A molecular survey reveals high occurrence of co-infections in intensive pork production farms with increased rates of mummified swine fetuses in Southern Brazil / [Pesquisa molecular revela alta ocorrência de coinfecções em granjas de produção intensiva de suínos com altas taxas de mumificação fetal no sul do Brasil]

Neste estudo, 308 amostras de fetos mumificados foram testadas para parvovírus suíno (PPV), circovírus suíno tipos 2 e 3 (PCV2 e PCV3) e leptospiras patogênicas. A idade gestacional no momento da perda gestacional e a frequência da mumificação fetal de acordo com a ordem de parto também foram investigadas. As amostras foram coletadas em granjas comerciais de criação de suínos da região sul do Brasil que apresentassem taxas de mumificação fetal igual ou maiores a 2,5%. Fragmentos de pulmão, rim, fígado e coração de fetos suínos mumificados foram coletados para análise molecular. Resultados da PCR foram classificados de acordo com a região de origem das amostras, tendo Santa Catarina, Paraná e Rio Grande do Sul contabilizado 87 (28,25%), 89 (28,90%) e 132 (42,86%) do total de amostras de fetos suínos mumificados, respectivamente. Coinfecções foram observadas na maioria dos casos e PCV3 foi o agente mais prevalente detectado, encontrado em 298 amostras (96,75%). A maioria das perdas gestacionais foi observada entre 50 e 70 dias de gestação (168; 54,5%) e a mumificação fetal não foi associada à ordem de parto das matrizes. Os achados sugerem que as altas taxas de fetos suínos mumificados na região Sul do Brasil podem ser explicadas pela infecção com esses agentes virais.(AU)

Monitoring of Leptospira in captive turtles by DNA analysis / [Monitoramento de Leptospira em quelônios mantidos em cativeiro pela análise de DNA]

Existem poucos estudos sobre doenças infecciosas em animais silvestres. O objetivo deste estudo foi pesquisar DNA de Leptospira spp. em sangue de tartarugas mantidas em cativeiro, pertencentes ao Bosque Rodrigues Alves (Jardim Zoobotânico da Amazônia). O DNA foi isolado das amostras de sangue coletadas de 148 tartarugas pertencentes a seis espécies diferentes. A reação em cadeia da polimerase (PCR) foi realizada utilizando-se iniciadores específicos para DNA de Leptospira spp. Nenhuma das amostras apresentou resultado positivo para Leptospira spp.(AU)

Spatial distribution of serologically reactive sheep to Leptospira spp. in the northeast region of Brazil

Considering the importance of leptospirosis in sheep farming and public health and the significance of identifying which serogroups circulate in sheep within each region, the objective of this study was to determine the seroprevalence and spatial distribution of the most frequent serogroups causing infection by Leptospira sp. in ovine herds in the Northeast region of Brazil. Blood samples were collected from 4197 sheep from 229 herds in 7 Northeastern States. Sera were analyzed via microscopic agglutination test (MAT). The frequency of seroreactive sheep for Leptospira sp. was 14.06%. The states of Alagoas, Ceará, Paraíba, Piauí, Rio Grande do Norte, and Sergipe, located in the Caatinga biome, had the highest frequencies of serologically reactive sheep, and Maranhão, in the Cerrado biome, had the lowest frequency. The most frequent serogroups were Autumnalis (19.49%), Australis (15.76%) and Serjoe (14.41%). In the states of Ceará, Paraíba, Rio Grande do Norte, and Sergipe, 100% of their municipalities had at least one seroreactive animal. The highest frequencies of seropositive animals were found in the municipalities of União (50%), Passagem (49.06%), Canindé (48.89%), Igaci (28.95%), Gararu (31.2%), Pirapemas (17.5%), and Angicos (16%) located in the states of Piauí, Paraíba, Ceará, Alagoas, Sergipe, Maranhão and Rio Grande do Norte, respectively. The animal-level prevalence (14.06%) obtained in the present study is significant, especially considering the rustic nature of the species and the adverse conditions of the region for the infectious agent. In semi-arid conditions, it has been suggested that perhaps sheep do not seroconvert detectable titers on MAT with a cut-off point of 1:100. It is important to highlight that the ovine population in the Northeast region of Brazil is composed of mixed animals, which have been considered more resistant to infection by Leptospira spp. Also, environmental factors hostile to the survival of the infectious agent in the studied region should be taken into consideration, since they may have influenced the seropositive animal-level prevalence. A noteworthy variation was observed in agglutinin titers, which ranged from 100 to 1,600, where 80.2% of the positive samples had titers ≤ 200. It is important to highlight that more elevated titers (≥ 400) were obtained in all seven states, which may suggest an acute infection caused by a non-adapted serovar, indicating that preventive and control measures focused on possible infection sources for sheep should be adopted. Although some states showed the same serogroups as the most frequent, a variety of serogroups was observed in municipalities, which may indicate different sources of infection, whether interspecies, intraspecies, or via alternative routes of transmission in semi-arid conditions, such as venereal. This indicates that even though sheep are more resistant to infection, they become exposed due to the environment or management conditions. As such, identification, isolation, and treatment of the affected animals are alternative measures recommended for prevention and control of leptospirosis in sheep in the semi-arid region. It is evident that despite the lack of rain observed in the last decade in the Northeast region of Brazil, which prevented the formation of favorable environments for the presence of Leptospira, the infectious agent remains among the sheep, as well as other production and wild animals in the region. Some factors may be contributing to this scenario, such as the fact that sheep farming in the region is = characterized mainly by subsistence systems, where veterinary assistance and adequate sanitary management are absent, thus increasing the possibility of contact with Leptospira. (AU)

Seroprevalence and incidence of Leptospira spp. in domestic dogs in the Southeast region of São Paulo State, Brazil / Soroprevalência e incidência de Leptospira spp. em cães domésticos na região sudeste do estado de São Paulo, Brasil

Leptospirosis is a zoonotic disease caused by Leptospira and domestic dogs can act as host of some serovars. In order to analyze the transmission dynamics in a dog population, with and without immunization, a longitudinal study was carried out with a focus to evaluate antibody response and to identify serovars. Blood samples were collected in three consecutive years (2015 to 2017) from 331, 373 and 347 dogs respectively. The dog seroprevalence in each year was 11%, 7% and 14%, respectively, and the incidence in 2016 was 5% and in 2017, 14%. The most frequent serovars were Cynopteri and Butembo in 2015, Cynopteri, Butembo and Hardjoprajitno in 2016, and Canicola and Butembo in 2017. Dogs can play a role as sentinel animals and hosts of Leptospira serovars. The percentage of seropositive dogs due to vaccination was higher than the previous years without immunization and lower than in previous years for other serovars, which we interpret as evidence for the importance of immunization. These parameters associated with active canine population control are important for prevention and control of leptospirosis not only in dogs but alsoto inhibit the transmission between dogs and humans.(AU)

A leptospirose é uma zoonose causada pelo agente etiológico Leptospira. Cães domésticos atuam como hospedeiro de diversos sorovares deste agente. Com intuito de analisar a dinâmica da leptospirose em uma população canina, com e sem imunização, um estudo longitudinal foi realizado avaliando a resposta sorológica destes animais e identificando seus sorovares. Foram coletadas amostras de 331, 373 e 347 cães em três anos consecutivos (2015 a 2017). As soroprevalências foram de 11%, 7% e 14%, respectivamente, e a incidência em 2016 foi de 5% e em 2017 de 14%. Os sorovares mais frequentes foram Cynopteri e Butembo em 2015, Cynopteri, Butembo e Hardjoprajitno em 2016, e Canicola e Butembo em 2017. Estes cães estão atuando como bio-indicadores da presença de Leptospira na região do estudo, incluindo sorovares zoonóticos, e contribuindo com a sua manutenção no ambiente. A soropositividade para sorovares protegidos pela vacina foi mais alta do que nos anos anteriores à imunização, enquanto para os sorovares não protegidos pela vacina diminuiu, demonstrando a importância da imunização para essa população de cães. Medidas de prevenção e controle para a leptospirose, como imunização e controle populacional canino, são recomendadas no local para inibir a transmissão do agente entre as populações de cães e humanos envolvidas.(AU)

Caracterización epidemiológica de la infección por Leptospira spp. en caballos de trabajo y en personas ocupacionalmente expuestas en seis unidades de la Policía Nacional de Colombia / Epidemiological characterization of Leptospira spp. infection in working horses and in an occupationally exposed population in six Colombian police stations

Resumen Introducción. Los caballos de trabajo de la Policía Nacional tienen un estrecho contacto con sus manejadores y la población en general durante las actividades recreativas y de patrullaje, lo cual puede favorecer la transmisión de la leptospirosis en los caballos y el personal ocupacionalmente expuesto. Objetivo. Caracterizar epidemiológicamente la leptospirosis mediante pruebas de serología, urocultivo y reacción en cadena de la polimerasa (Polymerase Chain Reaction, PCR) en caballos de trabajo y personal con riesgo ocupacional pertenecientes a seis unidades de la Policía Nacional de Colombia. Materiales y métodos. Se evaluaron 153 caballos machos castrados y 123 personas en las seis unidades en los municipios de Manizales, Pereira, Armenia, Ibagué, Tuluá y Cali. Se utilizaron tres formatos estructurados para recabar información y se obtuvieron muestras sanguíneas de las personas y de los caballos, las cuales se procesaron con la prueba de aglutinación microscópica (Macroscopic Agglutination Test, MAT) para 24 serogrupos. Se practicó el examen clínico de los caballos y se obtuvieron muestras de orina para el urocultivo y la PCR convencional. Resultados. La seroprevalencia de Leptospira spp. fue de 3,25 % (n=4) en las personas y de 85 % (n=130) en los caballos. Entre los caballos, los serogrupos Djasiman y Shermani fueron los más prevalentes. El urocultivo fue positivo en el 64,7 % (99/153) de las muestras, en tanto que los análisis de PCR fueron negativos. Se encontró una asociación estadísticamente significativa de la frecuencia de salida de las instalaciones (p=0,009) y la presencia de fauna silvestre (p=0,051) con la infección por el serogrupo Shermani. Conclusión. Las características epidemiológicas de la leptospirosis en los caballos sugieren una presentación endémica de la infección y su papel como reservorios de la bacteria; sin embargo, debe dilucidarse la patogenia de la enfermedad con estudios complementarios.

Abstract Introduction: Police working horses are in close contact with their managers and the general population during recreational and patrol activities, which can favor the transmission of leptospirosis among the horses and the occupationally exposed personnel. Objective. To characterize epidemiologically leptospirosis through serology, urine culture and PCR in working horses and in the occupationally exposed population in six police stations in Colombia. Materials and methods. We tested 153 castrated male horses and 123 people in six police stations in the municipalities of Manizales, Pereira, Armenia, Ibagué, Tuluá, and Cali. Three structured formats were applied and blood samples were obtained from people and horses, which were processed with the Macroscopic Agglutination Test, (MAT) for 24 serogroups. Horses were subject to a clinical examination, and urine samples were obtained for urine culture and conventional PCR. Results. The seroprevalence of human Leptospira spp. was 3.25% (n=4) while in horses it was 85% (n=130). Among the horses, serogroups Djasiman and Shermani were the most prevalent. The urine culture was positive in 64.7% (99/153) of the samples, whereas PCR analyzes were negative. A statistically significant association was found between the frequency of exiting the facilities (p=0.009) and the presence of wildlife (p=0.0051) with the infection by serogroup Shermani. Conclusion. The epidemiological characteristics of leptospirosis in horses suggest an endemic presentation of the infection and its role as reservoirs of the bacteria; however, it is necessary to elucidate the pathogenesis of the disease with complementary studies.

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)

Identification of pathogenic and nonpathogenic Leptospira species of Brazilian isolates by Matrix Assisted Laser Desorption/Ionization and Time Flight mass spectrometry

ABSTRACT Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools™ software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.

Occurrence of serological reactions for serogroup Sejroe (CTG and Prajtino) in female buffalo in the state of Pernambuco, Brazil

ABSTRACT The objective of this study was to evaluate the occurrence of anti-Leptospira spp. antibodies in female buffalo in the state of Pernambuco. A total of 123 female buffalo blood samples were collected from five properties distributed in the state of Pernambuco. The microscopic agglutination test was used to study anti-Leptospira spp. antibodies. The occurrence of anti-Leptospira spp. antibodies was 28.5% (35/123; CI 20.7-37.3%) and on different properties, the occurrence ranged from 28.6% to 80.0%, with 100% of the properties showing animals with positive results. The serovars of the serogroup Sejroe with a higher incidence were Hardjoprajtino (CTG strain, 49.1%) and Hardjo (Prajtino genotype, 43.2%), followed by serogroup Grippotyphosa with the Grippotyphosa serovar (3.9%), serogroup Pomona with the Pomona serovar (1.9%), and the Icterohaemorrhagiae serovar Copenhageni (1.9%). This was the first record of the occurrence of anti-Lepstospira spp. antibodies in female buffalo in the state of Pernambuco. Control measures are necessary to prevent health and economic losses, given that the agent involved affects animal reproduction, triggering drops in conception rates or even clinical cases of abortion.

Development and validation of a modified TaqMan based real-time PCR assay targeting the lipl32 gene for detection of pathogenic Leptospira in canine urine samples

Abstract A modified TaqMan real-time polymerase chain reaction targeting a 138 bp fragment within the lipl32 gene was developed to identify exclusively pathogenic Leptospira spp. in dog urine samples. Thirty-five samples from dogs with suspected clinical leptospirosis and 116 samples from apparently healthy dogs were tested for presence of leptospiral DNA using the TaqMan-based assay. The results were compared with those from a well-established conventional PCR targeting the 16S RNA encoding gene associated with nucleotide sequencing analysis. The overall agreement between the assays was 94.8% (confidence interval [CI] 95% 88-100%). The newly developed assay presented 91.6% (CI 95% 71.5-98.5%) relative sensitivity (22[+] lipl32 PCR/24[+] 16S RNA and sequencing), 100% (CI 95% 96.3-100%) relative specificity and 98.7% accuracy (CI 95% 94.8-100%). The lipl32 assay was able to detect and quantify at least 10 genome equivalents/reaction. DNA extracted from 17 pathogenic Leptospira spp., 8 intermediate/saprophytic strains and 21 different pathogenic microorganisms were also tested using the lipl32 assay, resulting in amplification exclusively for pathogenic leptospiral strains. The results also demonstrated high intra and inter-assay reproducibility (coefficient of variation 1.50 and 1.12, respectively), thereby qualifying the newly developed assay as a highly sensitive, specific and reliable diagnostic tool for leptospiral infection in dogs using urine specimens.

Isolation of saprophytic Leptospira spp. from a selected environmental water source of Argentina / Aislamiento de Leptospira spp. saprofitas desde fuentes de agua en un ambiente seleccionado de 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.

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.

Efficacy of leptospiral commercial vaccines on the protection against an autochtonous strain recovered in Brazil

Abstract In swine and bovines, leptospirosis prevention and control is carried out via vaccination of susceptible animals using bacterins. However, the efficiency of leptospirosis vaccines has been questioned. This work aimed to investigate the potency of five leptospirosis vaccines sold commercially in Brazil, challenging the animals with one autochthonous strain of Leptospira, Canicola serovar, denoted LO4, isolated from swine. The standard protocol was followed, and renal carriers of Leptospira were identified among the surviving animals by culture and PCR. Of the five vaccines tested, only two proved effective. None of the surviving animals was positive by culture; however, one animal was positive by PCR. Three of the five vaccines sold commercially in Brazil for the immunization of swine or bovines failed the test of the efficacy to protect the vaccinated animals following challenge with an autochthonous Leptospira strain, Canicola serovar. The two vaccines provided protection against the renal carrier state in the surviving animals. The criteria used to produce leptospirosis bacterins sold commercially in Brazil must be reviewed. The industry should support researches on leptospiral vaccinology to improve the quality of the present vaccines and discover new immunogenic strains, because it is known that vaccination is one of the most important tools to increase the reproduction rates in livestock.

Prevalência de leptospirose em rebanhos bovinos no Pantanal de Mato Grosso do Sul / Prevalence of leptospirosis in cattle herds in the Pantanal of Mato Grosso do Sul

Foi realizado um estudo epidemiológico da leptospirose em fêmeas acima de 24 meses, provenientes de 246 rebanhos, e 2.766 animais amostrados aleatoriamente nos nove municípios que compõem a região do Pantanal de Mato Grosso do Sul, bem como identificados os fatores de risco associados à doença. As amostras de sangue foram coletadas no período de setembro a novembro de 2009 e examinadas pelo teste de aglutinação microscópica ante uma coleção de 24 antígenos vivos de Leptospira spp., representantes dos sorovares Australis, Bratislava, Autumnalis, Butembo, Castellonis, Batavie, Canicola, Whitcombi, Cynopteri, Grippotyphosa, Hebdomadis, Copenhageni, Icterohaemorrhagiae, Javanica, Panamá, Pomona, Pyrogenes, Hardjo, Wolffi, Shermani, Tarassovi, Sentot, Andamana e Patoc. Adicionalmente, representantes de doze estirpes de leptospiras isoladas no Brasil foram adicionados à coleção de antígenos do teste de soroaglutinação microscópica (SAM). A prevalência aparente foi de 66% e a prevalência real de animais infectados, de 79,80%, com intervalo de confiança (IC) de 95% (78,3-81,3) e 241 rebanhos apresentando pelo menos um animal reagente. Os sorovares mais prováveis foram o Hardjo seguido pelo Wolffi. Os resultados demonstram que a leptospirose bovina continua presente no Pantanal, com alta prevalência tanto em rebanhos quanto em indivíduos, sendo os principais fatores de risco para a doença o tipo de exploração e a raça.(AU)

This is an epidemiological study of leptospirosis in 24 month-old females from 246 herds. Two thousand, seven hundred and sixty six (2,766) animals were randomly sampled in the nine counties comprising the region of Pantanal of Mato Grosso do Sul, Brazil. The risk factors associated with the disease were also identified. Blood samples were collected from September to November 2009 and examined by the microscopic agglutination test (MAT) against a collection of 24 live antigens of Leptospira spp., representatives of serovars Australis, Bratislava, Autumnalis, Butembo, Castellonis, Batavie, Canicola Whitcombi, Cynopteri, Grippotyphosa, Hebdomadis, Copenhageni, Icterohaemorrhagiae, Javanica, Panama, Pomona, Pyrogenes, Hardjo, Wolffi, Shermani, Tarassovi, Sentot, Andamana, and Patoc. Additionally, twelve representatives of Leptospira strains isolated in Brazil were added to the collection of antigens for the microscopic agglutination test (MAT). The apparent prevalence was 66% and the actual prevalence of infected animals was 79.80%, with a confidence interval of 95% (78.3 to 81.3) and 241 herds having at least one reactive animal. The most likely serovars were Hardjo followed by Wolffi. Results show that bovine leptospirosis is still present in Pantanal, with high prevalence both in animals and herds, the main risk factors for the disease being the type of cattle farming and breeding.(AU)

Surto de leptospirose em bezerros criados em resteva de arroz / Leptospirosis outbreak in calves maintained in a rice stubble field

A leptospirose é uma doença infecciosa causada por bactérias do gênero Leptospira, que afeta animais domésticos, selvagens e também humanos. De outubro a novembro de 2014, numa propriedade rural localizada em Glorinha, RS, em que bovinos eram mantidos em resteva de arroz, 13 bezerros manifestaram hemoglobinúria e apatia, nove dos quais morreram em menos de 24 horas após o início dos sinais clínicos. Foram necropsiados quatro bezerros (A, B, C e D). Fragmentos de tecido foram fixados em formalina a 10%. Amostras de rim, fígado e pulmão dos Bezerros B, C e D foram enviadas para análise de PCR para RNA ribossômico 16S e a proteína Lip 32 de Leptospira. No exame macroscópico foram observados mucosas e tecido subcutâneo amarelados, fígado alaranjado, pulmões com múltiplas petéquias, predominantemente nos lóbulos craniais. A cavidade torácica do Bezerro A estava repleta de um líquido vermelho-escuro. À avaliação microscópica foi observada hemorragia acentuada nos pulmões; no fígado havia necrose e vacuolização hepatocelular centrolobular difusa moderada, além de infiltrado linfocítico periportal discreto. Nos rins observou-se nefrite intersticial linfoplasmocítica discreta multifocal. A análise por PCR teve resultado positivo para os Bezerros B e D. O diagnóstico de leptospirose nos bezerros foi baseado nos achados epidemiológicos, clínicos e patológicos, associados ao resultado positivo na PCR. Este estudo demonstra a importância da investigação da doença quando animais jovens são criados em áreas inundadas e têm manifestações clínicas de doença septicêmica aguda.(AU)

Leptospirosis is an infectious disease caused by bacteria of the genus Leptospira, which affect domestic and wild animals, and also humans. From October to November 2014, in a rural property located in Glorinha, RS, where cattle were kept in the rice stubble, thirteen calves presented hemoglobinuria and apathy, nine of which died within less than 24 hours after the onset of clinical signs. Four calves were necropsied (A, B, C and D). Tissue samples were collected in 10% formalin. Samples of kidney, liver and lung from calves B, C and D were sent for PCR analysis for 16S ribosomal RNA and the protein Lip 32 genes of Leptospira. At macroscopic examination jaundiced mucosae and subcutaneous tissue, orange liver, and lungs with multiple petechiae, predominantly in cranial lobes, were observed. The thoracic cavity of calf A was filled with a reddish fluid. At microscopic examination, severe hemorrhage was observed in the lungs; in the liver there was moderate diffuse centrilobular hepatocellular necrosis and vacuolization, in addition to discrete periportal lymphocytic infiltrate. Discrete multifocal lymphoplasmocytic interstitial nephritis was observed in the kidneys. PCR analyzis resulted positive for calves B and D. The diagnosis of leptospirosis in the calves was based on epidemiological, clinical and pathological findings associated with positive PCR analysis. This study demonstrates the importance of investigation of the disease when young bovids are raised in flooded areas and have clinical signs of an acute septicemic disease.(AU)

Human leptospirosis: occurrence of serovars of Leptospira spp. in the state of Minas Gerais, Brazil, from 2008 to 2012

Abstract Background: Leptospirosis is an infectious and acute disease caused by Leptospira spp. that have high epidemic potential. This study verified the main Leptospira spp. serovars detected by MAT from serum of patients with suspicion of leptospirosis from 2008 to 2012 in Minas Gerais State. Methods: The laboratory received sera from 4654 patients. All serum were screened by IgM-ELISA according to the manufacturer's instructions. Each sample reactive or indeterminate were tested against twenty-four serovars of Leptospira by MAT. Results: In this study, 597 patients were classified as reactive on MAT. Only 301 patients were confirmed by laboratory test. It was not possible confirmation by laboratory diagnosis of 296 patients. Among the samples classified as reactive on MAT, 273 patients exhibited titers bigger than 800 for one or more serovars; seroconversion was detected in 28 cases. Percentage of 85.1% of the samples reactive on MAT corresponded to males, 39.4% corresponded to patients aged between 20 and 39 years old. The most common serovars found were Icterohaemorrhagiae, Andamana, Patoc, Tarassovi, Copenhageni, Hardjo and Australis. Concerning the samples that exhibited titers bigger than 800, serovar Icterohaemorrhagiae was also the most common, followed by Copenhageni, Andamana, Patoc, Tarassovi, Grippotyphosa and Canicola. In this study, 40% of the cases occurred to the metropolitan area, state capital and 34 neighboring towns. Conclusion: Our results show the possibly spreading serovars in Minas Gerais State and contribute to knowledge of human leptospirosis, aiming at improving the prevention, control of the disease, as well as the treatment of infected patients.

Culture and molecular identification of microorganisms from Digital Dermatitis lesions in dairy cattle: Leptospira, an unexpected finding / Cultivo e identificação molecular de microrganismos isolados de lesões de dermatite digital em bovinos leiteiros: Leptospira, um achado inesperado

Bovine digital dermatitis (BDD) is an infectious and contagious disease characterized by ulcerative and proliferative lesions affecting the skin on the bulbs of the heel or the interdigital cleft in dairy cattle, often associated with lameness. Evidences on the etiology of BDD indicate that it is multifactorial, involving environmental factors and multiple bacterial colonization. We isolated and identified microorganisms from BDD biopsy samples obtained from five Holstein Friesian and two Jersey cows by cultivation and molecular identification of bacterial isolates using 16S rRNA gene sequence analysis. We identified six bacterial species: Spirochetes as Treponema pedis and Leptospira broomi/L. fainei, L. licerasiae/L. wolffii; Corynebacterium appendicis, Cupriavidus gilardii and Enterococcus casseliflavus/E. gallinarum. It was quite surprising to have isolated and identified Leptospira species in three out of seven cultures, from different individual cows and two different farms. The species identified belong to the intermediate pathogenic clade, which is a group found to cause human and animal disease. Our findings indicate the need to further investigate the association of Leptospira of intermediate pathogenicity with BDD lesions and whether its presence would have any veterinary and medical significance both in Leptospirosis and with the pathogenesis of BDD lesions, especially in tropical countries.(AU)

Dermatite digital bovina (DDB) é uma doença infecciosa, contagiosa, caracterizada por lesões ulcerativas e proliferativas da região dos talões e/ou do espaço interdigital, frequentemente associada com claudicação. Evidências indicam que a etiologia da DDB é multifatorial, envolvendo fatores ambientais e colonização polimicrobiana. Relata-se aqui o isolamento e a identificação bacteriana em amostras de biópsias em lesões de DDB, obtidas de cinco vacas da raça Holandesa e duas da raça Jersey, por meio de cultivo e identificação molecular de isolados, com base na análise de sequências de genes 16S rRNA. São identificadas seis espécies bacterianas: as espiroquetas Treponema pedis e Leptospira broomi/L. fainei, L. licerasiae/L. wolffii; Corynebacterium appendicis, Cupriavidus gilardii e Enterococcus casseliflavus/E. gallinarum. O isolamento e a identificação de espécies de Leptospira surpreenderam, destacando-se sua presença em três dos sete cultivos obtidos em diferentes vacas, de duas fazendas distintas. As espécies identificadas pertencem ao grupo tipificado como de patogenicidade intermediária, causador de doenças em animais e no homem. Os resultados apresentados indicam a necessidade de maiores investigações sobre a associação entre Leptospira de patogenicidade intermediária e a patogênese das lesões DDB, investigando-se sua presença e significado nas medicinas veterinária e humana, especialmente em países tropicais.(AU)

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

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

Occurrence of antibodies against Leptospira spp in dogs from Teresina, Piauí, Brazil / Ocorrência de anticorpos contra Leptospira spp em cães de Teresina, Piauí, Brasil

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

A leptospirose é uma doença sistêmica causada por bactéria Leptospira spp. que afeta seres humanos, animais domésticos e selvagens. O presente trabalho investigou a presença de anticorpos anti-Leptospira spp. na população canina da cidade de Teresina-Piauí, e os respectivos sorovares predominantes. Amostras de sangue de 425 cães foram coletadas no Centro de Controle de Zoonoses de Teresina, no período de julho de 2010 a janeiro de 2012, e submetidas à prova de Soroaglutinação Microscópica (SAM). Este estudo encontrou uma taxa de infecção média com 17,41% (IC 95%; 13,8 ­ 21,0) e 11 sorovares reagentes, sendo os prevalentes Canicola (18,9%), Autumnalis (16,2%), Icterohaemorrhagiae (12,1%) e Butembo (12,1%). As questões levantadas neste estudo indicam a ocorrência de infecção por Leptospira spp em cães da cidade de Teresina-Piauí, Brasil.(AU)

Leptospirosis infantil en pacientes con síndrome febril en la Región de Urabá, Colombia / Childhood leptospirosis in patients with febrile syndrome in the Region of Urabá, Colombia

RESUMEN El objetivo del estudio fue describir los casos de leptospirosis infantil en cuatro municipios de la región de Urabá (Colombia) y factores relacionados con severidad de los cuadros clínicos. Se realizó un estudio transversal analítico retrospectivo de 74 pacientes menores de 17 años con síndrome febril por leptospirosis entre 2010 y 2012. El mayor número de casos correspondió a niños de áreas urbanas (57/74; 77%), entre 10 y 14 años (33/74; 44,5%). La respuesta por MAT más frecuente fue a los serogrupos Grippothyposa y Bratislava, asociados a reservorios silvestres y pecuarios respectivamente. Se encontró asociación entre la presencia de animales domésticos dentro de la vivienda (OR 2,73; IC95% 0,98-7,60; p=0,05) y la severidad de la enfermedad. El riesgo de leptospirosis infantil severa se relaciona con la tenencia de animales domésticos dentro de la vivienda.

ABSTRACT The aim of the study was to discover cases of childhood leptospirosis in four municipalities in the region of Urabá (Colombia) and the factors related to the severity of clinical manifestations. A retrospective cross-sectional study assessed 74 children aged younger than 17 years with febrile syndrome due to leptospirosis between 2010 and 2012. The majority of cases were in children from urban areas (57/74; 77%), between 10 and 14 years of age (33/74; 44.5%). The microscopic agglutination test (MAT) revealed that the most frequent serogroups were Grippotyphosa and Bratislava, which are associated with wildlife and livestock reservoirs, respectively. There was a association between the presence of household pets and the severity of the disease (odds ratio [OR] 2.73; 95% confidence interval [CI] = 0.98-7.60; p=0.05). The risk of severe childhood leptospirosis is linked to having household pets.

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

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