Discovery of Leptospira spp. seroreactive peptides using ORFeome phage display.

BACKGROUND: Leptospirosis is the most common zoonotic disease worldwide. The diagnostic performance of a serological test for human leptospirosis is mainly influenced by the antigen used in the test assay. An ideal serological test should cover all serovars of pathogenic leptospires with high sensitivity and specificity and use reagents that are relatively inexpensive to produce and can be used in tropical climates. Peptide-based tests fulfil at least the latter two requirements, and ORFeome phage display has been successfully used to identify immunogenic peptides from other pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Two ORFeome phage display libraries of the entire Leptospira spp. genomes from five local strains isolated in Malaysia and seven WHO reference strains were constructed. Subsequently, 18 unique Leptospira peptides were identified in a screen using a pool of sera from patients with acute leptospirosis. Five of these were validated by titration ELISA using different pools of patient or control sera. The diagnostic performance of these five peptides was then assessed against 16 individual sera from patients with acute leptospirosis and 16 healthy donors and was compared to that of two recombinant reference proteins from L. interrogans. This analysis revealed two peptides (SIR16-D1 and SIR16-H1) from the local isolates with good accuracy for the detection of acute leptospirosis (area under the ROC curve: 0.86 and 0.78, respectively; sensitivity: 0.88 and 0.94; specificity: 0.81 and 0.69), which was close to that of the reference proteins LipL32 and Loa22 (area under the ROC curve: 0.91 and 0.80; sensitivity: 0.94 and 0.81; specificity: 0.75 and 0.75). CONCLUSIONS/SIGNIFICANCE: This analysis lends further support for using ORFeome phage display to identify pathogen-associated immunogenic peptides, and it suggests that this technique holds promise for the development of peptide-based diagnostics for leptospirosis and, possibly, of vaccines against this pathogen.

Macrophages and Galectin 3 Control Bacterial Burden in Acute and Subacute Murine Leptospirosis That Determines Chronic Kidney Fibrosis.

Previous studies have suggested that macrophages may contribute to acute Leptospira dissemination, as well as having a major role in kidney fibrosis. Our aim was to characterize the role of macrophages and galectin 3 (Gal-3) on the survival, clinical course, bacterial burden, interstitial nephritis, and chronic kidney fibrosis in Leptospira interrogans serovar Copenhageni (LIC)-induced experimental murine leptospirosis. C57BL/6J mice depleted of macrophages by liposome-encapsulated clodronate treatment and infected with LIC presented a higher bacterial burden, had reduced subacute nephritis and enhanced chronic kidney fibrosis relative to untreated, infected mice. Moreover, LIC infection in mice whose Gal-3 was disrupted (Lgals3-/-) had a higher bacterial burden and enhanced subacute nephritis and chronic kidney fibrosis when compared to C57BL/6J wild-type mice. Chronic fibrosis did not correlate with higher transcription levels of TGF-ß1 or IL-13 in the kidneys. Kidney fibrosis was found in chronically infected rats as well as in wild infected rats. On the other hand, human fibroblast cultures exhibited enhanced differentiation to myofibroblasts after treatment with LIC. Our results demonstrate that macrophages and Gal-3 play a critical role in controlling the LIC burden but has a minor role in subsequent fibrosis. Instead, kidney fibrosis was better correlated with bacterial burden. Taken together, our results do not support a role for macrophages to disseminate leptospires during acute infection, nor in chronic kidney fibrosis.

Whole-genome sequencing of Leptospira interrogans from southern Brazil: genetic features of a highly virulent strain

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.

Primer informe de Leptospira interrogans en el roedor sigmodontino Scapteromys aquaticus / First report on Leptospira interrogans in the sigmodontine rodent Scapteromys aquaticus / Primeiro relato da presença de Leptospira interrogans em roedores sigmodontíneos Scapteromys aquaticus

RESUMEN La leptospirosis es una enfermedad zoonótica de distribución mundial que puede transmitirse por contacto directo o indirecto con orina o tejidos de animales infectados. En Argentina, la leptospirosis es endémica en la provincia de Santa Fe y presenta brotes epidémicos durante las inundaciones. Sin embargo, se sabe muy poco sobre el papel que cumplen los roedores silvestres en la diseminación de la enfermedad en el país. El objetivo de este estudio fue identificar las especies hospederas de leptospiras patógenas entre los roedores presentes en un asentamiento ribereño de la provincia de Santa Fe. Se realizó un muestreo de roedores durante octubre de 2015. Los riñones de los animales capturados se analizaron por real-time PCR para el gen LipL32 de leptospiras patógenas. En los animales que resultaron positivos, se realizó test de microaglutinación (MAT) y tipificación molecular por amplificación del gen 16S rRNA y dos esquemas de MLST. Se capturaron 37 roedores de las especies Akodon azarae, Cavia aperea, Oligoryzomys flavescens, Rattus rattus y Scapteromys aquaticus. En el análisis por real-time PCR resultó positivo un macho de Scapteromys aquaticus. El suero de este individuo y del resto de los S. aquaticus capturados (n = 18) se analizaron por test de microaglutinación (MAT), y fueron no reactivos para los 10 serovares probados. La amplificación del gen 16S rRNA identificó la especie infectante como Leptospira interrogans, mientras que no se obtuvo amplificación para los dos esquemas de MLST. El hallazgo de este estudio aporta nueva información acerca de presencia de leptospiras patógenas en roedores silvestres, que es relevante para la zona por tratarse de una especie ampliamente distribuida en ambientes pantanosos e inundables de América del Sur.(AU)

ABSTRACT Leptospirosis is a globally distributed zoonosis that can be transmitted through direct or indirect contact with the urine or tissues of infected animals. In Argentina, leptospirosis is endemic in the province of Santa Fe and epidemic outbreaks occur during floods. However, very little is known about the role that wild rodents play in the spread of the disease in Argentina. The objective of this study was to identify the host species of pathogenic Leptospira among rodents in a riverine settlement in the province of Santa Fe. We conducted a trapping session in October 2015. Kidneys of the captured animals were analyzed by real-time PCR for the LipL32 gene of pathogenic Leptospira. Animals that were positive were subjected to microscopic agglutination test (MAT) and molecular typing by amplification of the 16S rRNA gene and two multilocus sequence typing (MLST) schemes. A total of 37 rodents of the species Akodon azarae, Cavia aperea, Oligoryzomys flavescens, Rattus rattus, and Scapteromys aquaticus were captured. Real-time PCR found one male Scapteromys aquaticus that was positive. The serum of this individual and of the rest of the S. aquaticus captured (n = 18) were analyzed by MAT and were non-reactive for the 10 serovars tested. Amplification of the 16S rRNA gene identified the infective species as Leptospira interrogans, while amplification could not be obtained for the two MLST schemes. The findings of this study contribute new information concerning the presence of pathogenic Leptospira in wild rodents, which is relevant in this region because the species is widely distributed in swampy and flood-prone environments of South America.(AU)

RESUMO A leptospirose é uma doença zoonótica de distribuição mundial transmitida pelo contato direto ou indireto com a urina ou os tecidos de animais infectados. Na Argentina, a leptospirose é endêmica na Província de Santa Fé com surtos epidêmicos ocorrendo com as enchentes. Sabe-se pouco sobre o papel dos roedores silvestres na propagação da doença no país. O objetivo deste estudo foi identificar as espécies hospedeiras de leptospiras patogênicas em roedores encontrados em um núcleo de povoamento ribeirinho na Província de Santa Fé. A amostragem dos roedores foi feita no mês de outubro de 2015. Os tecidos dos rins dos animais capturados foram analisados com a técnica de reação em cadeia da polimerase em tempo real (PCR-RT) quanto à presença do gene LipL32 de leptospiras patógenas. Para os animais com resultados positivos, foi realizado o teste de microaglutinação (MAT) e tipagem molecular baseada na amplificação do gene 16S rRNA e dois esquemas de tipagem por sequenciamento de locos múltiplos (MLST). Ao todo, foram capturados 37 roedores das espécies Akodon azarae, Cavia aperea, Oligoryzomys flavescens, Rattus e Scapteromys aquaticus. O ensaio de PCR-RT foi positivo em um roedor macho da espécie Scapteromys aquaticus. Os soros deste animal e dos outros S. aquaticus capturados (n = 18) foram analisados com o MAT e os resultados foram não reagentes para os 10 sorovares testados. A amplificação do gene 16S rRNA permitiu identificar a espécie infetante como sendo Leptospira interrogans e não houve amplificação nos dois esquemas de MLST. O achado deste estudo fornece um novo dado quanto à presença de leptospiras patogênicas em roedores silvestres, importante para esta área por se tratar de uma espécie de ampla distribuição em terras pantanosas e inundáveis da América do Sul.(AU)

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

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

Search of leptospires and of antibodies against leptospires in animals and human beings in farms in Pantanal and Caatinga Brazilian biomes / Pesquisa de leptospiras e de anticorpos contra leptospiras em animais e humanos de propriedades rurais nos biomas brasileiros Pantanal e Caatinga

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 Fishers 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) Caatinga Copenhageni...

Foi investigada a ocorrência de leptospiras e de sororreatividade para leptospiras em animais e seres humanos de seis propriedades rurais localizadas em dois biomas brasileiros que apresentam condições geoclimáticas distintas: Pantanal municípios de Miranda (MS), Itiquira (MT) e Poconé (MT) e Caatinga municípios de Sobradinho (CE), Garanhuns (PE) e Sobral (BA). Em cada uma das propriedades, foram realizadas colheitas de sangue e de urina de animais selvagens de vida livre, animais domésticos e de seres humanos. As colheitas de materiais foram realizadas no período de fevereiro a abril de 2012 no bioma Caatinga e no período de julho a setembro de 2012 no bioma Pantanal. A reatividade sorológica contra Leptospira spp. foi verificada pela técnica de soroaglutinação microscópica (SAM) efetuada com uma coleção de antígenos constituída por 24 sorovares de Leptospira spp. A pesquisa de leptospiras foi efetuada por cultivos de amostras de urina semeadas nos meios Fletcher e de Ellinghausen McCullough Johnson Harris (EMJH). Os cultivos em que houve crescimento de leptospiras foram encaminhados ao Laboratório de Leptospirose do Instituto de Patobiologia, Instituto Nacional de Tecnologia Agropecuária, Buenos Aires, Argentina e as estirpes de leptospiras isoladas foram genotipadas com o emprego da técnica de Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). O procedimento de tipificação empregou os VNTR 4, 7, 9, 10, 19, 23, 31, Lb4 e Lb5, que discriminam estirpes de L. interrogans e L. borgpetersenii. No Pantanal, foram examinados 17 animais selvagens, 65 animais domésticos e dois humanos. Na Caatinga, foram examinados sete animais selvagens, 100 animais domésticos e 26 humanos. Das 84 amostras de sangue examinadas no Pantanal, 47 (55,95%) foram reagentes e, das 133 da Caatinga, 59 (44,36%) foram reagentes. Pelo teste exato de Fisher, considerando-se um nível de significância de 0,05, não houve diferença entre as proporções de animais...

Produção e purificação de imunoglobulinas Y policlonais anti-Leptospira spp / Production and purification of polyclonal anti-Leptospira immunoglobulin Y

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.

The aim was to determine whether hens immunized with an inactivated suspension of Leptospira and a solution of outer membrane proteins extracted from the serovar Hardjo, could produce specific polyclonal antibodies to Leptospira, detected in ELISA assay. Eight hens White Leghorn race with 25-weeks-old were immunized, three with an inactivated suspension of Leptospira, three with a solution of outer membrane proteins (OMP) extracted from the serovar Hardjo and two controls immunized with saline. Blood samples were collected fortnightly and eggs daily. The IgY was purified from the egg yolk using the method for the delipidation of dilution with water acidic and ammonium sulfate precipitation. The ELISA assay was performed to verify the specificity of the IgY, these was possible to observe the production of specific antibody to Leptospira both in serum and purified egg yolk. The specific antibody titers peaked in the fifth week post immunization. The production of polyclonal IgY was effective for producing high titers of specific antibodies.

Risk factors and predictors of severe leptospirosis in New Caledonia.

BACKGROUND: Leptospirosis is a major public health concern in New Caledonia (NC) and in other tropical countries. Severe manifestations of the disease are estimated to occur in 5-15% of all human infections worldwide and factors associated with these forms are poorly understood. Our objectives were to identify risk factors and predictors of severe forms of leptospirosis in adults. METHODS AND FINDINGS: We conducted a retrospective case-control study of inpatients with laboratory-confirmed leptospirosis who were admitted to two public hospitals in NC in 2008-2011. Cases were patients with fatal or severe leptospirosis, as determined by clinical criteria. This approach was meant to be pragmatic and to reflect the routine medical management of patients. Controls were defined as patients hospitalized for milder leptospirosis. Risk and prognostic factors were identified by multivariate logistic regression. Among the 176 patients enrolled in the study, 71 had criteria of severity including 10 deaths (Case Fatality Rate = 14.1%). Three risk factors were independently associated with severe leptospirosis: current cigarette smoking (OR = 2.94 [CI 1.45-5.96]); delays >2 days between the onset of symptoms and the initiation of antibiotherapy (OR = 2.78 [CI 1.31-5.91]); and Leptospira interrogans serogroup Icterohaemorrhagiae as the infecting strain (OR = 2.79 [CI 1.26-6.18]). The following post-admission laboratory results correlated with poor prognoses: platelet count ≤50,000/µL (OR = 6.36 [CI 1.79-22.62]), serum creatinine >200 mM (OR = 5.86 [CI 1.61-21.27]), serum lactate >2.5 mM (OR = 5.14 [CI 1.57-16.87]), serum amylase >250 UI/L (OR = 4.66 [CI 1.39-15.69]) and leptospiremia >1000 leptospires/mL (OR = 4.31 [CI 1.17-15.92]). CONCLUSIONS: To assess the risk of developing severe leptospirosis, our study illustrates the benefit for clinicians to have: i) the identification of the infective strain, ii) a critical threshold of qPCR-determined leptospiremia and iii) early laboratory results. In New Caledonia, preventative measures should focus on early presumptive antibacterial therapy and on rodent (reservoir of Icterohaemorrhagiae serogroup) control.

In vivo cell aggregations of a recent swine biofilm-forming isolate of Leptospira interrogans strain from Argentina / Agregaciones celulares in vivo de Leptospira interrogans producidas por un aislamiento porcino capaz de formar biofilm

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

La leptospirosis es una zoonosis de amplia distribución causada por el género Leptospira. Las leptospiras existen de manera saprófita asociadas a ambientes acuáticos o como patógenos animales que también pueden sobrevivir en el agua. Trabajos previos demostraron que tanto las leptospiras saprófitas como las patógenas tienen la capacidad de formar biofilms, que consisten en una comunidad de bacterias embebidas en una matriz extracelular adherida a una superficie. Esta estructura tendría la función de proveer protección contra el medioambiente. En este estudio, analizamos la capacidad de formar biofilm en un aislamiento obtenido recientemente de un feto porcino abortado, caracterizado como Leptospira interrogans serovar Pomona, y en la bacteria saprófita Leptospira biflexa serovar Patoc. Se estudió la formación de biofilm en distintas superficies (vidrio y poliestireno), las que se evaluaron por microscopía óptica, inmunofluorescencia y microscopía electrónica de barrido. La capacidad de formar agregaciones bacterianas in vivo se evaluó utilizando un modelo de cobayas preñadas infectadas con ambas cepas. Se obtuvieron biofilms tanto en las superficies plásticas como de vidrio. La microscopía de barrido mostró diferencias en la estructura del biofilm formado entre ambas cepas. Se observaron agregaciones celulares en vasos placentarios de los animales infectados con L. interrogans serovar Pomona. Los biofilms y las agregaciones celulares son compatibles con la vida saprofítica en el agua y podrían favorecer a los microorganismos patógenos en la colonización del hospedador, lo que podría llevar al aborto en los animales preñados.

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

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

Leptospirosis and tularaemia in raccoons (Procyon lotor) of Larimer County, [corrected] Colorado.

Raccoons (Procyon lotor) are commonly implicated as carriers of many zoonotic pathogens. The purpose of this cross-sectional study was to look for Leptospira interrogans and Francisella tularensis in opportunistically sampled, free-ranging raccoons of Larimer Country, Colorado, USA. Sixty-five animals were included in the study and testing consisted of gross post-mortem examination, histopathology, and both immunohistochemistry and PCR for L. interrogans and F. tularensis. No significant gross lesions were identified and the most common histological lesions were lymphoplasmacytic interstitial nephritis and pulmonary silicosis; rare periportal hepatitis, splenic lymphoid hyperplasia and small pulmonary granulomas were also identified. Of 65 animals, 20 (30%) were positive for Leptospira on IHC but only one by PCR. Animals with inflammation in their kidneys were seven times more likely to be positive for Leptospira than animals without inflammation. The severity of inflammation was variable but often mild with minimal associated renal pathology. One animal was positive for Francisella on both IHC and PCR; IHC staining was localized to histiocytic cells within a pulmonary granuloma. In Colorado the significance and epidemiology of Leptospira is poorly understood. The high prevalence of infection in raccoons in this study population suggests that this species may be important in the regional epidemiology or could be used to estimate risk to domestic animals and humans. Identification of a single Francisella positive animal is significant as this is an uncommon disease in terrestrial animals within the state; the apparently higher prevalence in this peridomestic species implies that raccoons may be good indicators of the pathogen in the region. The results of this study suggest that raccoons may serve as effective sentinels for both Leptospira and Francisella in the state of Colorado. Further studies are needed to better characterize the prevalence and epidemiology of both organisms within the region.

Survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction

This work reports a survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction (PCR). Seventy pampas deer were captured in the dry season and surveyed using PCR, microscopic agglutination test (MAT) (n = 51) and by both techniques (n = 47). PCR detected infections in two pampas deer and MAT detected infections in three. Through sequencing and phylogenetic analyses, the PCR-amplified fragment detected in deer was identified as Leptospira interrogans. Serovars Pomona and Butembo were detected using MAT and the highest titre was 200 for serovar Pomona. Epidemiological aspects of the findings are discussed.

Leptospirosis research: fast, easy and reliable enumeration of mobile leptospires

Leptospirosis caused by Leptospira interrogans is the most widespread zoonosis and a major public health problem worldwide. Based on light-scattering and absorption, quantification of leptospires using UV-VIS spectroscopy was used as an indirect counting technique by measuring the optical density and comparing this to automated direct counting using a counting chamber in combination with imaging and analyzing software. Two serovars, Bangkok and Copenhagenii, from log-phase growth were used for the establishment of standard curves. They were found to be linear and slightly different in gradient for each serovar. The ease, rapidity, and reliability of these two adapted and optimized counting techniques may provide a useful alternative enumeration technique for leptospirosis research.

Freqüência de aglutininas anti-Leptospira interrogans em eqüídeos, em Minas Gerais, 2003 a 2004 / Frequency of anti-Leptospira interrogans agglutinins in equidae in Minas Gerais, Brazil, from 2003 to 2004

The goal of this research was to find the frequency and spatial distribution of infection caused by Leptospira interrogans in equidae in Minas Gerais State from September 2003 to March 2004. Samples of blood serum (6,475) were analyzed by microscopic agglutination test. From the total, 381 samples were positive (5.9 percent), with title equal or superior to 1:200 for one or more serovars of leptospira. The most frequent serovars were Hardjo (Norma), Pomona, Bratislava, and Batavie. The higher frequency of equidae reagents were recorded at the North and Northeast region of Minas Gerais, followed by Triângulo Mineiro and Alto Paranaíba, then Central, West, Metropolitan area of Belo Horizonte, and South/Southwest.

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

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 SAM, em que houve isolamento de leptospiras do fígado e aparelho reprodutor. A presença do DNA de leptospira foi investigada pela técnica da PCR e foram observados resultados positivos nos rins de 11 fêmeas, no fígado de duas, no aparelho reprodutor de duas e na urina de uma delas. Nefrose, nefrite intersticial multifocal variando de moderada a severa, cilindros hialinos e focos hemorrágicos, congestão hepática e de cornos uterinos foram lesões histológicas evidenciadas com freqüência mais alta em animais positivos para leptospira. A impregnação argêntica (Warthin Starry) confirmou a presença de espiroquetas nos túbulos renais das quatro fêmeas onde houve cultura positiva para leptospiras dos rins. O sorogrupo dos cinco isolados foi identificado como Pomona pela técnica de aglutinação cruzada com anticorpos policlonais de referência. A caracterização molecular dos isolados foi realizada pela análise do número variável de repetições em tandem (VNTR). Os mesmos revelaram um padrão distinto da estirpe padrão de L. interrogans sorovar Pomona, porém idêntico a um padrão previamente identificado em estirpes isoladas na Argentina, pertencentes ao sorovar Pomona.

Diagnostic potential of an iron-regulated hemin-binding protein HbpA that is widely conserved in Leptospira interrogans.

Like most bacteria, Leptospira spp. requires iron for growth. However, they face conditions of iron limitation due to the low solubility of the ferric iron and additionally due to the observation that most of the available iron is held as protein-bound iron by the mammalian host. Our experimental observations with pathogenic Leptospira showed that they do not elaborate siderophores upon iron limitation. In Leptospira interrogans serovar Lai, we demonstrated direct acquisition of iron via an iron-regulated hemin-binding protein HbpA. PCR analysis and Southern hybridization studies revealed that the hbpA gene was conserved in the serovars belonging to L. interrogans species and was absent in the non-pathogenic Leptospira biflexa serovar Patoc and Leptospira meyeri serovar Ranarum. Here, we extended the PCR-based detection of the hbpA gene to clinical isolates and demonstrated the hbpA amplicon in all the serovars belonging to L. interrogans species from a total collection of 91 clinical isolates obtained from different geographical regions. In addition, we detected anti-HbpA antibodies in the serum of patients with leptospirosis. PCR-based detection of hbpA in clinical isolates of serovars and the in vivo expression of HbpA reflects the diagnostic potential of this antigen.

Leptospira interrogans is recognized through DC-SIGN and induces maturation and cytokine production by human dendritic cells.

Leptospirosis is a global zoonotic disease, caused by pathogenic Leptospira species including Leptospira interrogans, that causes public health and livestock problems. Pathogenesis, immune response and cellular receptors for Leptospira are not well understood. Interaction of dendritic cells (DCs) with L. interrogans serovar Autumnalis L-643 and BL-6 isolated from leptospirosis patients, and both virulent and avirulent serovar Pyrogenes 2317 strains isolated from animal were investigated. Carbohydrate analysis using lectins showed that all of these leptospires contained high mannose components as a common backbone and DC-SIGN was involved in leptospires' attachment. Interaction of the L. interrogans strains with DCs induced maturation, but had different effects on IL-10, IL-12p70 and tumor necrosis factor (TNF)-alpha production. Both virulent and avirulent Pyrogenes 2317 and Autumnalis BL-6 but not L-643 strains induced IL-12p70 and TNF-alpha production, but minimal IL-10 secretion. These data demonstrated that L. interrogans binds DC-SIGN and induces DCs maturation and cytokine production, which should provide new insights into cellular immune processes during leptospirosis.