Abstract Leptospirosis is a worldwide zoonose and is endemic in many countries. This disease is maintained in nature by chronic renal infection of carrier animals, being rodents and other small mammals the most important reservoirs. Also, significant sources of human infection are domestic animals, such as livestock and dogs. The wild boar (Sus scrofa) is an introduced species from Europe which is widespread in America and particularly in the Center and South of Argentina. This species is highly valued by hunters in the region and their meat is consumed more often. In many countries of the world (Europe, USA and Australia) studies of seroprevalence have been carried out, the seroreactivity does not mean that the wild boar has clinical symptoms of leptospirosis or that wild boars are maintenance host of this pathogen. However, these animals have been in contact to leptospiras in their environment in the past. This study has the objective to molecularly genotype the pathogenic Leptospira sp. isolated strain obtained from fetuses of wild boars from Patagonia Argentina. Four fetuses were aborted from a wild boar (Sus scrofa) in Patagonia, the southern region of Argentina. Necropsy was performed of the 4 fetuses and samples of kidneys, liver, spleen and lung were cultivated in Fletcher and EMJH mediums. Also, samples of liver, spleen, lung and kidney of fetuses were processed and analyzed by direct immunofluorescence. Multiple Locus Variable number tandem repeats Analysis (MLVA) was used to characterize the isolated strain. The genetic profile of the isolated pathogenic Leptospira sp. strain was identical to the profile of Leptospira borgpetersenii serovar Castellonis Castellon 3, serogroup Ballum. To the best of our knowledge this is the first isolation of Leptospira spp. from a wild boar fetus.

Resumen La leptospirosis es una zoonosis mundial y es endémica en muchos países. Esta enfermedad se mantiene en la naturaleza por la infección renal crónica de los animales portadores; los roedores y otros mamíferos pequeños son los reservorios más importantes. Además, otras fuentes importantes de infección para el humano son los animales domésticos, como el ganado y los perros. El jabalí (Sus scrofa) es una especie introducida de Europa que está muy extendida en América y particularmente en el centro y sur de Argentina. Esta especie es muy valorada por los cazadores de la región y su carne se consume con mayor frecuencia. En muchos países del mundo (Europa, EE. UU. y Australia) se han realizado estudios sobre seroprevalencia, la seroreactividad no significa que el jabalí manifieste síntomas clínicos de leptospirosis o que los jabalíes sean reservorios de este patógeno bacteriano. Sin embargo, estos animales han estado en contacto con leptospiras en su entorno en el pasado. Este estudio tiene el objetivo de tipificar molecularmente la cepa aislada obtenida a partir de 4 fetos de jabalíes (Sus scrofa) provenientes de la Patagonia argentina. Se realizaron necropsias de los 4 fetos y se cultivaron muestras de riñones, hígado, bazo y pulmón en medios Fletcher y EMJH. Además, las muestras de hígado, bazo, pulmón y riñón de los fetos se procesaron y analizaron por inmunofluorescencia directa. Se utilizó el análisis de repeticiones en tándem de número variable de locus múltiple (MLVA) para caracterizar la cepa aislada. El perfil genético de la cepa de leptospira fue idéntica al perfil de Leptospira borgpetersenii serovar Castellonis Castellon 3 perteneciente al serogrupo Ballum. Hasta donde sabemos, este es el primer aislamiento de Leptospira spp. de un feto de jabalí.

ABSTRACT Leptospirosis is an endemic disease caused byLeptospiraspp., a bacterium that affects animals and humans. In recent years, the number of reports of leptospirosis in wild animals has increased, which highlights the need to study the infectious agents in these animals. In this study, a duplex PCR for the detection of leptospiral DNA was performed on 50 kidney samples from bats, and a MAT (Microscopic Agglutination Test) for serological detection of anti-leptospiral antibodies was applied to 47 serum samples from bats from different regions of Buenos Aires Province, Argentina. DNA was extracted using Chelex-100 and duplex PCR was performed by targeting the detection of genessecYandflaB, of pathogenicLeptospiraspp. Of the 50 kidney samples, 3 were positive forEumopssp. andTadaridabrasiliensisby duplex PCR. Of the 47 serum samples, 12 were positive for different serovars:Leptospira interrogansserovars Icterohaemorrhagiae, Cynopteri and Bataviae, andLeptospira borgpeterseniiserovar Ballum. This is the first report of the detection of pathogenic leptospires by serology in bats belonging to theT. brasiliensisandEptesicus furinalisspecies in Argentina. In addition, this is the first report of the detection of pathogenic leptospiral DNA by PCR inT. brasiliensisspecies. The detection ofLeptospiraspp. in these wild animals shows that they may play an important role as wildlife reservoirs of leptospires.

RESUMEN La leptospirosis es una enfermedad endémica causada porLeptospiraspp., una bacteria que afecta a animales y a humanos. En los últimos años, el número de reportes de leptospirosis en animales silvestres ha aumentado, lo que resalta la necesidad de analizar los agentes infecciosos en estos animales. En este estudio, se aplicó una reacción en cadena de la polimerasa (PCR) dúplex para la identificación del ADN leptospiral en 50 muestras de riñones de murciélagos y la prueba de aglutinación microscópica (MAT) para la detección serológica de anticuerpos antileptospira en 47 muestras de suero de murciélagos de diferentes regiones de la provincia de Buenos Aires, Argentina. El ADN fue extraído usando Chelex-100 y la PCR dúplex estuvo dirigida a la detección de los genessecYyflaBdeLeptospiraspp. patógena. De las 50 muestras de riñón, tres resultaron positivas por PCR dúplex paraEumopssp. yTadaridabrasiliensis. De las 47 muestras de suero, 12 fueron positivas a diferentes serovares:LeptospirainterrogansserovaresIcterohaemorrhagiae, Cynopteri y Bataviae, yLeptospiraborgpeterseniiserovarBallum. Este es el primer reporte de detección de leptospiras patógenas por serología en murciélagos pertenecientes a las especiesT. brasiliensisyEptesicusfurinalisen Argentina. Además, también es el primero en la localización de ADN leptospiral por PCR en la especieT. brasiliensis.La identificación deLeptospiraspp. en estos animales silvestres muestra que pueden desempeñar un papel importante como reservorios de leptospiras en la fauna silvestre.

Abstract We here describe the first isolation and molecular typing of Leptospira borgpe-tersenii serovar Hardjo Bovis in Argentina, obtained from urine of aborted cows from abreeding herd located in Saladillo, Buenos Aires Province. The abortions occurred in coincidence with important floods with many cows presenting suspicious serological titers and subsequentseroconversion. The percentage of abortions was 3.5% of a herd of 1700 cows and the microor-ganism was isolated from 7 of the 20 urine samples obtained.

Resumen Se describe el primer aislamiento y la tipificación molecular de Leptospira borgpetersenii serovar Hardjo Bovis en Argentina, obtenido a partir de orina de vacas abortadas de unrodeo de cría ubicado en Saladillo, provincia de Buenos Aires. Los abortos coincidieron con unperíodo de importantes inundaciones, en el que varios animales presentaron títulos serológicossospechosos y posterior seroconversión. El porcentaje de abortos alcanzó el 3,5% del total delrodeo, compuesto por 1700 vacas, y se aisló el microorganismo en 7 de 20 muestras de orinaobtenidas.

RESUMO: O diagnóstico definitivo da leptospirose canina é geralmente realizado demonstrando a seroconversão em amostras do paciente no período agudo e de convalescença por serologia. No entanto, a aplicação de técnicas de PCR pode contribuir para a confirmação de casos suspeitos num período de tempo mais curto. O objetivo deste estudo foi avaliar dois ensaios de PCR publicados em humanos (PCR-lipL32 em tempo real e PCR-rrs convencional) em culturas puras e em amostras de sangue com anticoagulante, soro e urina experimentalmente contaminados. Posteriormente, investigamos a utilidade de ambos os ensaios de PCR em amostras clínicas de cães com suspeita de leptospirose tomando a técnica de microaglutinação (MAT) como referência. A sensibilidade analítica foi de 1 e 10 genoma equivalente por reação para PCR-lipL32 em tempo real e para PCR-rrs convencional, respectivamente. Ambos os ensaios amplificaram corretamente as 14 estirpes patogênicas, mas foram negativos para avaliar o ADN de outros microrganismos que poderiam estar presentes em amostras clinicas. Em nas amostras experimentalmente contaminadas PCR-LipL32 em tempo real detectou 100 bactérias/mL em sangue total, 1000 bactérias/mL em soro e 10 bactérias/mL em urina. Enquanto o PCR-rrs convencional detectou 1000 bactérias/mL em sangue total e soro e 100 bactérias/mL na urina. Dos 51 cães suspeitos, sete foram considerados casos confirmados pela MAT. O PCR-lipL 32 em tempo real detectou seis dos sete casos confirmados, enquanto o PCR-rrs convencional foi positivo em quatro deles. As técnicas de PCR avaliadas provaram ser uma ferramenta de diagnóstico útil na confirmação de casos clínicos caninos quando utilizados em conjunto com a técnica MAT.

ABSTRACT: Canine leptospirosis is definitely diagnosed by demonstrating seroconversion in paired serum samples from the acute and convalescent period by the microagglutination test (MAT). However, the application of a polymerase chain reaction (PCR) assay can provide earlier confirmation of suspected cases. The objective of this study was to evaluate two PCR assays used in diagnosis of human leptospirosis (lipL32 real-time PCR and rrs conventional PCR) in cultured microorganisms and experimentally contaminated samples (whole blood, serum, urine), and investigate their applicability in clinical samples from dogs with presumptive diagnosis of leptospirosis by using the MAT as a reference. The analytical sensitivity of the lipL32 real-time PCR was 1 genome equivalent per reaction, whereas that for the rrs conventional PCR was 10 genome equivalents per reaction. Both assays amplified the pathogenic strains but were negative when evaluating the DNA of other microorganisms that may be present in clinical samples. The lipL32 real-time PCR detected 100 bacteria/mL in whole blood samples, 1000 bacteria/mL in serum samples and 10 bacteria/mL in urine samples, whereas the rrs conventional PCR detected 1000 bacteria/mL in whole blood and serum samples and 100 bacteria/mL in urine samples. Seven out of the 51 samples from dogs with presumptive diagnosis of leptospirosis were considered as confirmed cases. ThelipL32 real-time PCR detected positive results in six of the seven confirmed cases, whereas the rrs conventional PCR detected four. The PCR assays evaluated proved to be useful diagnostic tools in the confirmation of canine leptospirosis when used together with the MAT.

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.

Se aislaron 10 cepas de Leptospira spp. a partir de muestras de agua del arroyo Nievas, partido de Olavarría (provincia de Buenos Aires, Argentina). Los aislamientos mostraron motilidad y morfología típica del género Leptospira bajo microscopía de campo oscuro y se desarrollaron en medio líquido EMJH después de 8 días de incubación a 13 y 30°C. Todos los aislamientos fueron negativos por MLVA, y mediante la secuenciación del gen 16S del ARNr se identificaron como leptospiras no patógenas. Cuatro de estos aislamientos mostraron un perfil genético idéntico a la cepa Leptospira biflexa serovar Patoc de referencia, en tanto que 6 de ellos presentaron similitudes de secuencias estrechamente relacionadas con las especies Leptospira yanagawae y Leptospira meyeri dentro del intervalo del 97 y 98%, respectivamente. Las cepas ScialfaASA42, ScialfaASA45, ScialfaASA44, ScialfaASA47, ScialfaASA49, ScialfaASA50 y ScialfaASA51 posiblemente representen una nueva especie del género Leptospira.

Leptospirosis is a zoonosis having worldwide distribution. The objective of this work was to develop a molecular technique to differentiate pathogenic Leptospira spp. A region of adhesin ligB, present only in the pathogenic species was amplified by PCR and sequenced. ligBRpet and ligBFpet primers were used, which amplified the target DNA from pathogenic L. interrogans reference strains serovars Pomona strain Pomona, Canicola strain Hond Utrecht IV, Copenhageni strain M 20, Wolffi strain 3705, Pyrogenes strain Salinem, Hardjo strain Hardjoprajitmo, L. borgpetersenii serovar Castellonis strain Castellon 3 and 4 pathogenic strains isolated from bovines, pigs, rats and opossums. L. biflexa serovars Patoc strain Patoc i and Andamana strain Andamana were not amplified. Sequencing of the amplified products exhibited sufficient variation among serovars, which differentiates them.

La leptospirosis es la zoonosis de mayor distribución mundial. El objetivo del preReacción en cadena sente trabajo fue desarrollar una técnica molecular que diferencie leptospiras patógenas. Se amplificó mediante PCR y se secuenció una región de la adhesina ligB, presente solo en las especies patógenas. Se utilizaron los iniciadores ligBFpet y ligBRpet. Dichos iniciadores lograron amplificar el ADN blanco de 6 cepas patógenas referenciales de Leptospira interrogans (serovar Pomona cepa Pomona, serovar Canicola cepa Hond Utrecht IV, serovar Copenhageni cepa M 20, serovar Wolffi cepa 3705, serovar Pyrogenes cepa Salinem y serovar Hardjo cepa Hardjoprajitmo) y el de Leptospira borgpetersenii serovar Castellonis cepa Castellon 3; también el de 4 cepas patógenas aisladas de bovino, porcino, rata y comadreja. En las 2 cepas referenciales no patógenas utilizadas, Leptospira biflexa serovar Patoc cepa Patoc i y L. biflexa serovar Andamana cepa Andamana, no hubo amplificación. La secuenciación de los productos amplificados expuso una suficiente variación entre los serovares estudiados, que permite diferenciarlos.

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.

La leptospirosis es una zoonosis de distribución mundial. Nuestro objetivo fue examinar la presencia de anticuerpos contra 21 serovares reactivos de Leptospira en Chaetopractus villosus en la provincia de La Pampa, Argentina, mediante la prueba de aglutinación microscópica (MAT). Se realizó el estudio histopatológico y la detección in situ del agente por inmunohistoquímica en 24 y 3 individuos, respectivamente. Solo 35/150 (23,3%) muestras de suero presentaron anticuerpos contra Leptospira sp. Seis por ciento reaccionaron al serovar Canicola; 4,7% a Castellonis; 1,3% a Icterohemorrhagieae y 0,7% a Hardjo. Dieciséis (10,6%) sueros aglutinaron con Canicola-Castellonis y Castellonis-Icterohemorrhagiae, ambos con 4,7%, y con Canicola-Hardjo y Castellonis-Canicola-Icterohemorrhagiae, ambos con 0,6%. En 14 animales se encontraron lesiones compatibles, las que resultaron más graves en animales con títulos serológicos elevados (3200). En 3 animales estudiados se detectó el agente causal por inmunohistoquímica. Estos resultados constituyen los primeros registros de la presencia de Leptospira en C. villosus en La Pampa.

O objetivo do presente estudo foi descrever um caso de isolamento de espécie patogênica de Leptospira interrogans em amostra de urina de um humano morador da zona rural do Município de Cruz Alta, Estado do Rio Grande do Sul. De cada amostra de urina, uma alíquota foi semeada nos meios Fletcher e Ellinghausen - McCullough - Johnson - Harris (EMJH). As amostras, nas quais houve crescimento de espiroquetas, foram encaminhadas para o Laboratório de Leptospirose do Instituto de Patobiologia do Instituto Nacional de Tecnologia Agropecuária, Buenos Aires, Argentina e foram tipificadas pela técnica Multiple Locus of Variable Number Tandem Repeat (MLVA). De um residente do sexo masculino da área rural do município de Cruz Alta, foi isolada Leptospira interrogans sorovariedade Copenhageni cepa Fiocruz L1-130, uma descoberta muito importante e que serve como um alerta por caracterizar uma situação de risco de epidemia de leptospirose por uma cepa patogênica. Os profissionais de saúde precisam ser mais comprometidos com a atenção primária à saúde no Brasil e rotineiramente aplicar ações de medicina preventiva nas comunidades rurais, a fim de obter sucesso no controle da leptospirose e de outras importantes zoonoses

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

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.

The production of recombinant LipL32 protein using Escherichia coli has been used extensively for the development of vaccines and diagnostic tests for leptospirosis. However, E. coli has demonstrated limitations, including low yield and lack of post-translational modifications. In this study, rLipL32 was produced in eukaryotic expression system (Pichia pastoris) and evaluated the antigen by enzyme-linked immunosorbent assay (ELISA). The yield obtained from the culture supernatant reached 270 mg/L and ELISA showed an accuracy of 95.34%. In summary, the production of rLipL32 using P. pastoris did not impair the antigenic characteristics of this antigen and ensured its use for detecting the leptospiral antibodies in swine sera.

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

Introduction: the Leptospira genus species are causative agents of leptospirosis, a disease that is considered the most widely spread zoonotic disease worldwide. In Argentina, leptospirosis is endemic and Santa Fe province has the highest number of human cases. Since mid-1980's, the pathogenic leptospira species isolated from animals and humans have been differenciated through DNA-DNA hybridization tests, resulting in new species: L. interrogans, L. kirschneri, L. weilii, L. noguchii, L. borgpetersenii, L. santarosai, L. meyeri, L. inadai, L. faineri y L. alexanderi. Objectives: to isolate and to characterize by molecular test leptospiras from water poured into a channel that runs through Casilda City in Santa Fe Province, Argentina. Methods: six samples of water from the channel were cultured after having been filtered through 0.22 µm, Millpore filtres in EMJH and Fletcher media to isolate leptospires. They were incubated at 30 °C for 15 days, and weekly observed through dark field microscopy. Polymerase chain reaction assay was used under specific conditions (Sugathan, 2005), with two sets of primers (Gravekamp, 1993), to determine whether the isolates were pathogenic. The molecular technique for genotyping was Multiple-Locus Variable-number tandem repeats Analysis (MLVA). Results: five Leptospira spp. isolates were obtained of which 2 were positive to PCR, all of which determined that they were pathogenic leptospiras. MLVA genotyping allowed the observation of a pattern similar to that of L. borgpetersenii species in one pathogenic isolates, but the other isolate was not identified. Conclusions: in the City where the study was carried out, with a population of about 40,000 inhabitants, a L. borgpetersenii species was identified for the first time in urban water sources, with the potential risk that it may pose for human and animal populations.

Introducción: las especies del género Leptospira son los agentes causales de la leptospirosis, enfermedad considerada como la zoonosis de mayor distribución en el mundo.En Argentina reviste carácter endémico. La provincia de Santa Fe registra el mayor número de casos humanos. Desde mediados de la década de 1980, las especies patógenas de leptospiras aisladas de animales y humanos fueron diferenciadas sobre la base de estudios de hibridización ADN-ADN, surgiendo nuevas especies: L. interrogans, L. kirschneri, L. weilii, L. noguchii, L. borgpetersenii, L. santarosai, L. meyeri, L. inadai, L. faineri y L. alexanderi. Objetivo: aislar y caracterizar mediante métodos moleculares, leptospiras de fuentes de agua que vierten en un canal que atraviesa la ciudad de Casilda, Santa Fe, en Argentina. Métodos: se sembraron 6 muestras de agua de las vertientes al canal, previa filtración con filtros Millipore de 0,22 µm, en medios EMJH y Fletcher para aislamiento de leptospiras. Se incubaron en estufa a 30 °C durante 15 días, se observaron semanalmente mediante microscopia de campo oscuro. Se implementó la técnica de reacción en cadena de la polimerasa bajo las condiciones específicas (Sugathan, 2005), con dos juegos de cebadores (Gravekamp, 1993), que permiten detectar la presencia de ADN de leptospiras patógenas. La técnica molecular utilizada para genotipificar fue multiple-locus variable-number tandem repeats analysis (MLVA). Resultados: se obtuvieron 5 aislamientos de Leptospira spp., de los cuales 2 resultaron positivos a la reacción en cadena de la polimerasa, determinando que se trataba de leptospiras patógenas. Mediante la genotipificación por MLVA se pudo observar que uno de los aislamientos patógenos mostró un patrón correspondiente a la especie Leptospira borgpetersenii, no siendo identificable la otra cepa. Conclusiones: en la ciudad del estudio, que tiene alrededor de 40 000 habitantes, se logró identificar por primera vez una cepa de Leptospira borgpetersenii de fuentes de aguas urbanas, con el peligro potencial que esto representa para la población humana y animal.

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.

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 is a worldwide zoonosis caused by a spirochete that belongs to the genus Leptospira. In the last years, new methods, such as the PCR-based multiple-locus variable-number tandem repeat analysis (MLVA), have been developed for the genotyping of leptospires. In the present work, the MLVA patterns for all reference strains used in Argentina for bovine, ovine, porcine, equine, caprine and canine leptospirosis diagnosis, as well as in human and wild animal diagnosis, were obtained. MLVA results are presented in such a way that they can be readily used for the identifcation of these strains by the simple and direct comparison of agarose gels. Making the use and interpretation of the MLVA for leptospires typing easier will help increase the use of this method as a routine procedure for human and animal diagnosis, for epidemiological studies, vaccine control and other applications.

La leptospirosis es una zoonosis de distribución global causada por una espiroqueta perteneciente al género Leptospira. En los últimos años se han desarrollado nuevos métodos para la genotipifcación de las leptospiras, entre ellos el denominado multiple-locus variable-number tandem repeat analysis (MLVA). En este trabajo se obtuvieron los patrones de MLVA de todas las cepas de referencia utilizadas en la Argentina para el diagnóstico de leptospirosis en bovinos, ovinos, porcinos, equinos, caprinos y perros, y que también son utilizadas en el diagnóstico de leptospirosis en humanos y en animales salvajes. Los resultados del MLVA se muestran de manera tal que pueden ser fácilmente utilizados para la identifcación de estas cepas por simple comparación visual de geles de agarosa. Al facilitar el uso y la interpretación del MLVA para la tipifcación de leptospiras, se ayudará a difundir la utilización rutinaria de este método en el diagnóstico humano y animal, en estudios epidemiológicos y para el control de vacunas, entre otras aplicaciones.