptospira interrogans ATCC...2% on the nucleotide level and 0.6% on the protein level. Similar strain discrimination was obtained in case of Machupo virus strains Carvallo and

Comparison of Mucosal, Subcutaneous and Intraperitoneal Routes of Rat Leptospira Infection

Science.gov (United States)

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

Lai-like leptospira from the Andaman Islands

NARCIS (Netherlands)

Sehgal, S. C.; Vijayachari, P.; Smythe, L. D.; Norris, M.; Symonds, M.; Dohnt, M.; Korver, H.; v d Kemp, H.; Hartskeerl, R. A.; Terpstra, W. J.

2000-01-01

Leptospirosis has been an important public health problem in the Andaman Islands since 1988. As information about the exact etiological agent is not available, the present study was undertaken to isolate and identify Leptospira from human patients. An isolate coded AF61 was recovered from the blood

The prevalence of Leptospira sp in sewer rats (Rattus norvegicus)

DEFF Research Database (Denmark)

Krøigaard, Louise; Villumsen, Steen; Markussen, Mette Drude

Test). Preliminary results based on one fourth of the captured rats indicate, that the prevalence of pathogenic leptospira infected sewer rats are relatively high, as 28 out 48 rats were positive by PCR. This suggests that the sewer could be an environment representing high prevalence of leptospira...... among rats and thus an environment with high risk of transmission....

[MALDI-TOF MASS-SPECTROMETRIC ANAIYSIS OF LEPTOSPIRA SPP. USED IN SERODIAGNOSTICS OF LEPTOSPIROSIS].

Science.gov (United States)

Zyeva, E V; Stoyanova, N A; Tokarevich, N K; Totolyan, Areg A

2015-01-01

Creation of a classification model of Leptospira spp. serovar model using ClinProTools 3.0 software and evaluation of use of MALDI-TOF MS as a method of quality control of reference strains of leptospira. 10 reference strains of Leptospira spp. were used in the study according to microscopic agglutination reaction from the collection of Pasteur RIEM. All the strains were cultivated for 10 days in Terskikh medium at 28 degrees C. Cell extracts were obtained by ethanol/formic acid method. α-cyano-4-hydroxycinnamic acid solution was used as a matrix. Mass-spectra were obtained in Microflex mass-spectrometer (Bruker Daltonics, Germany). External validation of the test-model was carried out using novel spectra of every reference strain during their repeated reseeding. Values of cross-validation and confirmatory ability of the optimal model, built on a genetic algorithm, was 99.14 and 100%, respectively. This model contained 11 biomarker peaks (m/z 2959, 3447, 3548, 3764, 3895, 5221, 5917, 6173, 6701, 7013, 8364) for serovar classification. Results of the external validation have shown a 100% correct classification in serovar classesin Sejroe, Ballum, Tarassovi; Copenhageni, Mozdoc, Grippotyphosa and Patoc, that indicates a high prognostic ability of the model in these classes. However, data from verification matrix have shown, that 50%.of the spectra from Canicola and Pomona serovars were classified as Patoc class, that could be associated with cross serological activity of Patoc serovar L. biflexa with pathogenic leptospirae. MALDI-TOF mass-spectrometry method combined with building and using the classification model could be a useful instrument for intra-laboratory control of leptospira reseeding.

Detection of Leptospira DNA in urine and presence of specific antibodies in outdoor cats in Germany.

Science.gov (United States)

Weis, Sonia; Rettinger, Anna; Bergmann, Michele; Llewellyn, Julia R; Pantchev, Nikola; Straubinger, Reinhard K; Hartmann, Katrin

2017-04-01

Objectives Clinical manifestation of infection with Leptospira species in cats is rare. Nevertheless, cats can develop specific antibodies against the spirochetes after infection. In Canada, Taiwan and the USA it was recently demonstrated that naturally infected cats can also shed DNA from pathogenic Leptospira species in their urine, but the zoonotic potential of infected cats is still unclear. The objective of this study was to demonstrate if outdoor cats in Germany shed DNA from pathogenic Leptospira species in their urine. As a second aim, antibody prevalence was determined. Methods Two hundred and fifteen outdoor cats were prospectively recruited. Urine samples were tested by real-time PCR targeting the lipL32 gene of pathogenic Leptospira species. Antibody titres against eight serovars (Australis, Autumnalis, Bratislava, Canicola, Copenhageni, Grippotyphosa, Pomona, Saxkoebing) belonging to seven serogroups (Australis, Autumnalis, Canicola, Grippotyphosa, Icterohaemorrhagiae, Pomona, Sejroe) were determined by microscopic agglutination test. Results Urine samples from 7/215 cats (3.3%; 95% confidence interval [CI] 0.9-5.7) were PCR-positive. Specific antibodies were detected in 35/195 cats (17.9%; 95% CI: 12.5-23.3) with titres ranging from 1:100 to 1:6400. Australis, Bratislava and Grippotyphosa were the most common serovars. Conclusions and relevance Outdoor cats in Germany can shed DNA from pathogenic Leptospira species. Therefore, outdoor cats should be considered as a possible source of infection for dogs or humans. Further studies are needed to determine the role of Leptospira species as a cause of disease in cats.

Molecular epidemiology of pathogenic Leptospira spp. in the straw-colored fruit bat (Eidolon helvum) migrating to Zambia from the Democratic Republic of Congo.

Science.gov (United States)

Ogawa, Hirohito; Koizumi, Nobuo; Ohnuma, Aiko; Mutemwa, Alisheke; Hang'ombe, Bernard M; Mweene, Aaron S; Takada, Ayato; Sugimoto, Chihiro; Suzuki, Yasuhiko; Kida, Hiroshi; Sawa, Hirofumi

2015-06-01

The role played by bats as a potential source of transmission of Leptospira spp. to humans is poorly understood, despite various pathogenic Leptospira spp. being identified in these mammals. Here, we investigated the prevalence and diversity of pathogenic Leptospira spp. that infect the straw-colored fruit bat (Eidolon helvum). We captured this bat species, which is widely distributed in Africa, in Zambia during 2008-2013. We detected the flagellin B gene (flaB) from pathogenic Leptospira spp. in kidney samples from 79 of 529 E. helvum (14.9%) bats. Phylogenetic analysis of 70 flaB fragments amplified from E. helvum samples and previously reported sequences, revealed that 12 of the fragments grouped with Leptospira borgpetersenii and Leptospira kirschneri; however, the remaining 58 flaB fragments appeared not to be associated with any reported species. Additionally, the 16S ribosomal RNA gene (rrs) amplified from 27 randomly chosen flaB-positive samples was compared with previously reported sequences, including bat-derived Leptospira spp. All 27 rrs fragments clustered into a pathogenic group. Eight fragments were located in unique branches, the other 19 fragments were closely related to Leptospira spp. detected in bats. These results show that rrs sequences in bats are genetically related to each other without regional variation, suggesting that Leptospira are evolutionarily well-adapted to bats and have uniquely evolved in the bat population. Our study indicates that pathogenic Leptospira spp. in E. helvum in Zambia have unique genotypes. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Expanding the Genetic Toolbox for Leptospira Species by Generation of Fluorescent Bacteria ▿

OpenAIRE

Aviat, Florence; Slamti, Leyla; Cerqueira, Gustavo M.; Lourdault, Kristel; Picardeau, Mathieu

2010-01-01

Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence ...

Leptospira Contamination in Household and Environmental Water in Rural Communities in Southern Chile

OpenAIRE

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 an...

The isolated Leptospira Spp. Identification by molecular biological techniques

Directory of Open Access Journals (Sweden)

Duangjai Suwancharoen

2017-01-01

Full Text Available Leptospirosis is a zoonotic disease caused by the bacteria of Leptospira spp. Identification of this bacterium relies on serotyping and genotyping. Data base for animal causative serovars in Thailand is limited. As the unknown serovars are found in the laboratory, they need to be sent overseas for referent identification. To reduce the cost, this research intended to develop a leptospiral identification method which is user–friendly and able to classify efficiently. Ten Leptospira isolations were cultured from urine samples. They were identified by three molecular biological techniques, including Pulsed-Field Gel Electrophoresis (PFGE, Variable Number Tandem Repeat (VNTR and Multilocus Sequence Typing (MLST. These methods were developed and compared to find the most suitable one for leptospiral identification. VNTR was found to be inappropriate since it could not identify the agents and it did not show the PCR product. PFGE and MLST gave the same results of the unknown 1 and 2 which were L.weilii sv Samin st Samin. Unknown 4 showed different results by each technique. Unknown 5 to 10 were likely to be L.meyeri sv Ranarum st ICF and Leptonema illini sv Illini st 3055 by PFGE but MLST could not identify the serovar. However, molecular biological technique for Leptospira identification should be done by several methods in order to confirm the result of each other.

Prevalencia de anticuerpos contra Leptospira en personas asintomáticos y en perros de Chancay, Lima 2001

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Manuel Céspedes

2007-10-01

Full Text Available Objetivos. Determinar la prevalencia de anticuerpos contra Leptospiras en personas asintomáticas dedicadas a la agricultura, pesca y comercio y en perros domésticos de localidades de Chancay (Huaral, Lima. Material y métodos. Estudio transversal analítico, se tomó muestras de suero de 268 pobladores de tres localidades, en quienes se evaluó la presencia de anticuerpos totales contra Leptospiras por el método de ELISA IgG y la prueba de microaglutinación (MAT. Se buscaron los factores asociados con la positividad a Leptospiras calculando el OR con su intervalo de confianza al 95%. De la misma manera, se tomó muestras de suero de 241 perros a los que se realizó la prueba de MAT. Resultados. La prevalencia de anticuerpos contra Leptospira en población asintomática fue de 10,1% (IC95%: 6,3-13,9, la cual estuvo asociada con el abastecimiento de agua para consumo en quebrada o pozo (OR: 3,48, IC95%: 1,39-8,74, con el antecedente de nadar en el río o acequia (OR: 4,75, IC95%: 1,51-14,92 y con tener una edad entre 21-40 años (OR: 2,47, IC95%: 1,10-5,52. Los serovares más frecuentes fueron Icterohaemorrhagiae y Canicola según MAT. En canes, 27,8% (67/241 tuvieron serología positiva a leptospiras. Conclusiones. Existe una mediana prevalencia de serología positiva para Leptospiras en la población general asintomática y condiciones favorables para la presencia de Leptospiras en las localidades estudiadas. En estas zonas se recomienda realizar actividades educativas preventivas frecuentes, tomando en cuenta los resultados de este estudio y el personal de salud debe sospechar de la leptospirosis como una causa de enfermedad febril.

Preliminary Investigations on the Distribution of Leptospira Serovars in Domestic Animals in North-west Morocco.

Science.gov (United States)

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. © 2014

Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing.

Science.gov (United States)

Waggoner, Jesse J; Balassiano, Ilana; Mohamed-Hadley, Alisha; Vital-Brazil, Juliana Magalhães; Sahoo, Malaya K; Pinsky, Benjamin A

2015-01-01

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. 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%). 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 case detection.

Critical Knowledge Gaps in Our Understanding of Environmental Cycling and Transmission of Leptospira spp.

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Barragan, Veronica; Olivas, Sonora; Keim, Paul; Pearson, Talima

2017-10-01

Exposure to soil or water contaminated with the urine of Leptospira -infected animals is the most common way in which humans contract leptospirosis. Entire populations can be at high risk of leptospirosis while working in inundated fields, when engaging in aquatic sports, or after periods of heavy rainfall. The risk of infection after contact with these environmental sources depends on the ability of Leptospira bacteria to survive, persist, and infect new hosts. Multiple variables such as soil and water pH, temperature, and even environmental microbial communities are likely to shape the environmental conditions needed by the pathogen to persist. Here we review what is known about the environmental phase of the infectious Leptospira transmission cycle and identify knowledge gaps that will serve as a guide for future research. Copyright © 2017 Barragan et al.

Antibodies to parvovirus, distemper virus and adenovirus conferred to household dogs using commercial combination vaccines containing Leptospira bacterin.

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Taguchi, M; Namikawa, K; Maruo, T; Lynch, J; Sahara, H

2010-12-11

To examine how the inclusion (+) or exclusion (-) of inactivated Leptospira antigens in a vaccine for canine parvovirus type 2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type 2 (CAdV-2) affects antibody titres to CPV-2, CDV and CAdV-1 antigens, household dogs were vaccinated with commercially available vaccines from one of three manufacturers. CPV-2, CDV and CAdV-1 antibody titres were measured 11 to 13 months later and compared within three different age groups and three different bodyweight groups. There were significant differences between CPV-2 antibody titres in dogs vaccinated with (+) vaccine and those vaccinated with (-) vaccine for two products in the two-year-old group and for one product in the greater than seven-year-old group; no significant differences were seen that could be attributed to bodyweight. No differences in CDV antibody titres were observed within age groups, but a significant difference was seen in the 11 to 20 kg weight group for one product. Significant differences in CAdV-1 antibody titres were seen for one product in both the two-year-old group and the ≤10 kg weight group.

Identification of Pathogenic Leptospira in Rat and Shrew Populations Using rpoB Gene and Its Spatial Distribution in Boyolali District

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Dyah Widiastuti

2016-08-01

Ngemplak dan Desa Jeron Kecamatan Nogosari, Kabupaten Boyolali. Pemeriksaan Polymerase Chain Reaction dilakukan pada 104 sampel ginjal tikus dari dua lokasi penelitian. Analisis spasial sederhana dilakukan untuk memetakan sebaran tikus yang positif Leptospira. Terdapat enam sampel positif gen rpoB Leptospira pada Rattus tanezumi, Rattus argentiventer dan Suncus murinus. Lima dari keenam sampel menunjukkan hubungan kekerabatan yang paling dekat dengan Leptospira borgpetersenii serovar Sejroe berdasarkan gen rpoB. Satu isolat tidak memiliki hubungan kekerabatan yang dekat dengan serovar manapun yang masuk dalam cluster. Analisis spasial berdasarkan jarak aktivitas harian tikus menunjukkan tikus positif Leptospira ditemukan berada dalam kisaran 30 meter dan 150 meter dari penderita leptospirosis.

Leptospira borgpetersenii hybrid leucine-rich repeat protein: Cloning and expression, immunogenic identification and molecular docking evaluation.

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Sritrakul, Tepyuda; Nitipan, Supachai; Wajjwalku, Worawidh; La-Ard, Anchalee; Suphatpahirapol, Chattip; Petkarnjanapong, Wimol; Ongphiphadhanakul, Boonsong; Prapong, Siriwan

2017-11-01

Leptospirosis is an important zoonotic disease, and the major outbreak of this disease in Thailand in 1999 was due largely to the Leptospira borgpetersenii serovar Sejroe. Identification of the leucine-rich repeat (LRR) LBJ_2271 protein containing immunogenic epitopes and the discovery of the LBJ_2271 ortholog in Leptospira serovar Sejroe, KU_Sej_R21_2271, led to further studies of the antigenic immune properties of KU_Sej_LRR_2271. The recombinant hybrid (rh) protein was created and expressed from a hybrid PCR fragment of KU_Sej_R21_2271 fused with DNA encoding the LBJ_2271 signal sequence for targeting protein as a membrane-anchoring protein. The fusion DNA was cloned into pET160/GW/D-TOPO® to form the pET160_hKU_R21_2271 plasmid. The plasmid was used to express the rhKU_Sej_LRR_2271 protein in Escherichia coli BL21 Star™ (DE3). The expressed protein was immunologically detected by Western blotting and immunoreactivity detection with hyperimmune sera, T cell epitope prediction by HLA allele and epitope peptide binding affinity, and potential T cell reactivity analysis. The immunogenic epitopes of the protein were evaluated and verified by HLA allele and epitope peptide complex structure molecular docking. Among fourteen best allele epitopes of this protein, binding affinity values of 12 allele epitopes remained unchanged compared to LBJ_2271. Two epitopes for alleles HLA-A0202 and -A0301 had higher IC 50 values, while T cell reactivity values of these peptides were better than values from LBJ_2271 epitopes. Eight of twelve epitope peptides had positive T-cell reactivity scores. Although the molecular docking of two epitopes, 3FPLLKEFLV11/47FPLLKEFLV55 and 50KLSTVPEGV58, into an HLA-A0202 model revealed a good fit in the docked structures, 50KLSTVPEGV58 and 94KLSTVPEEV102 are still considered as the proteins' best epitopes for allele HLA-A0202. The results of this study showed that rhKU_Sej_LRR_2271 protein contained natural immunological properties that should

Preliminary studies on pathogenic Leptospira spp . In slaughtered ...

African Journals Online (AJOL)

This study was undertaken to investigate the occurrence of pathogenic Leptospira spp. and the associated renal morphological changes in pigs slaughtered in slaughter slabs within Abeokuta metropolis, Nigeria. A total of 42 pigs' kidney samples were randomly collected for the study. The samples were examined using ...

Utilization of Exocellular Mannan from Rhodotorula glutinis as an Immunoreactive Antigen in Diagnosis of Leptospirosis

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Matsuo, Kouki; Isogai, Emiko; Araki, Yoshio

2000-01-01

Previously, Rhodotorula glutinis was reported to produce a large amount of exocellular mannan, having a repeating unit of →3)-d-Manp-(1→4)-d-Manp-(1→. Recently, we found that antigenic polysaccharides of Leptospira biflexa serovar patoc strain Patoc I have the same repeating unit and cross-react with antisera raised against extended strains of other leptospires (K. Matsuo, E. Isogai, and Y. Araki, Carbohydr. Res., in press). This structural identity and the difficulty of producing and isolating antigens led us to confirm the usefulness of Rhodotorula mannan as an immunoreactive antigen in a serological diagnosis of leptospirosis. In the present investigation, we confirmed the structural identity of an exocellular mannan isolated from R. glutinis AHU 3479 and tried to use it as an immunoreactive antigen in a serological diagnosis of leptospirosis. From its chemical analysis and 1H- and 13C-labeled nuclear magnetic resonance spectrometry, the Rhodotorula mannan was confirmed to consist of the same disaccharide units. Furthermore, such a preparation was shown to immunoreact to various sera from patients suffering with leptospirosis as well as to most rabbit antiserum preparations obtained from immunization with various strains of pathogenic leptospires. Therefore, the Rhodotorula mannan preparation is useful as an immunoreactive antigen in the serological diagnosis for leptospirosis. PMID:11015396

Presence of antibodies against Leptospira serovars in Chaetophractus villosus (Mammalia, Dasypodidae), La Pampa province, Argentina.

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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. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Technical improvement to prevent DNA degradation of Leptospira spp. in pulsed field gel electrophoresis.

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Ribeiro, R L; Machry, L; Brazil, J M V; Ramos, T M V; Avelar, K E S; Pereira, M M

2009-08-01

Leptospirosis is a public health problem. Infection with pathogenic Leptospira occurs by exposure to many environments and is traditionally associated with occupational risk activities. Pulsed-field gel electrophoresis was used to investigate the epidemiological relatedness among Leptospira isolates. However, analysis by PFGE yielded inconclusive data as a result of extensive DNA degradation. This degradation can be significantly reduced by the inclusion of thiourea in the electrophoresis buffer, improving the analysis of DNA banding patterns.

Sensitive real-time PCR detection of pathogenic Leptospira spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis.

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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

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. 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 (pLeptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay. 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.

Global patterns of Leptospira prevalence in vertebrate reservoir hosts

DEFF Research Database (Denmark)

Andersen-Ranberg, Emilie U.; Pipper, Christian Bressen; Jensen, Per Moestrup

2016-01-01

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...

Seroprevalence and Risk Factors for Leptospira Seropositivity in Beef Cattle, Sheep and Deer Farmers in New Zealand.

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Sanhueza, J M; Heuer, C; Wilson, P R; Benschop, J; Collins-Emerson, J M

2017-08-01

Leptospirosis is a global zoonosis that in New Zealand affects primarily people occupationally exposed to livestock. The objective of this study was to estimate the seroprevalence of five Leptospira serovars in farmers working on cattle, sheep and deer farms that had the serological status of animals previously assessed and to identify risk factors for farmer seropositivity. A total of 178 farmers from 127 properties participated in the study. Blood samples were tested using the microscopic agglutination test (MAT) for the presence of antibodies to Leptospira. Samples with a MAT titre ≥48 were considered seropositive. Using Bayesian statistical analysis, the median seroprevalence of Leptospira, all serovars combined, was estimated to be 6.6% (95% probability interval (PI) 3.6-10.9%). Risk factors associated with seropositivity were assisting deer or cattle calving, farming deer, having ≥25% of flat terrain and high abundance of wild deer on farm, while high possum abundance on farm was negatively associated with seropositivity. No association was observed between farmer serostatus and previously recorded livestock serology. Leptospira seropositivity was associated with influenza-like illness of farmers (RR = 1.7; 95% PI 1.0-2.5). Assuming a causal relationship, this suggested an annual risk of 1.3% (95% PI 0.0-3.0%) of influenza-like illnesses due to Leptospira infection in the population of farmers. The association between seropositivity and disease can be used to estimate the public health burden of leptospirosis in New Zealand. Identifying and understanding risk factors for Leptospira seropositivity can inform preventive measures, hence contributing to the reduction of leptospirosis incidence in farmers. © 2016 Blackwell Verlag GmbH.

Prevalencia de anticuerpos contra dengue y leptospira en la población de Jáltipan, Veracruz Prevalence of dengue and leptospira antibodies in the state of Veracruz, Mexico

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Joel Navarrete-Espinosa

2006-06-01

Full Text Available OBJETIVO: Conocer la prevalencia de anticuerpos contra dengue y leptospira en Jáltipan, Veracruz, México MATERIAL Y MÉTODOS: Estudio transversal en 500 habitantes de Jáltipan, Veracruz, mediante un muestreo por hogares, aplicación de cuestionario y toma de muestras sanguíneas. Se buscaron anticuerpos IgG y se evaluaron factores asociados a sus seroprevalencias mediante regresión logística RESULTADOS: La seroprevalencia global para denguevirus fue 79.6% (IC95% 76-81; los principales factores vinculados con la infección fueron la edad (pOBJECTIVE: To discover the prevalence of antibodies against denguevirus and leptospira. MATERIAL AND METHODS: A cross-sectional study was carried out in 500 inhabitants of Veracruz, Mexico through household sampling, administration of a questionnaire and collection of blood samples. The presence of IgG antibodies was determined and factors associated with seroprevalence were evaluated using a logistic regression model. RESULTS: Total denguevirus seroprevalence was 79.6% (IC95% 76-81. The main factors associated with infection were age and residence in neighborhoods with higher vectorial indices (RMP=2.94, IC95% 1.46-6.1. Overall seroprevalence against leptospira was 4% (IC95% 2-6. Prevalence was higher in the 25 to 44 years age group (35%, IC95% 15-59. Seroprevalence in individuals who referred to living with dogs, pigs, cows and rats was 25% (IC95% 0.63-80. Eighty-five percent of individuals who were leptospira positive were also dengue positive. CONCLUSIONS: The prevalence of denguevirus antibodies suggests the risk of serious dengue hemorrhagic outbreaks. Although leptospirosis prevalence was low, the coexistence of both agents was confirmed.

Leptospira Seroprevalence and Risk Factors in Health Centre Patients in Hoima District, Western Uganda.

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Dreyfus, Anou; Dyal, Jonathan W; Pearson, Raewynne; Kankya, Clovice; Kajura, Charles; Alinaitwe, Lordrick; Kakooza, Steven; Pelican, Katharine M; Travis, Dominic A; Mahero, Michael; Boulware, David R; Mugisha, Lawrence

2016-08-01

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. 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. 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.

Leptospira Seroprevalence and Risk Factors in Health Centre Patients in Hoima District, Western Uganda.

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Anou Dreyfus

2016-08-01

Full Text Available 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.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.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.

Influence of household rat infestation on leptospira transmission in the urban slum environment.

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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

2014-12-01

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. 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). 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 therefore be an effective risk stratification strategy for targeting control

Contrasting Patterns in Mammal–Bacteria Coevolution: Bartonella and Leptospira in Bats and Rodents

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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

Meta-analysis to estimate the load of Leptospira excreted in urine: beyond rats as important sources of transmission in low-income rural communities.

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Barragan, Veronica; Nieto, Nathan; Keim, Paul; Pearson, Talima

2017-01-28

Leptospirosis is a major zoonotic disease with widespread distribution and a large impact on human health. Carrier animals excrete pathogenic Leptospira primarily in their urine. Infection occurs when the pathogen enters a host through mucosa or small skin abrasions. Humans and other animals are exposed to the pathogen by direct contact with urine, contaminated soil or water. While many factors influence environmental cycling and the transmission of Leptospira to humans, the load of pathogenic Leptospira in the environment is likely to play a major role. Peridomestic rats are often implicated as a potential source of human disease; however exposure to other animals is a risk factor as well. The aim of this report is to highlight the importance of various carrier animals in terms of the quantity of Leptospira shed into the environment. For this, we performed a systematic literature review and a meta-analysis of the amount of pathogen that various animal species shed in their urine. The quantity of pathogen has been reported for cows, deer, dogs, humans, mice, and rats, in a total of 14 research articles. We estimated the average Leptospira per unit volume shed by each animal species, and the daily environmental contribution by considering the total volume of urine excreted by each carrier animal. Rats excrete the highest quantity of Leptospira per millilitre of urine (median = 5.7 × 10 6  cells), but large mammals excrete much more urine and thus shed significantly more Leptospira per day (5.1 × 10 8 to 1.3 × 10 9  cells). Here we illustrate how, in a low-income rural Ecuadorian community, host population demographics, and prevalence of Leptospira infection can be integrated with estimates of shed Leptospira to suggest that peridomestic cattle may be more important than rats in environmental cycling and ultimately, transmission to humans.

Anti-Leptospira spp. antibodies in captive animals in Paraíba State

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Arthur Brasil

2013-12-01

Full Text Available The aim of this survey was to verify the occurrence of anti-Leptospira spp. antibodies in captive animals in the Parque Zoobotânico Arruda Câmara, João Pessoa, Paraíba State, Northeastern Brazil. Blood samples were collected from 49 animals: 26 mammals of the species Sapajus libidinosus, Cebus flavius, Saimiri sciureu, Coendu sp., Pseudalopex vetulus, Leopardus pardalis, Leopardus tigrinus, Galactitis vitata, Eira barbara, Nasua nasua, Tayassu tajacu and Ratus norvegicus; 10 birds of the species Penelope jacucaca, Pavo cristatus, Anodorhynchus hyacinthinus, Ara chlorothpterus, Pionites leucogaster, Polyborus plancus, Geranoaetus melanoleucus and Urubitinga urubitinga; and 13 reptiles of the species Caiman latirostris, Paleosuchus trigonatus, Caiman crocodilus, Tupinabis merinae, Tupinambis teguixin, Boa constrictor, Corallus hortulanus, Python molurus, Bufocephala vanderhaegei, Geochelone denticulata and Geochelone carboraria. Sera were examined by the microscopic agglutination teste (MAT using 24 serovars as antigens and cut-off point of 1:100. One ocelot (Leopardo pardalis presented positive reaction for the Icterohaemorrhagiae serovar with titer of 100, however, it did not show any clinical sign of the infection. Sinantropic rodents are the main reservoirs of this serovar, which suggests the need of maintenance and continuous evaluation of rodent control programs.

Inmunogenicidad y capacidad protectora en hamsters de vacunas antileptospirósicas monovalentes de células enteras del serogrupo Ballum Immunogenicity and protective capacity of leptospiral whole-cell monovalent serogroup Ballum vaccines in hamsters

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A. González

2005-12-01

Full Text Available El serogrupo Ballum de Leptospira constituye en la actualidad la primera causa de leptospirosis humana en Cuba. Vacunas de células enteras químicamente inactivadas fueron formuladas a partir de dos cepas clínicas de Leptospira interrogans serogrupo Ballum empleando como adyuvante hidróxido de aluminio. Los niveles de aglutininas inducidos en hamsters por una u otra preparación vacunal fueron estimados mediante aglutinación microscópica y la actividad IgG específica fue cuantificada mediante ELISA. La capacidad de protección homóloga y heteróloga contra la infección letal y subletal se determinó mediante el desafío con 100 y 10 000 DL50 de cinco cepas virulentas pertenecientes a los serogrupos Ballum, Canicola, Icterohaemorrhagiae y Pomona. Las evaluaciones realizadas demostraron que ambas vacunas fueron inmunogénicas e indujeron una completa protección homóloga en el modelo animal empleado. La protección cruzada frente a serogrupos heterólogos solo fue significativa en una de las preparaciones monovalentes frente al desafío con 100 DL50 de Canicola. Como resultado de este estudio se pudo comprobar la alta inmunogenicidad y capacidad protectora en hamsters de vacunas monovalentes de células enteras formuladas a partir de dos cepas candidatas vacunales del serogrupo de Leptospira de mayor circulación en humanos en Cuba no incluido en la vacuna actualmente disponible.Leptospira serogroup Ballum is at present the first cause of human leptospirosis in Cuba. Killed whole-cell vaccines were formulated with two clinical isolates of Leptospira interrogans serogroup Ballum using aluminum hydroxide as adjuvant. Agglutinins levels induced by each vaccine in hamsters were estimated by microscopic agglutination test and specific IgG activities were quantified by a whole cell-based enzyme-linked immunosorbent assay. Homologous and cross protective capacity against lethal and sublethal infection were determined in vaccinated animals by

A simple and rapid molecular method for Leptospira species identification

NARCIS (Netherlands)

Ahmed, Ahmed; Anthony, Richard M.; Hartskeerl, Rudy A.

2010-01-01

Serological and DNA-based classification systems only have little correlation. Currently serological and molecular methods for characterizing Leptospira are complex and costly restricting their world-wide distribution and use. Ligation mediated amplification combined with microarray analysis

Detection of Leptospira in Rats Trapped from Households in Phraroj Village, Muang Sam Sip District, Ubon Ratchathani Province Using Polymerase Chain Reaction Technique.

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Wongbutdee, Jaruwan; Jittimanee, Jutharat

2016-01-01

Leptospirosis, a zoonotic disease caused by Leptospira, has been a health problem in Thailand for several years. Rats are a major reservoir host for Leptospira, and the people who are usually in contact with environments contaminated with rats' urine are at risk of infection. The prevalence rate of Leptospira infection in rats may result in the spread of leptospirosis in humans. This study aimed to determine the prevalence rate ofLeptospira infection in a total of 28 rats and develop a spatial database for leptospirosis surveillance in Phraroj village in Muang Sam Sip District, Ubon Ratchathani Province. The positions of the households and the rat-trapping area were tagged by using of a Global Positioning System (GPS). DNA samples were isolated fromn rats' kidneys. The polymerase chain reaction (PCR) technique was used for the detection of 16s rRNA and LipL32 genes specific to genus and pathogenic Leptospira respectively. All of the data were used to develop a geo-data base by the connection of spatial data and attributed data to be used for query and retrieval. A map of the positions of the households and the rat-trapping area in Phraroj village was created. No rats were found to be infected in the Leptospira survey. There was no trapped rat infected with Leptospira in Phraroj village. This result may involve unreported leptospirosis in patients in this village. The Leptospira survey in rats and the geo-database will be used as a primary resource to support and make decisions about surveillance, prevention, and control of leptospirosis.

Is Leptospira able to survive in raw milk? Study on the inactivation at different storage times and temperatures.

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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.

Seroprevalence of Brucella abortus and Leptospira hardjo in cattle

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S. Jegaveera Pandian

2015-02-01

Full Text Available 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: 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.

A zymography analysis of proteinase activity present in Leptospira.

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Madathiparambil, Madanan G; Cattavarayane, Sandhanakrishnan; Manickam, Gayathri D; Singh, Kavita; Perumana, Sudhakaran R; Sehgal, Subhash C

2011-03-01

Leptospirosis is a major public health problem caused by spirochete Leptospira which is an extracellular pathogen. During infection and invasion, the bacteria cross the physical barriers and later it encounter with the host defence mechanism. These processes may involve proteolytic degradation of the host tissue biomatrix. In an effort to understand the production and nature of Leptospiral proteinases, investigations were carried out using zymograpic methods. The results showed that the leptospires degrades different kind of protein substances such as gelatin, casein, and albumin. Gelatin zymography reveals that different serovars contain multiple gelatinases in the molecular weight range from 240 to 32 kDa. Studies using inhibitors suggested that the Leptospiral proteinases include metalloproteinases, serine or cysteine proteinases. The temperature sensitivity suggests that some of these proteinases are stable even at high temperatures. The presence of multiple gelatinases in Leptospira serovars suggests a critical role for these enzymes in Leptospiral invasion and pathogenesis.

Leptospira spp. y leptospirosis humana

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Claudia M. Romero-Vivas

2016-01-01

Full Text Available La leptospirosis, la enfermedad bacteriana zoonótica y emergente más importante en el mundo, es causada por las especies patógenas de Leptospira spp. Han sido descritas veinte especies de Leptospira spp.; se ha determinado la secuencia del ADN genómico de algunas cepas patógenas; la función de la mayoría de los genes involucrados en su patogénesis permanece desconocida. La leptospirosis humana presenta un rango de síntomas que van desde una fiebre indiferenciada hasta una ictericia, hemorragia, fallas renales y pulmonares severas. La administración temprana e intravenosa de penicilina G es requerida para reducir las tasas de mortalidad, pero los antibióticos pueden no ser efectivos en la enfermedad pulmonar severa. En las Américas, las áreas de alto riesgo son Brasil, Centroamérica y el Caribe. En Colombia se han realizado pocos estudios. La prueba serológica de oro, la microaglutinación, tiene alta sesibilidad y especificidad cuando se usan baterías de serovariedades locales, pero es serogrupo específica. Las vacunas generan respuestas específicas para la serovariedad usada, pero no previenen la infección o trasmisión. Problemas en el diagnóstico de laboratorio de la leptospirosis conllevan a un subregistro en el número de casos; altas tasas de mortalidad asociadas a fallas renal y pulmonar son resultado de las dificultades en el manejo de los casos.

Discovery of Novel Leptospirosis Vaccine Candidates Using Reverse and Structural Vaccinology

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Alan John Alexander McBride

2017-04-01

Full Text Available Leptospira spp. are diderm (two membranes bacteria that infect mammals causing leptospirosis, a public health problem with global implications. Thousands of people die every year due to leptospirosis, especially in developing countries with tropical climates. Prophylaxis is difficult due to multiple factors, including the large number of asymptomatic hosts that transmit the bacteria, poor sanitation, increasing numbers of slum dwellers, and the lack of an effective vaccine. Several leptospiral recombinant antigens were evaluated as a replacement for the inactivated (bacterin vaccine; however, success has been limited. A prospective vaccine candidate is likely to be a surface-related protein that can stimulate the host immune response to clear leptospires from blood and organs. In this study, a comprehensive bioinformatics approach based on reverse and structural vaccinology was applied toward the discovery of novel leptospiral vaccine candidates. The Leptospira interrogans serovar Copenhageni strain L1-130 genome was mined in silico for the enhanced identification of conserved β-barrel (βb transmembrane proteins and outer membrane (OM lipoproteins. Orthologs of the prospective vaccine candidates were screened in the genomes of 20 additional Leptospira spp. Three-dimensional structural models, with a high degree of confidence, were created for each of the surface-exposed proteins. Major histocompatibility complex II (MHC-II epitopes were identified, and their locations were mapped on the structural models. A total of 18 βb transmembrane proteins and 8 OM lipoproteins were identified. These proteins were conserved among the pathogenic Leptospira spp. and were predicted to have epitopes for several variants of MHC-II receptors. A structural and functional analysis of the sequence of these surface proteins demonstrated that most βb transmembrane proteins seem to be TonB-dependent receptors associated with transportation. Other proteins

Serological investigation of Leptospira infection and its circulation in one intensive-type water buffalo farm in the Philippines.

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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 (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.

High Leptospira seroprevalence in captive and wild-caught vervet monkeys ( Chlorocebus sabeus) on the Caribbean island of Saint Kitts.

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Rajeev, Sreekumari; Conan, Anne; Pratt, Nicola; Beierschmitt, Amy; Palmour, Roberta

2017-11-01

Leptospirosis is a zoonotic disease of global importance. Very little information is available on Leptospira infection in nonhuman primates. We report herein a high seroprevalence (49.4%; 95% confidence interval: 41.6-57.2%) to Leptospira serovars in vervet monkeys ( Chlorocebus sabeus) on the Caribbean island of Saint Kitts. Monkeys bred in captivity ( n = 81) had a significantly higher seroprevalence compared to wild-caught monkeys ( n = 81; p Leptospira serovars and seroconversion occurs in wild and captive vervet monkeys on the Caribbean island of Saint Kitts. Further studies are warranted to better understand epidemiology, transmission, pathology, and possible reservoir status in this species.

Presence of Antibodies to Leptospira spp. in Black-tailed Prairie Dogs ( Cynomys ludovicianus ) and Beavers ( Castor canadensis ) in Northwestern Mexico.

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López-Pérez, Andrés M; Carreón-Arroyo, Gerardo; Atilano, Daniel; Vigueras-Galván, Ana L; Valdez, Carlos; Toyos, Daniel; Mendizabal, Daniel; López-Islas, Jonathan; Suzán, Gerardo

2017-10-01

Leptospires are widespread spirochete bacteria that infect mammals, including rodents and humans. We investigated the presence of Leptospira antibodies in two species of rodents from San Pedro River Basin (SPRB) in northwestern Mexico as part of the black-tailed prairie dog ( Cynomys ludovicianus ) monitoring plan and the North American beaver ( Castor canadensis ) reintroduction program. We sampled a total of 26 black-tailed prairie dogs and three beavers during October-November 2015. We detected antibodies against Leptospira spp. by microagglutination test in 12 (46%) prairie dogs and in two (67%) beavers. The antibody titers for seropositive rodents varied from 1:100 to 1:200, but none of the animals showed clinical signs of disease. We found seven Leptospira spp. serogroups (Autumnalis, Australis, Bataviae, Canicola, Celledoni, Grippotyphosa, and Sejroe) circulating in rodent species in SPRB. We did not find any differences between sex and age concerning Leptospira-positive rodents. Our findings suggest the presence of endemic cycles and potential risks of Leptospira infection in both species from SPRB. Although the impact of this infection on threatened species remains unclear, human activities and environmental stress might facilitate the emergence or reemergence of leptospirosis disease as has been reported elsewhere.

Comparison of four antibiotics for inactivating leptospires in bull semen diluted in egg yolk extender and experimentally inoculated with Leptospira santarosai serovar guaricura Comparação de quatro antibióticos para inativar leptospiras em sêmen bovino diluído em gema-citrato e experimentalmente contaminado com Leptospira santarosai sorovar guaricura

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Fabiana Miraglia

2003-06-01

Full Text Available Inactivation of leptospires in pools of semen from three Holstein Friesian bulls, collected in an artificial vagina, was investigated. Spermatic concentration was adjusted in egg yolk citrate extender, submitted to the following treatments: A (control; without antibiotics; B (penicillin, 1,000 UI/mL - streptomycin, 1,000 µg/mL; C (amoxicillin, 1,000 µg/mL; D (ceptiofur sodium, 1,000 µg/mL; E (amoxicillin 1,000 µg/mL - ceptiofur sodium 1,000 µg/mL. Leptospires (2.0 x 10(6 leptospires/mL were added into the diluted semen. Recovery of leptospires was obtained in modified EMJH semi-solid medium with and without antibiotics. The antibiotics in the concentrations used did not affect means of percentage of progressive motility and individual progressive motility of spermatozoids. Penicillin-streptomycin presented the best results in leptospire inactivation (97.1%. Amoxicillin, ceptiofur sodium and their combination at the concentrations studied presented poor results: 59.29%; 32.5% and 60.36% of inactivation, being less effective in leptospire inactivation than penicillin-streptomycin.A inativação de leptospiras em misturas de sêmen, obtidas através de vagina artificial, de três touros holandeses, foi estudada. A concentração espermática foi ajustada em diluidor gema-citrato utilizando os seguintes tratamentos: A (controle; sem antibióticos; B (penicilina, 1000 UI/mL - estreptomicina, 1000 µg/mL; C (amoxicilina, 1000 µg/mL; D (ceftiofur sódico, 1000 µg/mL; E (amoxicilina 1000 µg/mL - ceftiofur sódico 1000 µg/mL. Leptospiras (2,0x10(6 leptospiras/mL foram adicionadas ao sêmen diluído. A recuperação das leptospiras foi obtida em meio EMJH modificado semi-sólido, com e sem antibióticos. As médias da porcentagem de motilidade progressiva e a de motilidade individual progressiva dos espermatozóides não foram afetadas pelos antibióticos nas concentrações usadas. Penicilina-estreptomicina apresentou os melhores resultados na

Colony-level assessment of Brucella and Leptospira in the Guadalupe fur seal, Isla Guadalupe, Mexico.

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Ziehl-Quirós, E Carolina; García-Aguilar, María C; Mellink, Eric

2017-01-24

The relatively small population size and restricted distribution of the Guadalupe fur seal Arctocephalus townsendi could make it highly vulnerable to infectious diseases. We performed a colony-level assessment in this species of the prevalence and presence of Brucella spp. and Leptospira spp., pathogenic bacteria that have been reported in several pinniped species worldwide. Forty-six serum samples were collected in 2014 from pups at Isla Guadalupe, the only place where the species effectively reproduces. Samples were tested for Brucella using 3 consecutive serological tests, and for Leptospira using the microscopic agglutination test. For each bacterium, a Bayesian approach was used to estimate prevalence to exposure, and an epidemiological model was used to test the null hypothesis that the bacterium was present in the colony. No serum sample tested positive for Brucella, and the statistical analyses concluded that the colony was bacterium-free with a 96.3% confidence level. However, a Brucella surveillance program would be highly recommendable. Twelve samples were positive (titers 1:50) to 1 or more serovars of Leptospira. The prevalence was calculated at 27.1% (95% credible interval: 15.6-40.3%), and the posterior analyses indicated that the colony was not Leptospira-free with a 100% confidence level. Serovars Icterohaemorrhagiae, Canicola, and Bratislava were detected, but only further research can unveil whether they affect the fur seal population.

Ocorrência de anticorpos contra Leptospira spp em cães de Teresina, Piauí, Brasil

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Elís Rosélia Dutra de Freitas Siqueira Silva

2017-05-01

Full Text Available Leptospirosis is a systemic disease caused by the species of bacteria Leptospira spp., which affects human beings, domestic and wild animals. The present study searched the presence of antibodies against Leptospira spp. in the canine population of the city Teresina, Piauí, and the most common serovars. Blood samples from 425 stray dogs were collected in the local zoonosis center in Teresina from July 2010 to January 2012 and submitted to the Microscopic Seroagglutination Test (MAT. This study found an average infection rate of 17.41% (IC 95%; 13,8 – 21,0 by 11 different serovars; the four most frequent were Canicola (18.9%, Autumnalis (16.2%, Icterohaemorrhagiae (12.1%, and Butembo (12.1%. The questions raised in this study indicated the occurrence of Leptospira spp infection in dogs of Terezina- Piaui, Brazil.

Evaluation of the Leptospira species microscopic agglutination test in experimentally vaccinated cats and Leptospira species seropositivity in aged azotemic client-owned cats.

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Shropshire, Sarah B; Veir, Julia K; Morris, Arianne K; Lappin, Michael R

2016-10-01

The objectives of this study were to validate the microscopic agglutination test (MAT) using feline sera, determine cross-reactivity of Borrelia burgdorferi antibodies in the MAT, and evaluate if there is an association between Leptospira species seropositivity in aged (⩾10 years) client-owned cats with and without azotemia (creatinine >2 g/dl). A four-serovar canine leptospiral vaccine was administered to two specific pathogen-free (SPF) cats on days 0 and 14. The MAT was performed intermittently until day 42 for the serovars Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona and Bratislava, with a cut-off value of ⩾1:100. Five purpose-bred cats were infested with wild-caught Ixodes scapularis adults with an average B burgdorferi infection rate of 50%, and tested for antibodies against B burgdorferi C6 peptide and DNA in skin biopsies, as well as by MAT. Sera from 66 azotemic and 75 non-azotemic cats ⩾10 years of age were tested for Leptospira species antibodies using the MAT and results were compared by the χ(2) test. Both SPF cats seroconverted by week 3 and formed antibodies against at least one serovar. There was no cross-reactivity in the MAT using samples from cats with antibodies to B burgdorferi. MAT results were positive for 4/66 azotemic cats and 8/75 non-azotemic cats; these results were not statistically different. The MAT can be interpreted using feline serum and does not appear to cross-react in cats with B burgdorferi antibodies. There was no association between Leptospira species MAT results and azotemia in this group of aged client-owned cats but further studies are needed to determine if leptospirosis contributes to feline chronic kidney disease. © The Author(s) 2015.

Descripción de un brote de leptospirosis en la ciudad de Santa Fe, Argentina, marzo-abril de 1998 Description of a leptospirosis outbreak in the city of Santa Fe, Argentina, March-April 1998

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Norma B. Vanasco

2000-01-01

Full Text Available En marzo-abril de 1998 se identificó en un barrio de la ciudad de Santa Fe (Argentina un brote de una enfermedad aguda caracterizada por fiebre, cefaleas y mialgias intensas. Se presentan los estudios realizados en relación con este brote y los intentos de identificación de la fuente y del modo de transmisión. Los hallazgos epidemiológicos, serológicos y clínicos indicaron que el agente causal fue Leptospira interrogans. Como prueba de tamizaje se aplicó la técnica de aglutinación macroscópica con antígeno termorresistente, seguida de la prueba de ELISA y, como prueba de confirmación, la aglutinación microscópica frente a 10 serovariedades de L. interrogans. Se estudiaron 32 individuos, 8 perros y 8 muestras de agua. Se registraron 12 casos confirmados, 2 probables y 18 negativos. En seis perros se demostró la existencia de infección y en las muestras de agua se detectó la presencia de espiroquetas móviles. Los sueros humanos reaccionaron con las serovariedades ballum, canicola, icterohaemorrhagiae y pyrogenes, y los caninos con ballum, canicola y pomona. La coaglutinación observada en todos los casos confirmados indica que se trató de casos agudos de leptospirosis, pero no permite identificar la serovariedad causal. Salvo en el caso índice, no se reconoció clínicamente la enfermedad. Varios hechos sugieren que la causa del brote fue la inundación pluvial de la zona estudiada. Los resultados de este estudio resaltan la necesidad de una vigilancia activa de la leptospirosis ante desastres naturales como las inundaciones.In March-April 1998 in a neighborhood in the city of Santa Fe, Argentina, there was an outbreak of an acute disease characterized by fever, headaches, and intense myalgias. This article presents the studies surrounding this outbreak and the attempts to identify the source and the mode of transmission. The epidemiological, serological, and clinical findings indicated that the causative agent was Leptospira

Intestinal spirochetosis: first cases reported in Brazil and the use of immunohistochemistry as an aid in histopathological diagnosis Espiroquetose intestinal: primeiros casos no Brasil e o uso de técnicas imunohistoquímicas como ajuda no diagnóstico histopatológico

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T. de Brito

1996-02-01

Full Text Available Colonization of the colon and rectum by intestinal spirochetes is detected for the first time in Brazil in 4 of 282 (1.41% patients who had undergone sigmoidoscopy and/or colonoscopy with a histopathological diagnosis of chronic non specific-colitis. This frequency is probably understimated, since surgically obtained specimens were not considered in the present study. Histopathological diagnosis was performed using routine stains like hematoxylin-eosin which showed the typical, of 3-µm thick hematoxyphilic fringe on the brush border of the surface epithelium, and by silver stains like the Warthin-Starry stain. Immunohistochemical procedures using two, polyclonal, primary antibodies, one against Treponema pallidum and the other against Leptospira interrogans serovar copenhageni serogroup Icterohaemorrhagiae cross-reacted with spirochetal antigen/s producing a marked contrast of the fringe over the colonic epithelium, preserving the spiral-shaped morphology of the parasite. In one case with marked diarrhea, immunohistochemistry detected spirochetal antigen/s within a cell in an intestinal crypt, thus demonstrating that the infection can be more widely disseminated than suspected using routine stains. Immunohistochemical procedures, thus, greatly facilitate the histological diagnosis of intestinal spirochetosis and may contribute to a better understanding of the pathogenesis of the disease. Transmission and scanning electron microscopy performed in one case showed that the spirochete closely resembled the species designated as Brachyspira aalborgi.A colonização do colo e reto por espiroquetas intestinais foi detectada pela primeira vez no Brasil em 4 de 282 (1,41% pacientes submetidos a sigmoidoscopia e/ou colonoscopia com o diagnóstico histopatológico de colite crônica inespecífica. A frequência é provavelmente menor que a real, urna vez que espécimens cirúrgicos não foram considerados no presente estudo. O diagnóstico histopatol

Transcriptional Response of Leptospira interrogans to Different Iron Sources

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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...

PATHOGENIC LEPTOSPIRA SEROVARS IN FREE-LIVING SEA LIONS IN THE GULF OF CALIFORNIA AND ALONG THE BAJA CALIFORNIA COAST OF MEXICO.

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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.

Aplicación de las pruebas de PCR convencional simple y múltiple para la identificación de aislamientos de Leptospira spp. en Colombia Application of conventional and multiplex PCR assays for identification of isolates of Leptospira spp. in Colombia

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Natali Moreno

2010-12-01

Full Text Available Debido a las dificultadas asociadas con la identificación serológica de aislamientos de Leptospira ssp, se genera gran interés en la pruebas moleculares por su poder discriminatorio, reproducibilidad y fácil interpretación. Objetivo. Aplicar y validar la prueba de PCR convencional, usando dos pares de iniciadores descritos previamente y dirigidos a los genes lipL32 (PCR simple y secY/flaB (PCR múltiple, con el fin de evaluar su aplicación para identificar especies patógenas y saprófitas de Leptospira spp. Materiales y métodos. Para la estandarización de las pruebas de PCR se usó 22 cepas de referencia internacional y 12 aislamientos colombianos. Se determinó el nivel de detección de cada pareja de iniciadores, su especificidad frente a otros microorganismos causantes de enfermedades endémicas en Colombia y su capacidad de identificar especies dentro del grupo de Leptospira. Resultados. El límite de detección de la PCR simple lipL32 fue una dilución 1:10000 y para la PCR múltiple secY/flaB fue una dilución 1:100 para el gen secY y 1:1000 para flaB. La especificidad de todos los iniciadores fue de 100%. La PCR simple lipL32, mostró amplificado específico para 21/22 cepas de referencia mientras que la PCR multiple secY/flaB lo fue para 18/22 cepas. De los 12 aislamientos colombianos, siete fueron positivos por PCR lipL32 y seis lo fueron por PCR secY/flaB. Conclusiones. Los resultados más consistentes fueron obtenidos con la PCR simple lipL32 tanto en límite de detección, especificidad y utilidad para la identificación de Leptospira spp, por lo que esta prueba es aplicable a la identificación molecular de aislamientos patógenos de Leptospira spp de diversas fuentes.Serological identification of Leptospira ssp isolates is difficult to achieve. Thus, molecular testing may be of great interest thanks to its high discrimination power, reproducibility and easy interpretation. Objective. To implement and validate conventional

Estudio transversal de leptospirosis y muerte fetal en Yucatán, México

OpenAIRE

María Fidelia Cárdenas-Marrufo; Ignacio Vado-Solis; Gaspar Fernando Peniche-Lara; Carlos Perez-Osorio; José Correa-Segura

2016-01-01

Introducción: Leptospirosis es una enfermedad zoonótica que afecta principalmente la población humana de bajos recursos. Infección aguda por leptospirosis durante el embarazo se ha asociado con aborto espontáneo y muerte fetal durante el primer trimestre del embarazo.Objetivo: Estimar la frecuencia de infección por Leptospira interrogans en mujeres con aborto espontáneo en el estado de Yucatán, México.Métodos: Se efectuó un estudio trasversal en 81 mujeres con aborto espontáneo. La prueba de ...

Leptospira species molecular epidemiology in the genomic era.

Science.gov (United States)

Caimi, K; Repetto, S A; Varni, V; Ruybal, P

2017-10-01

Leptospirosis is a zoonotic disease which global burden is increasing often related to climatic change. Hundreds of whole genome sequences from worldwide isolates of Leptospira spp. are available nowadays, together with online tools that permit to assign MLST sequence types (STs) directly from raw sequence data. In this work we have applied R7L-MLST to near 500 genomes and strains collection globally distributed. All 10 pathogenic species as well as intermediate were typed using this MLST scheme. The correlation observed between STs and serogroups in our previous work, is still satisfied with this higher dataset sustaining the implementation of MLST to assist serological classification as a complementary approach. Bayesian phylogenetic analysis of concatenated sequences from R7-MLST loci allowed us to resolve taxonomic inconsistencies but also showed that events such as recombination, gene conversion or lateral gene transfer played an important role in the evolution of Leptospira genus. Whole genome sequencing allows us to contribute with suitable epidemiologic information useful to apply in the design of control strategies and also in diagnostic methods for this illness. Copyright © 2017 Elsevier B.V. All rights reserved.

WC1 is a hybrid γδ TCR coreceptor and pattern recognition receptor for pathogenic bacteria.

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Hsu, Haoting; Chen, Chuang; Nenninger, Ariel; Holz, Lauren; Baldwin, Cynthia L; Telfer, Janice C

2015-03-01

WC1 proteins are uniquely expressed on γδ T cells and belong to the scavenger receptor cysteine-rich (SRCR) superfamily. While present in variable, and sometimes high, numbers in the genomes of mammals and birds, in cattle there are 13 distinct genes (WC1-1 to WC1-13). All bovine WC1 proteins can serve as coreceptors for the TCR in a tyrosine phosphorylation dependent manner, and some are required for the γδ T cell response to Leptospira. We hypothesized that individual WC1 receptors encode Ag specificity via coligation of bacteria with the γδ TCR. SRCR domain binding was directly correlated with γδ T cell response, as WC1-3 SRCR domains from Leptospira-responsive cells, but not WC1-4 SRCR domains from Leptospira-nonresponsive cells, bound to multiple serovars of two Leptospira species, L. borgpetersenii, and L. interrogans. Three to five of eleven WC1-3 SRCR domains, but none of the eleven WC1-4 SRCR domains, interacted with Leptospira spp. and Borrelia burgdorferi, but not with Escherichia coli or Staphylococcus aureus. Mutational analysis indicated that the active site for bacterial binding in one of the SRCR domains is composed of amino acids in three discontinuous regions. Recombinant WC1 SRCR domains with the ability to bind leptospires inhibited Leptospira growth. Our data suggest that WC1 gene arrays play a multifaceted role in the γδ T cell response to bacteria, including acting as hybrid pattern recognition receptors and TCR coreceptors, and they may function as antimicrobials. Copyright © 2015 by The American Association of Immunologists, Inc.

77 FR 43827 - International Workshop on Alternative Methods for Leptospira

Science.gov (United States)

2012-07-26

... recent advances and innovations in science and technology that can be applied to Leptospira vaccine... alternative methods. The workshop is open to the public at no charge with attendance limited only by the... priority. Leptospirosis affects numerous animal species including livestock, pets, and wildlife. Vaccines...

Cloning and Expression of Leptospira LipL32 Antigen as a Candidate for Rapid Diagnosis

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Nooshin Sohrabi

2013-09-01

Full Text Available Background and Objective: Leptospirosis as an important emerging infectious zoonotic disease caused by spirochetes of the genus Leptospira. Given the low sensitivity and long duration of its culture, the diagnosis of leptospirosis is mainly based on serological methods. The microscopic agglutination test (MAT is considered as the reference method. Because of the complexity of the MAT, there is an urgent need for the development of new reliable and rapid screening tests for leptospirosis. Major leptospiral outer membrane proteins (OMPs, present only in pathologic strains, could be regarded as a good candidate for diagnostic studies. Here we report the cloning and expression of LipL32, as a prominent immunogenic protein, in a prokaryotic system. Materials and Methods: After the amplification of LipL32 gene, it was cloned into the pQE30 vector. The insertion of LipL32 gene into the vector was screened and confirmed with restriction analysis and sequencing. The recombinant plasmid was transformed into E. coli M15 strain, and the expressed protein was identified by SDS-PAGE and western blotting. This recombinant protein with 6× His-tagged sequence was purified using Ni-NTA affinity column chromatography. Results: The results revealed that the selected gene was successfully cloned in pQE30 vector and recombinant protein (rLipL32 of approximately ~32 kDa was produced, purified and confirmed by western blotting. Conclusion: This recombinant protein could be potentially used for the development of serodiagnosis tests for the diagnosis of leptospirosis in humans and animals.

COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

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Tülin GÜVEN GÖKMEN

Full Text Available SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM, serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively. The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01% than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis.

Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

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Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

2009-12-08

Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS). We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. This is the first study to compare transcriptional and translational responses to temperature shift in L. interrogans. The results thus provide an insight into the mechanisms used by L

Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

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Miranda Lo

Full Text Available BACKGROUND: Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. METHODOLOGY/PRINCIPAL FINDINGS: To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS. We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. CONCLUSIONS/SIGNIFICANCE: This is the first study to compare transcriptional and translational responses to temperature

Frequency of Leptospira spp. in sheep from Brazilian slaughterhouses and its association with epidemiological variables

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Rodrigo Costa da Silva

2012-03-01

Full Text Available Leptospirosis is a worldwide anthropozoonosis that infects livestock, including sheep as the carriers to other animals and humans. The present study aimed to determine the prevalence of Leptospira spp. in sheep from two slaughterhouses in the state of São Paulo, Brazil and its association with epidemiological variables. Serum samples from 182 sheep were evaluated for Leptospira spp. antibodies by microscopic agglutination test (MAT. Results indicated 34/182 (18.68%; CI95% 13.70-24.98% positive serum samples, mainly to the serovar Copenhageni (17/34; 50%; CI95% 33.99-66.01%. Bacterial growth in the Fletcher medium was detected for 13/34 (38.24%; CI95% 23.87-55.08% animals, and confirmed by Polymerase Chain Reaction (PCR and sequencing for only two kidney samples from two animals. Thus, treatment and vaccination of sheep, besides rodent control, can be useful to prevent the infection in the studied region since sheep are important Leptospira spp. carriers, and its transmission to slaughterhouse workers is mainly through the manipulation of visceral tissues.

Orientia, rickettsia, and leptospira pathogens as causes of CNS infections in Laos

DEFF Research Database (Denmark)

Dittrich, Sabine; Rattanavong, Sayaphet; Lee, Sue J

2015-01-01

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......, 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...

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

Science.gov (United States)

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

Prevalence of renal lesions in slaughtered cattle in Shiraz, Iran, and detection of Leptospira in them by nested PCR-RFLP.

Science.gov (United States)

Taghadosi, Vahideh; Hosseinzadeh, Saeid; Shekarforoush, Seyed Shahram; Samiei, Azadeh

2016-12-01

Renal diseases in cattle are frequently not recognized due to the subclinical conditions. Some species of Leptospira are the main cause of infectious agents that damage the kidneys and lead to abortion and economic losses in cattle and are also of major concern in the public health. This study was aimed to assess the prevalence of renal lesions of slaughtered cattle in the Shiraz abattoir and to determine the correlation between rejected kidneys and infection with Leptospira using nested PCR-restriction fragment length polymorphism (RFLP) techniques. Out of 1000 inspected animals, 205 (20.5 %) revealed the renal lesions. Chronic nephritis (7.5 %), white-spotted kidney (7.3 %), and petechial hemorrhage (3.5 %) were the most prevalent forms of the lesions. A direct correlation between increasing the age and significant increase in the rate of lesions was also observed (P = 0.03). Using nested PCR-RFLP assay, 40.8 % of the tested kidneys were turned to be infected to the pathogenic species of Leptospira. The risk of infection of the kidneys with white spot to pathogenic species of Leptospira (53.8 %) was more than that of the kidneys with other lesions (25.0 %) (P = 0.014). The odd ratio indicates that the kidneys with white spot lesions are likely to be infected with pathogenic species of Leptospira, five times greater than other lesions. This study showed that renal lesions especially white-spotted kidney, which were considerably associated with Leptospira in slaughtered cattle in Shiraz, were very high. This is important in terms of public health and in particular, increases the risk of transmission of disease to human specially in the high-risk careers including farmers, veterinarians, and abattoir workers.

Identification of Tenrec ecaudatus, a Wild Mammal Introduced to Mayotte Island, as a Reservoir of the Newly Identified Human Pathogenic Leptospira mayottensis.

Science.gov (United States)

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.

Identification of Tenrec ecaudatus, a Wild Mammal Introduced to Mayotte Island, as a Reservoir of the Newly Identified Human Pathogenic Leptospira mayottensis.

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Erwan Lagadec

2016-08-01

Full Text Available 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.

An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples.

Science.gov (United States)

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 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.

Serological survey on Leptospira infection in slaughtered swine in North-Central Italy.

Science.gov (United States)

Bertelloni, F; Turchi, B; Vattiata, E; Viola, P; Pardini, S; Cerri, D; Fratini, F

2018-05-30

Swine can act as asymptomatic carriers of some Leptospira serovars. In this study, 1194 sera from 61 farms located in five different Regions of North-West Italy were collected from slaughtered healthy pigs. Presence of antibody against four Leptospira serovars was evaluated. Overall, 52.5% of analysed farms presented at least one positive animal and 34.4% presented at least one positive swine with titre ⩾1:400. A percentage of 16.6% sera was positive and 5.9% samples presented a positive titre ⩾1:400. Tuscany and Lombardy showed the highest percentage of positive farms (64.3% and 54.6%, respectively) and sera (28.5% and 13.3%, respectively), probably due to environmental conditions and potential risk factors, which promote maintenance and spreading of Leptospira in these areas. The main represented serogroups were Australis (21.3% positive farms, 8.2% positive sera) and Pomona (18.0% positive farms, 8.1% positive sera). In swine, these serogroups are the most detected worldwide; however, our results seem to highlight a reemerging of serogroup Pomona in pigs in investigated areas. A low percentage of sera (0.6%) scored positive to Canicola, leaving an open question on the role of pigs in the epidemiology of this serovar. Higher antibody titres were detected for serogroups Australis and Pomona. Swine leptospirosis is probably underestimated in Italy and could represent a potential risk for animal and human health.

Epidemiology of Leptospirosis in Africa: A Systematic Review of a Neglected Zoonosis and a Paradigm for 'One Health' in Africa.

Science.gov (United States)

Allan, Kathryn J; Biggs, Holly M; Halliday, Jo E B; Kazwala, Rudovick R; Maro, Venance P; Cleaveland, Sarah; Crump, John A

2015-01-01

Leptospirosis is an important but neglected bacterial zoonosis that has been largely overlooked in Africa. In this systematic review, we aimed to summarise and compare current knowledge of: (1) the geographic distribution, prevalence, incidence and diversity of acute human leptospirosis in Africa; and (2) the geographic distribution, host range, prevalence and diversity of Leptospira spp. infection in animal hosts in Africa. Following Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, we searched for studies that described (1) acute human leptospirosis and (2) pathogenic Leptospira spp. infection in animals. We performed a literature search using eight international and regional databases for English and non-English articles published between January 1930 to October 2014 that met out pre-defined inclusion criteria and strict case definitions. We identified 97 studies that described acute human leptospirosis (n = 46) or animal Leptospira infection (n = 51) in 26 African countries. The prevalence of acute human leptospirosis ranged from 2 3% to 19 8% (n = 11) in hospital patients with febrile illness. Incidence estimates were largely restricted to the Indian Ocean islands (3 to 101 cases per 100,000 per year (n = 6)). Data from Tanzania indicate that human disease incidence is also high in mainland Africa (75 to 102 cases per 100,000 per year). Three major species (Leptospira borgpetersenii, L. interrogans and L. kirschneri) are predominant in reports from Africa and isolates from a diverse range of serogroups have been reported in human and animal infections. Cattle appear to be important hosts of a large number of Leptospira serogroups in Africa, but few data are available to allow comparison of Leptospira infection in linked human and animal populations. We advocate a 'One Health' approach to promote multidisciplinary research efforts to improve understanding of the animal to human transmission of leptospirosis on the African

Epidemiology of Leptospirosis in Africa: A Systematic Review of a Neglected Zoonosis and a Paradigm for 'One Health' in Africa.

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Kathryn J Allan

Full Text Available Leptospirosis is an important but neglected bacterial zoonosis that has been largely overlooked in Africa. In this systematic review, we aimed to summarise and compare current knowledge of: (1 the geographic distribution, prevalence, incidence and diversity of acute human leptospirosis in Africa; and (2 the geographic distribution, host range, prevalence and diversity of Leptospira spp. infection in animal hosts in Africa.Following Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA guidelines, we searched for studies that described (1 acute human leptospirosis and (2 pathogenic Leptospira spp. infection in animals. We performed a literature search using eight international and regional databases for English and non-English articles published between January 1930 to October 2014 that met out pre-defined inclusion criteria and strict case definitions.We identified 97 studies that described acute human leptospirosis (n = 46 or animal Leptospira infection (n = 51 in 26 African countries. The prevalence of acute human leptospirosis ranged from 2 3% to 19 8% (n = 11 in hospital patients with febrile illness. Incidence estimates were largely restricted to the Indian Ocean islands (3 to 101 cases per 100,000 per year (n = 6. Data from Tanzania indicate that human disease incidence is also high in mainland Africa (75 to 102 cases per 100,000 per year. Three major species (Leptospira borgpetersenii, L. interrogans and L. kirschneri are predominant in reports from Africa and isolates from a diverse range of serogroups have been reported in human and animal infections. Cattle appear to be important hosts of a large number of Leptospira serogroups in Africa, but few data are available to allow comparison of Leptospira infection in linked human and animal populations. We advocate a 'One Health' approach to promote multidisciplinary research efforts to improve understanding of the animal to human transmission of leptospirosis on the

Prolonged Leptospira Urinary Shedding in a 10-Year-Old Girl

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Emilie Chow

2012-01-01

Full Text Available We present a case of leptospirosis in a previously healthy girl following a trip to Costa Rica. While she was clinically asymptomatic, she had spirochetes cultured from her urine six weeks following her trip. Prolonged urinary shedding following infection with Leptospira is possible in humans and often has subtle manifestations in children.

Comparative genomic analysis of Brazilian Leptospira kirschneri serogroup Pomona serovar Mozdok

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Luisa Z Moreno

2016-08-01

Full Text Available 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.

Prevalencia de anticuerpos anti-Leptospira spp. en personas con exposición laboral en el departamento del Tolima / Prevalence of anti-Leptospira spp. antibodies among people with occupational exposure in Tolima Department

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Blanca L. Guzmán-Barragán

2016-05-01

Full Text Available Resumen Objetivo: estimar la prevalencia de anticuerpos IgM contra Leptospira spp., mediante el Ensayo de Inmunoabsorción Ligado a Enzimas (elisa, en la población de riesgo laboral de 8 municipios del Tolima. Metodología: se obtuvieron muestras de sangre de 261 empleados, las cuales fueron analizadas mediante la técnica de elisa para la detección de anticuerpos IgM anti-Leptospira spp., seguido de mat y serotipificación. Resultado: se estimó una seroprevalencia del 25,29%, con una seroreactividad mayor en trabajadores de plantas de beneficio animal (34,2%, recolección de residuos sólidos (27,1% y trabajadores de acueducto y alcantarillado (14,8%. La actividad en plantas de beneficio animal se identificó como factor de riesgo de Leptospira spp. (OR=1,86. Los serovares identificados fueron L. Bratislava (16, Ballum (5, Tarassovi (3, Hebdomadis (2, Sejroe (2 y Icterhemorragiae (1. El municipio de Libano presento el mayor porcentaje de positividad (36,96%, seguido de Espinal y Guamo con 28,57% cada uno. Discusión: la evaluación del sistema de vigilancia indicó deficiencia en recursos y debilidades de los profesionales de la salud al desconocer los procedimientos, investigación, diagnóstico y notificación de la enfermedad. Conclusiones: la leptospirosis está presente en poblaciones de riesgo laboral en el Tolima y se hace necesario abordar esta problemática en la población de otros municipios y los animales transmisores de la enfermedad. / Abstract Objective: to estimate the prevalence of IgM antibodies against Leptospira spp. Using the enzyme-linked immunosorbent assay (elisa in a population at occupational risk from 8 municipalities of the Tolima department, Colombia. Methodology: blood samples were collected from 261 employees and analyzed with the elisa technique to detect IgM and anti-Leptospira spp. antibodies. This was followed by mat and serotyping. Results: a seroprevalence of 25.29% was estimated, with higher

Complete genome sequence of Leptospira alstonii serovar room 22, strain GWTS#1

Science.gov (United States)

We report the complete genome sequence of Leptospira alstonii serovar room 22 strain GWTS#1. This is the first isolate of L. alstonii to be cultured from a mammal, in Western Europe, and represents a new serovar of pathogenic leptospires....

Plasminogen Binding Proteins and Plasmin Generation on the Surface of Leptospira spp.: The Contribution to the Bacteria-Host Interactions

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Monica L. Vieira

2012-01-01

Full Text Available 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.

Deteksi Leptospira Patogen Pada Tersangka Penderita Leptospirosis Di Kabupaten Ponorogo

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Dyah Widiastuti

2017-02-01

Full Text Available AbstractUp to March 2012, there were increasing leptospirosis cases in Ponorogo. A total of 11 cases of leptospirosis were reported by the Ponorogo District Health Center. This study aims to detect pathogenic Leptospira bacteria using Polymerase Chain Reaction (PCR in suspected leptospirosis in Ponorogo. Collection of blood samples carried out by the local health care medical personnel (doctor, midwife or nurse was accompanied by a team of researchers. Subjects were people with fever (temperature >380C or fever accompanied by headache, muscle aches, and rash conjunctivitis. Blood and urine samples were taken purposively. Active case detection was also conducted in communities around the previous patients who had a history of infection risk and relationship of leptospirosis. Further blood samples examined by two different methods, Lepto Tek Lateral Flow and Polymerase Chain Reaction (PCR. Ninety blood and urine samples was collected. Four samples were positive for pathogenic Leptospira DNA consisted of two samples of blood (whole blood and two samples of urine. PCR-positive samples in the blood has negative results of Lepto Tek examination. While the PCR-positive samples in the urine, Lepto Tek was positive. Lepto tek showed lower sensitivity in early detection of leptospirosis. Examination of blood and urine samples using PCR can support the early discovery of leptospirosis cases.Keywords: Leptospirosis, Ponorogo, PCRAbstrakSampai dengan bulan Maret 2012 terjadi peningkatan kasus leptospirosis di Ponorogo. Sebanyak 11 kasus leptospirosis dilaporkan oleh Dinas Kesehatan Kabupaten Ponorogo. Penelitian ini bertujuan untuk mendeteksi bakteri Leptospira patogen menggunakan teknik Polimerase Chain Reaction (PCR pada tersangka penderita leptospirosis di Kabupaten Ponorogo. Pengumpulan sampel darah dilakukan oleh tenaga medis Puskesmas setempat (dokter, bidan atau perawat didampingi oleh tim peneliti terhadap penduduk yang mengalami demam (suhu badan

Emergency caesarean delivery in a patient with cerebral malaria-leptospira co infection: Anaesthetic and critical care considerations

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Sukhen Samanta

2014-01-01

Full Text Available Malaria-leptospira co-infection is rarely detected. Emergency surgery in such patients has not been reported. We describe such a case of a 24-year-old primigravida at term pregnancy posted for emergency caesarean delivery who developed pulmonary haemorrhage, acute respiratory distress syndrome, acute kidney injury, and cerebral oedema. Here, we discuss the perioperative management, pain management (with transverse abdominis plane block, intensive care management (special reference to management of pulmonary haemorrhage with intra pulmonary factor VIIa and the role of plasmapheresis in leptospira related jaundice with renal failure.

Solution structure of leptospiral LigA4 Big domain

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Mei, Song; Zhang, Jiahai [Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026 (China); Zhang, Xuecheng [School of Life Sciences, Anhui University, Hefei, Anhui 230039 (China); Tu, Xiaoming, E-mail: xmtu@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026 (China)

2015-11-13

Pathogenic Leptospiraspecies express immunoglobulin-like proteins which serve as adhesins to bind to the extracellular matrices of host cells. Leptospiral immunoglobulin-like protein A (LigA), a surface exposed protein containing tandem repeats of bacterial immunoglobulin-like (Big) domains, has been proved to be involved in the interaction of pathogenic Leptospira with mammalian host. In this study, the solution structure of the fourth Big domain of LigA (LigA4 Big domain) from Leptospira interrogans was solved by nuclear magnetic resonance (NMR). The structure of LigA4 Big domain displays a similar bacterial immunoglobulin-like fold compared with other Big domains, implying some common structural aspects of Big domain family. On the other hand, it displays some structural characteristics significantly different from classic Ig-like domain. Furthermore, Stains-all assay and NMR chemical shift perturbation revealed the Ca{sup 2+} binding property of LigA4 Big domain. - Highlights: • Determining the solution structure of a bacterial immunoglobulin-like domain from a surface protein of Leptospira. • The solution structure shows some structural characteristics significantly different from the classic Ig-like domains. • A potential Ca{sup 2+}-binding site was identified by strains-all and NMR chemical shift perturbation.

Solution structure of leptospiral LigA4 Big domain

International Nuclear Information System (INIS)

Mei, Song; Zhang, Jiahai; Zhang, Xuecheng; Tu, Xiaoming

2015-01-01

Pathogenic Leptospiraspecies express immunoglobulin-like proteins which serve as adhesins to bind to the extracellular matrices of host cells. Leptospiral immunoglobulin-like protein A (LigA), a surface exposed protein containing tandem repeats of bacterial immunoglobulin-like (Big) domains, has been proved to be involved in the interaction of pathogenic Leptospira with mammalian host. In this study, the solution structure of the fourth Big domain of LigA (LigA4 Big domain) from Leptospira interrogans was solved by nuclear magnetic resonance (NMR). The structure of LigA4 Big domain displays a similar bacterial immunoglobulin-like fold compared with other Big domains, implying some common structural aspects of Big domain family. On the other hand, it displays some structural characteristics significantly different from classic Ig-like domain. Furthermore, Stains-all assay and NMR chemical shift perturbation revealed the Ca"2"+ binding property of LigA4 Big domain. - Highlights: • Determining the solution structure of a bacterial immunoglobulin-like domain from a surface protein of Leptospira. • The solution structure shows some structural characteristics significantly different from the classic Ig-like domains. • A potential Ca"2"+-binding site was identified by strains-all and NMR chemical shift perturbation.

Gene expression profiles of immune mediators and histopathological findings in animal models of leptospirosis: comparison between susceptible hamsters and resistant mice.

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Matsui, Mariko; Rouleau, Vincent; Bruyère-Ostells, Lilian; Goarant, Cyrille

2011-11-01

Leptospirosis is a widespread zoonosis characterized by multiple organ failure and variable host susceptibility toward pathogenic Leptospira strains. In this study, we put the role of inflammatory mediators in parallel with bacterial burdens and organ lesions by comparing a susceptible animal model, the hamster, and a resistant one, the Oncins France 1 (OF1) mouse, both infected with virulent Leptospira interrogans serovar Icterohaemorrhagiae strain Verdun. Histological observations evidenced edema, congestion, hemorrhage, and inflammatory infiltration in the organs of hamsters, in contrast to limited changes in mice. Using reverse transcription-quantitative PCR techniques, we showed that the relative Leptospira burden progressively increased in hamster tissues, while a rapid clearance was observed in mouse tissues. The early regulation of the proinflammatory mediators interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, and cyclo-oxygenase-2 and the chemokines gamma interferon-inducible protein 10 kDa/CXCL10 and macrophage inflammatory protein-1α/CCL3 in mouse tissues contrasted with their delayed and massive overexpression in hamster tissues. Conversely, the induction of the anti-inflammatory cytokine IL-10 was faster in the resistant than in the susceptible animal model. The role of these cytokines in the pathophysiology of leptospirosis and the implications of their differential regulation in the development of this disease are discussed.

Molecular Survey of Bacterial Zoonotic Agents in Bats from the Country of Georgia (Caucasus.

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Ying Bai

Full Text Available Bats are important reservoirs for many zoonotic pathogens. However, no surveys of bacterial pathogens in bats have been performed in the Caucasus region. To understand the occurrence and distribution of bacterial infections in these mammals, 218 bats belonging to eight species collected from four regions of Georgia were examined for Bartonella, Brucella, Leptospira, and Yersinia using molecular approaches. Bartonella DNA was detected in 77 (35% bats from all eight species and was distributed in all four regions. The prevalence ranged 6-50% per bat species. The Bartonella DNA represented 25 unique genetic variants that clustered into 21 lineages. Brucella DNA was detected in two Miniopterus schreibersii bats and in two Myotis blythii bats, all of which were from Imereti (west-central region. Leptospira DNA was detected in 25 (13% bats that included four M. schreibersii bats and 21 M. blythii bats collected from two regions. The Leptospira sequences represented five genetic variants with one of them being closely related to the zoonotic pathogen L. interrogans (98.6% genetic identity. No Yersinia DNA was detected in the bats. Mixed infections were observed in several cases. One M. blythii bat and one M. schreibersii bat were co-infected with Bartonella, Brucella, and Leptospira; one M. blythii bat and one M. schreibersii bat were co-infected with Bartonella and Brucella; 15 M. blythii bats and three M. schreibersii bats were co-infected with Bartonella and Leptospira. Our results suggest that bats in Georgia are exposed to multiple bacterial infections. Further studies are needed to evaluate pathogenicity of these agents to bats and their zoonotic potential.

Evolution of the RNase P RNA structural domain in Leptospira spp

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Ravishankar, Vigneshwaran; Ahmed, Ahmed; Sivagnanam, Ulaganathan; Muthuraman, Krishnaraja; Karthikaichamy, Anbarasu; Wilson, Herald A.; Devendran, Ajay; Hartskeerl, Rudy A.; Raj, Stephen M. L.

2014-01-01

We have employed the RNase P RNA (RPR) gene, which is present as single copy in chromosome I of Leptospira spp. to investigate the phylogeny of structural domains present in the RNA subunit of the tRNA processing enzyme, RNase P. RPR gene sequences of 150 strains derived from NCBI database along

Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

International Nuclear Information System (INIS)

Nurul Najian, A.B.; Engku Nur Syafirah, E.A.R.; Ismail, Nabilah; Mohamed, Maizan; Yean, Chan Yean

2016-01-01

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. - Highlights: • We develop multiplex LAMP label-based lateral flow dipstick biosensor for detection of pathogenic Leptospira. • We design primers for multiplex LAMP targeting the conserved LipL32 gene of pathogenic Leptospira and LAMP internal

Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

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Nurul Najian, A.B.; Engku Nur Syafirah, E.A.R.; Ismail, Nabilah [Department of Medical Microbiology & Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Mohamed, Maizan [Faculty of Veterinary Medicine, Universiti Malaysia Kelantan, City Campus, Pengkalan Chepa, Locked Bag 36, 16100 Kota Bharu, Kelantan (Malaysia); Yean, Chan Yean, E-mail: yeancyn@yahoo.com [Department of Medical Microbiology & Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

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{sup −1} genomic equivalent ml{sup −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. - Highlights: • We develop multiplex LAMP label-based lateral flow dipstick biosensor for detection of pathogenic Leptospira. • We design primers for multiplex LAMP targeting the conserved LipL32 gene of pathogenic Leptospira and LAMP

Potent innate immune response to pathogenic leptospira in human whole blood.

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Marga G A Goris

Full Text Available 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.

The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis.

Science.gov (United States)

Silva, Everton F; Medeiros, Marco A; McBride, Alan J A; Matsunaga, Jim; Esteves, Gabriela S; Ramos, João G R; Santos, Cleiton S; Croda, Júlio; Homma, Akira; Dellagostin, Odir A; Haake, David A; Reis, Mitermayer G; Ko, Albert I

2007-08-14

Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund's adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P<0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis.

Identification of cattle, buffaloes and rodents as reservoir animals of Leptospira in the District of Gampaha, Sri Lanka.

Science.gov (United States)

Denipitiya, D T H; Chandrasekharan, N V; Abeyewickreme, W; Hartskeerl, R A; Hapugoda, M D

2017-03-23

Leptospirosis is an important emerging infectious disease in Sri Lanka. Rats are the most important reservoir of Leptospira but domestic and wild mammals may also act as important maintenance or accidental hosts. In Sri Lanka, knowledge of reservoir animals of leptospires is poor. The objective of this study was to identify potential reservoir animals of Leptospira in the District of Gampaha, Sri Lanka. Blood and kidney samples were collected from 38 rodents and mid-stream urine samples were randomly collected from 45 cattle and five buffaloes in the District of Gampaha. Kidney and urine samples were tested by real-time polymerase chain reaction (PCR) and serum samples were tested by the microscopic agglutination test (MAT). Of the 38 rodent kidney samples, 11% (4/38) were positive by real-time PCR. The prevalence of leptospiral carriage was 11% (3/26) and 8% (1/12) in female and male rodents, respectively. Three rodent serum samples were positive by MAT. Of the 50 cattle/buffalo urine samples tested, 10% (5/50) were positive by real-time PCR. The prevalence of leptospiral carriage was 9% (4/45) and 20% (1/5) in cattle and buffaloes, respectively. Results of PCR and MAT showed that Leptospira were present in a significant proportion of the rodents and farm animals tested in this study and suggest that these (semi-) domestic animals form an infection reservoir for Leptospira. Therefore, there is a potential zoonotic risk to public health, most notably to farmers in this area.

Differential in vivo gene expression of major Leptospira proteins in resistant or susceptible animal models.

Science.gov (United States)

Matsui, Mariko; Soupé, Marie-Estelle; Becam, Jérôme; Goarant, Cyrille

2012-09-01

Transcripts of Leptospira 16S rRNA, FlaB, LigB, LipL21, LipL32, LipL36, LipL41, and OmpL37 were quantified in the blood of susceptible (hamsters) and resistant (mice) animal models of leptospirosis. We first validated adequate reference genes and then evaluated expression patterns in vivo compared to in vitro cultures. LipL32 expression was downregulated in vivo and differentially regulated in resistant and susceptible animals. FlaB expression was also repressed in mice but not in hamsters. In contrast, LigB and OmpL37 were upregulated in vivo. Thus, we demonstrated that a virulent strain of Leptospira differentially adapts its gene expression in the blood of infected animals.

Incidence rates and risk factors for owner-reported adverse events following vaccination of dogs that did or did not receive a Leptospira vaccine.

Science.gov (United States)

Yao, Peng Ju; Stephenson, Nicole; Foley, Janet E; Toussieng, Chuck R; Farver, Thomas B; Sykes, Jane E; Fleer, Katryna A

2015-11-15

To determine incidence rates (IRs) and potential risk factors for owner-reported adverse events (AEs) following vaccination of dogs that did or did not receive a Leptospira vaccine. Observational, retrospective cohort study. 130,557 dogs. Electronic records of mobile veterinary clinics from June 2012 to March 2013 were searched to identify dogs that received ≥ 1 vaccine in a given visit. Signalment data, vaccinations received, medications administered, and owner-reported clinical signs consistent with AEs that developed ≤ 5 days after vaccination were recorded. Associations between potential risk factors and owner-reported AEs were evaluated by logistic regression analysis. The IR/10,000 dogs for owner-reported postvaccination AEs was 26.3 (95% CI, 23.6 to 29.2), whereas that for dogs that received a Leptospira vaccine alone or with other vaccines was 53.0 (95% CI, 42.8 to 64.9). Significant factors for increasing or decreasing risk of AEs were as follows: receiving a Leptospira vaccine (adjusted OR, 2.13), age at vaccination 1 to dogs), and IRs for these events did not differ significantly between dogs vaccinated with or without a Leptospira component. The overall IR for owner-reported postvaccination AEs was low. Results suggested vaccination against Leptospira (an organism that can cause fatal disease) is safe in the majority of cases, slightly increasing the risk of owner-reported AEs but not associated with a significant increase in hypersensitivity reactions, compared with other vaccinations administered.

Isolation of Leptospira borgpetersenii in synanthropic Didelphis albiventris in Jaboticabal, São Paulo, Brazil

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Felipe Jorge da Silva

2013-12-01

Full Text Available Leptospirosis is a waterborne disease and, therefore, stands out for the possibility of environmental contamination, the cross transmission between domestic and wild animals and humans. Opossum species are important reservoirs of this disease making them potential pathogen spreaders. Aiming to verify the presence of Leptospira spp. and the antibodies against Leptospira spp. in the Campus of São Paulo State University, in Jaboticabal, São Paulo, Brazil, freeliving wild life opossum (Didelphis albiventris were captured for blood and urine sampling. Serological analysis was performed Microscopic Agglutination Test (MAT. Aliquots of urine were seeded in media Ellinghausen-McCullough- Johnson-Harris (EMJH and Fletcher without antibiotics. The samples in which there was growth of leptospires were forwarded to the Leptospirosis Laboratory of the Institute of Pathobiology in the National Institute of Agricultural Technology, Buenos Aires, Argentina and were genotyped using Multiple Locus Variable number tandem repeat Analysis (MLVA. Of the 15 analyzed animals, nine (60.0% were reactant to Patoc serovar. The pathogenic specie Leptospira borgpetersenii was isolated and identified in three Didelphis albiventris. The isolation findings of pathogenic specie Leptopsira borgpetersenii in the urine culture of three Didelphis albiventris in a university campus are a major discovery in the area of preventive veterinary medicine and public health and open a discussion about the important role of free-living wild animals as reservoirs of this agent to domestic animals and humans, a condition that serves as a warning for the improvement of health practices.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB

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Lenice Roteia Cardoso Jung

2015-06-01

Full Text Available 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.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB).

Science.gov (United States)

Jung, Lenice Roteia Cardoso; Bomfim, Maria Rosa Quaresma; Kroon, Erna Geessien; Nunes, Álvaro Cantini

2015-06-01

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.

New serovars of Leptospira isolated from patients in Costa Rica: implications for public health

NARCIS (Netherlands)

de los Angeles Valverde, Maria; Goris, M. G. A.; González, V.; Anchia, M. E.; Díaz, P.; Ahmed, A.; Hartskeerl, R. A.

2013-01-01

Leptospira strains JICH 05 and INCIENSA 04 were isolated from hospitalized leptospirosis patients in the province of Puntarenas, Costa Rica. The isolates produced agglutination titres notably against members of serogroups Pyrogenes and Tarassovi, respectively, but appeared serologically unique in

Effect of metal ion on the structure and function of LiPDF: The study of the fine structure around the metal site using XANES

International Nuclear Information System (INIS)

Wang Yu; Chu Wangsheng; Yang Feifei; Yu Meijuan; Zhao Haifeng; Gong Weimin; Dong Yuhui; Xie Yaning; Wu, Ziyu

2010-01-01

We used X-ray absorption near edge structure (XANES) spectroscopy to investigate the metal-dependent enzymatic activity of the peptide deformylase from Leptospira interrogans (LiPDF). Ab initio full multiple scattering calculations performed by MXAN are applied to obtain the local structure of the cobalt-containing LiPDF (Co-LiPDF) and zinc-containing LiPDF (Zn-LiPDF) around the metal sites in pH9.0 buffer solution. The result shows the cobalt-wat1 (the bond water molecule) distance of Co-LiPDF is 1.89 A, much shorter than that of Zn-LiPDF, 2.50 A. That is an essential factor for its low catalytic activity.

Effect of metal ion on the structure and function of LiPDF: The study of the fine structure around the metal site using XANES

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Wang Yu [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei 230029 (China); Chu Wangsheng, E-mail: cws@ihep.ac.c [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); Yang Feifei; Yu Meijuan; Zhao Haifeng [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); Gong Weimin [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Dong Yuhui; Xie Yaning [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); Wu, Ziyu, E-mail: wuzy@ustc.edu.c [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei 230029 (China); Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China)

2010-07-21

We used X-ray absorption near edge structure (XANES) spectroscopy to investigate the metal-dependent enzymatic activity of the peptide deformylase from Leptospira interrogans (LiPDF). Ab initio full multiple scattering calculations performed by MXAN are applied to obtain the local structure of the cobalt-containing LiPDF (Co-LiPDF) and zinc-containing LiPDF (Zn-LiPDF) around the metal sites in pH9.0 buffer solution. The result shows the cobalt-wat1 (the bond water molecule) distance of Co-LiPDF is 1.89 A, much shorter than that of Zn-LiPDF, 2.50 A. That is an essential factor for its low catalytic activity.

Serologic survey in a chamois population of Abruzzo

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Leonardo Gentile

2000-09-01

Full Text Available Abstract As part of the Abruzzo National Park wildlife health management program, serum samples from 62 free-living and captive Abruzzo chamois, Rupicapra (pyrenaica ornata, were tested to estimate antibody presence to some pathogenous agents. The serum, drawn during chemical immobilisations for introductions or population study programs, were tested for: bovine herpes virus 1 (BHV-1, parainfluenza virus (PI3, pesti virus, foot and mouth disease virus (FMD type O-A-C, bovine leukemia virus (BLV, ovicaprini lentivirus, encephalomyocarditis virus (EMCV, S-phase brucellae, leptospira interrogans, mycobacterium paratuberculosis, chlamydia psittaci, coxiella burnetii, rickettsia mooseri and conori and toxoplasrna gondii. Twenty-three subjects were found positive to BHV-1 (7/62, pestivirus (6/62, EMCV (8/62, leptospira (5/62 and toxoplasma (4/62. Results did not indicate any active infection but only contact with some pathogenic agents.