Optimization and comparative analysis of LAMP and PCR techniques for the detection of leptospiral DNA in Golden Syrian hamsters.

Leptospirosis is a zoonotic disease with significant public health and economic impact worldwide. Rapid and accurate diagnosis is essential for effective prevention and treatment. This study optimized a loop-mediated isothermal amplification (LAMP) assay using BFo isothermal DNA polymerase with different colorimetric indicators. LAMP was able to detect DNA from pathogenic and intermediate leptospires, while non-pathogenic leptospires and other non-leptospiral microorganisms were negative. LAMP assay combined with calcein showed a tenfold higher limit of detection (1 ng of leptospiral DNA per reaction) than LAMP combined with hydroxynaphthol blue or end-point PCR lipL32 (10 ng of DNA per reaction). Animal samples were collected from infected and non-infected Golden Syrian hamsters (Mesocricetus auratus) to evaluate and compare the performance of LAMP and PCR. These techniques showed a substantial agreement according to Cohen's kappa statistic, being both useful techniques for detecting leptospiral DNA in clinical samples. Overall, this study demonstrates that the LAMP assay is a sensitive, specific, rapid, and simple tool for the detection of leptospiral DNA. It has the potential to facilitate the diagnosis of leptospirosis, particularly in low-income regions with limited diagnosis resources.

First report of pathogenic Leptospira spp. in Tadaridabrasiliensis bats (family Molossidae) and Eptesicus furinalis (family Vespertilionidae) of Argentina. New host species in this country? / Primer reporte de Leptospira spp. patógena en murciélagos Tadaridabrasiliensis (familia: Molossidae) y Eptesicusfurinalis (familia: Vespertilionidae) en Argentina. ¿Nuevas especies hospedadoras en el país?

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.

New enzyme-linked immunoassay for the detection of specific antibodies against multiple Leptospira serogroups in bovine sera.

Leptospirosis is a zoonotic disease with worldwide endemicity in Argentina, it is a significant public health problem in low-income populations. Bovine leptospirosis is a serious economic problem for cattle production, causing abortions, reduced milk yield, mortality in calves and decreased daily weight gain. We developed an enzyme-linked immunosorbent assay (ELISA) with sonicated Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhageni M 20. We evaluated its performance for the detection of specific antibodies against multiple Leptospira serogroups in bovine. The microscopic agglutination test (MAT) was used as the gold standard. The performance of this ELISA was evaluated with a panel of sera (118 MAT confirmed positive and 97 MAT negative). The overall sensitivity was close to 85.6 % and the specificity was 83.5 %, according to the MAT reference method. Analytical specificity of the IgG-ELISA was evaluated using 50 bovine serum samples from animals showing serum antibodies against other pathogens that cause abortion in bovine, such as Brucella sp., Neospora sp. and Bovine Viral Diarrhea (BVD), and no cross-reaction was observed. This IgG-ELISA can be an alternative to the MAT for diagnosis of leptospiral infection in bovine.

First report of pathogenic Leptospira spp. in Tadarida brasiliensis bats (family Molossidae) and Eptesicus furinalis (family Vespertilionidae) of Argentina. New host species in this country?

Leptospirosis is an endemic disease caused by Leptospira spp., 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 genes secY and flaB, of pathogenic Leptospira spp. Of the 50 kidney samples, 3 were positive for Eumops sp. and Tadarida brasiliensis by duplex PCR. Of the 47 serum samples, 12 were positive for different serovars: Leptospira interrogans serovars Icterohaemorrhagiae, Cynopteri and Bataviae, and Leptospira borgpetersenii serovar Ballum. This is the first report of the detection of pathogenic leptospires by serology in bats belonging to the T. brasiliensis and Eptesicus furinalis species in Argentina. In addition, this is the first report of the detection of pathogenic leptospiral DNA by PCR in T. brasiliensis species. The detection of Leptospira spp. in these wild animals shows that they may play an important role as wildlife reservoirs of leptospires.

Primer aislamiento de Leptospira borgpetersenii serovar Hardjo tipo Hardjo Bovis a partir de un caso clínico en Argentina / First isolation of Leptospira borgpetersenii serovar Hardjo type Hardjo Bovis from aclinical case in cattle in Argentina

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.

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.

[First isolation of Leptospira borgpetersenii serovar Hardjo type Hardjo Bovis from a clinical case in cattle in Argentina]. / Primer aislamiento de Leptospira borgpetersenii serovar Hardjo tipo Hardjo Bovis a partir de un caso clínico en Argentina.

We here describe the first isolation and molecular typing of Leptospira borgpetersenii serovar Hardjo Bovis in Argentina, obtained from urine of aborted cows from a breeding herd located in Saladillo, Buenos Aires Province. The abortions occurred in coincidence with important floods with many cows presenting suspicious serological titers and subsequent seroconversion. The percentage of abortions was 3.5% of a herd of 1700 cows and the microorganism was isolated from 7 of the 20 urine samples obtained.

Diagnosis of canine leptospirosis: evaluation of two PCR assays in comparison with the microagglutination test / Diagnóstico de leptospirose canina: avaliação de dois ensaios de PCR em comparação com o teste de microaglutinação

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.(AU)

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.(AU)

Diagnosis of canine leptospirosis: evaluation of two PCR assays in comparison with the microagglutination test / Diagnóstico de leptospirose canina: avaliação de dois ensaios de PCR em comparação com o teste de microaglutinação

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.(AU)

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.(AU)

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

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.

[Differentiation of pathogenic leptospires spp by PCR of ligB gene and sequencing]. / Diferenciación de serovares de leptospiras patógenas mediante PCR del gen ligB y secuenciación.

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.

Intrinsic and extrinsic factors related to pathogen infection in wild small mammals in intensive milk cattle and swine production systems.

BACKGROUND: Understanding the ecological processes that are involved in the transmission of zoonotic pathogens by small mammals may aid adequate and effective management measures. Few attempts have been made to analyze the ecological aspects that influence pathogen infection in small mammals in livestock production systems. We describe the infection of small mammals with Leptospira spp., Brucella spp., Trichinella spp. and Cysticercus fasciolaris and assess the related intrinsic and extrinsic factors in livestock production systems in central Argentina at the small mammal community, population and individual levels. METHODOLOGY/PRINCIPAL FINDINGS: Ten pig farms and eight dairy farms were studied by removal trapping of small mammals from 2008 to 2011. Each farm was sampled seasonally over the course of one year with cage and Sherman live traps. The 505 small mammals captured (14,359 trap-nights) included three introduced murine rodents, four native rodents and two opossums. Leptospira spp., anti-Brucella spp. antibodies and Trichinella spp. were found in the three murine rodents and both opossums. Rattus norvegicus was also infected with C. fasciolaris; Akodon azarae and Oligoryzomys flavescens with Leptospira spp.; anti-Brucella spp. antibodies were found in A. azarae. Two or more pathogens occurred simultaneously on 89% of the farms, and each pathogen was found on at least 50% of the farms. Pathogen infections increased with host abundance. Infection by Leptospira spp. also increased with precipitation and during warm seasons. The occurrence of anti-Brucella spp. antibodies was higher on dairy farms and during the winter and summer. The host abundances limit values, from which farms are expected to be free of the studied pathogens, are reported. CONCLUSIONS/SIGNIFICANCE: Murine rodents maintain pathogens within farms, whereas other native species are likely dispersing pathogens among farms. Hence, we recommend preventing and controlling murines in farm dwellings and isolating farms from their surroundings to avoid contact with other wild mammals.

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

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 Muggia of L. biflexa, a saprophytic species. To the best of our knowledge, this is the first isolation of a Leptospira sp. from cetaceans. Our phenotypic data indicate that strain Manara represents a novel species of the genus Leptospira, for which the name Leptospira brihuegai sp. nov. is proposed.

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

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.

Presencia de anticuerpos contra serovares de Leptospira en Chaetophractus villosus (Mammalia, Dasypodidae), provincia de La Pampa, Argentina / Presence of antibodies against Leptospira serovars in Chaetophractus villosus (Mammalia, Dasypodidae), La Pampa province, Argentina

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 Castellonis­Icterohemorrhagiae and Canicola­Castellonis serovars, both with 4.7%, and Canicola­Hardjo and Castellonis­Canicola­Icterohemorrhagiae 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 Pamp

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

Isolation of Leptospira spp. from a man living in a rural area of the Municipality of Cruz Alta, RS, Brazil / Isolamento de Leptospira spp. em um morador da zona rural do Município de Cruz Alta, RS, Brasil

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.

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.

Isolation of Leptospira spp. from a man living in a rural area of the Municipality of Cruz Alta, RS, Brazil / Isolamento de Leptospira spp. em um morador da zona rural do Município de Cruz Alta, RS, Brasil

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.(AU)

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.(AU)

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...

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 (humans and dogs), Patoc (horses and cattle), Panama (sheep and goats), Patoc, Copenhageni and Australis (wildlife). Four strains of Leptospira were isolated: two in Sobradinho, BA, L. interrogans serogroup Pomona in Cavea aperea and L. interrogans in Euphractus sexcinctus; and two in Sobral, CE, L. interrogans in Cerdocyon thous and L. interrogans serogroup Pomona in Euphractus sexcinctus.(AU)

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 sororreagentes para Leptospira spp. nos dois biomas avaliados (p = 0,063). Os sorovares predominantes nas reações de SAM foram: 1) Pantanal Bratislava (animais selvagens, cães e humanos); Grippotyphosa (equinos e bovinos); 2) Caatinga Copenhageni (humanos e cães), Patoc (equinos e bovinos), Panama (ovinos e caprinos), Patoc, Copenhageni e Australis (animais selvagens). Houve isolamento de quatro estirpes de leptospiras: duas em Sobradinho, BA, L. interrogans sorogrupo Pomona em Cavea aperea e L. interrogans em Euphractus sexcinctus; e duas em Sobral, CE, L. interrogans em Cerdocyon thous e L. interrogans sorogrupo Pomona em Euphractus sexcinctus.(AU)

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

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.

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

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.