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.

RESUMO: 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.

ABSTRACT: 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.

RESUMO: O objetivo do trabalho foi determinar a prevalência de leptospirose e brucelose por Brucella canis e determinar os fatores de risco associados com a positividade em cães da Estância Turística de Ibiúna, estado de São Paulo, Brasil. Foram examinados 570 animais distribuídos em 4 regiões nos 48 bairros do município, no período de setembro de 2007 a março de 2008. O diagnóstico sorológico da leptospirose foi efetuado com o teste de soroaglutinação microscópica (SAM), e para o diagnóstico de brucelose foi realizado hemocultivo. Dos 570 animais examinados, 187 (32,8%; IC95% 28,9 - 36,8) foram soropositivos para leptospirose, com predomínio de reações para os sorovares Pyrogenes, Autumnalis e Canicola, e 6 (1,05%; IC95% 0,4 - 2,2) foram positivos para brucelose. A variável atividade sexual (OR = 1,73) foi identificada como fator de risco associado à positividade para leptospirose, e o manejo do tipo solto foi considerado fator de risco tanto para leptospirose (OR = 1,96) quanto para brucelose (OR = 10,85). Conclui-se que a leptospirose e a brucelose estão presentes em cães da Estância Turística de Ibiúna, São Paulo, e que a atividade sexual e o acesso irrestrito à rua são condições associadas com a prevalência das infecções.

ABSTRACT: The aim of this survey was to determine the prevalence of leptospirosis and brucellosis due to Brucella canis and to determine the risk factors associated with positivity in dogs of the Tourist Resort of Ibiúna, State of São Paulo, Brazil. A total of 570 blood samples were collected from dogs from 4 regions of 48 districts of the county of Ibiúna during the period of September 2007 to March 2008. Serological diagnosis of leptospirosis was performed with the microscopic agglutination test (MAT), and blood culture was used for the diagnosis of brucellosis. Of the 570 dogs used 187 (32.8%; 95%CI 28.9 - 36.8) were seropositive to leptospirosis, with predominance of reactions to serovars Pyrogenes, Autumnalis and Canicola, and 6 (1.05%; 95%CI 0.4 - 2.2) were positive to brucellosis. Variable sexual activity (OR = 1.73) was identified as risk factor associated with the positivity to leptospirosis, and free access to street was considered risk factor for both leptospirosis (OR = 1.96) and brucellosis (OR = 10.85). It is concluded that leptospirosis and brucellosis are present in dogs of the Tourist Resort of Ibiúna, State of São Paulo, and sexual activity and free access to street are conditions associated with the prevalence of infections.

Este estudo teve como objetivo avaliar a exposição de onças-pintadas de vida livre (Panthera onca) para Leptospira spp. e Brucella abortus em duas unidades de conservação no Pantanal de Mato Grosso, Brasil. A presença de anticorpos em amostras de sangue de onze onças foi investigada utilizando antígenos autóctones isoladas no Brasil adicionais a coleção de antígenos de referência aplicada usualmente ao diagnóstico da leptospirose pelo teste de soroaglutinação microscópica (MAT). Para os anticorpos de B. abortus, foi utilizado o teste de Rosa Bengala. Duas onças-pintadas (18,2%) foram reagentes para Leptospira spp. e o sorotipo considerado como o mais provável pela infecção em ambos os animais foi um isolado brasileiro do sorovar Canicola (L01). Todas as onças-pintadas foram soronegativas para B. abortus. Estes dados indicam que a inclusão de antígenos autóctones em estudos sorológicos pode aumentar significativamente o número de animais reativos, assim como modificar a caracterização do sorotipo mais prevalente.

 This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection.

We investigated the existence of cross-protection between two anti-leptospirosis monovalent experimental bacterins produced with two strains of Leptospira serogroup Pomona: Fromm strain of serovar Kennewicky, isolated from pigs in the United States, and strain GR6 of serovar Pomona isolated from pigs in Brazil. Both were added of aluminum hydroxide as an adjuvant. Experimental bacterins were tested with the hamster potency test in order to assess protection provided against the disease and against the establishment of kidney infection. Controls were polyvalent commercial vaccine produced with Leptospira strains isolated outside Brazil, which included a representative of Pomona serovar, or Sorensen solution added of aluminum hydroxide adjuvant. The challenge was performed with cross-strains of serogroup Pomona tested in accordance with international standards established for the potency test. After 21 days of the challenge, survivors were killed to evaluate the condition of Leptospira renal carrier. Experimental bacterins protected hamsters against homologous and heterologous strains, demonstrating the existence of cross-protection. The commercial vaccine protected the hamsters challenged with both strains, but there was a high proportion of animals diagnosed as renal carriers when the challenge was performed with strain GR6, isolated from pigs in Brazil.

Leptospirosis is a zoonotic disease affecting most mammals and is distributed throughout the world. Several species of domestic and wild animals may act as reservoirs for this disease. The purpose of this study was to assess the exposure of free-ranging wild carnivores, horses and domestic dogs on a private reserve located in the northern Pantanal (Brazil) and the surrounding areas to Leptospira spp from 2002-2006, 75 free-ranging wild carnivores were captured in the Pantanal and serum samples were collected. In addition, samples from 103 domestic dogs and 23 horses in the region were collected. Serum samples were tested for the presence of Leptospira antibodies using the microscopic agglutination test. Thirty-two wild carnivores (42.7%) were considered positive with titres > 100, and 18 domestic dogs (17.5%) and 20 horses (74.1%) were also found to be positive. Our study showed that horses, dogs and several species of free-ranging wild carnivores have been exposed to Leptospira spp in the Pantanal, suggesting that the peculiar characteristics of this biome, such as high temperatures and an extended period of flooding, may favour bacterial persistence and transmission. In this region, wild carnivores and horses seem to be important hosts for the epidemiology of Leptospira species.

A Leptospirose é uma importante zoonose que causa transtornos reprodutivos em suínos. O isolamento de leptospiras de fetos abortados, natimortos e leitões fracos foi obtido em várias ocasiões, porém, a bactéria nunca foi isolada de leitões aparentemente saudáveis nascidos de matrizes com infecção subclínica. Seis matrizes da raça Landrace com data de serviço conhecida e gravidez confirmada por ultrasonografia foram infectadas entre 76 e 90 dias de gestação por via intravenosa com 5 ml de inóculo de Leptospira interrogans sorovar Canicola, e uma semana após o parto, pelo menos um leitão por matriz foi eutanaziado e foram colhidas amostras de fígado, rins, pulmões, coração, baço e conteúdo gástrico para exame por PCR. Transtornos reprodutivos ou qualquer sinal clínico de infecção não foram observados nas matrizes inoculadas. Os leitões nascidos destes animais não apresentaram sinais clínicos ou lesões macroscópicas que poderiam ser atribuídos à leptospirose. Todas as matrizes inoculadas apresentaram anticorpos anti-leptospiras no teste de soroaglutinação microscópica (MAT) nas amostras de soros colhidas após a inoculação, e não foram encontradas leptospiras na urina como também não foram detectadas pela PCR aplicada neste material, porém, a PCR realizada em rins e fígado de uma matriz eutanaziada apresentou resultado positivo. Dos 12 leitões eutanaziados, 10 (83,3%) apresentaram resultados positivos na PCR em pelo menos uma das amostras de rim, fígado, coração, baço, pulmão e conteúdo gástrico. O presente estudo relata a transmissão vertical da infecção e a detecção de leptospiras em leitões clinicamente saudáveis nascidos de matrizes infectadas experimentalmente, o que é importante do ponto de vista epidemiológico, pois a manutenção de animais com infecção subclínica pode permitir a persistência da bactéria no rebanho, expondo outros animais ao risco de infecção.

Leptospirosis is an important zoonosis that causes reproductive disorders in swine. The isolation of leptospires from aborted fetuses, stillbirths and weak piglets was obtained in several occasions, however, the bacteria was never isolated from apparently healthy piglets born from apparently healthy infected dams. Six sows of the Landrace breed with a known date of service and pregnancy confirmed by ultrasonography were infected intravenously with 5ml of Leptospira interrogans serovar Canicola inoculum between 76 and 90 days of gestation, and one week after farrowing, at least one piglet per sow was euthanized and samples of liver, kidneys, lungs, heart, spleen and gastric content were taken and examined by PCR. Reproductive disorders or any clinical sign of infection were not observed in the inoculated sows. The piglets born from these animals presented no clinical signs or macroscopic lesions that could be attributed to leptospirosis. All inoculated sows presented anti-leptospires antibodies by microscopic serum-agglutination test (MAT) in the postinoculation serum samples and leptospires were not found in the urine as well as were not detected by PCR applied in this material, however, PCR accomplished in kidneys and liver from a euthanized sow presented positive results. Of the total of 12 euthanized piglets, 10 (83.3%) presented positive results by PCR in at least one of the kidney, liver, heart, spleen, lung and gastric content samples. The present study reports the vertical transmission of the infection and the detection of leptospires in clinically healthy piglets born from experimentally infected sows, which is important of the epidemiological point of view as the maintenance of clinically healthy infected animals may allow the persistence of the bacteria in the herd, exposing other animals to the risk of infection.

O presente trabalho teve como objetivo investigar a prevalência de leptospirose em cães errantes da cidade de Patos, Estado da Paraíba, Brasil. O experimento foi conduzido durante o período de fevereiro a abril de 2003, com a colheita de 130 amostras de soro canino. O diagnóstico da leptospirose foi determinado pela técnica de soroaglutinação microscópica, utilizando-se uma coleção de 22 variantes sorológicas. Para a caracterização dos sorovares mais prováveis, levou-se em conta a titulação e a freqüência. A prevalência encontrada foi de 20%, com maior frequência dos sorovares autumnalis (20%), pomona (17,5%), grippotyphosa (10%) e patoc (10%).

This work aimed to survey the prevalence of leptospirosis in stray dogs from Patos city, state of Paraíba, Brazil. This study was held during the period of February to April of 2003, when 130 serum samples were collected. The diagnostic method run for leptospirosis was the microscopic agglutination test, using a batch of 22 leptospiral serovars. The most prevalent serovars were found crossing the results of frequency and titer of agglutinins. The results obtained indicated a prevalence of 20% and most frequent reactant serovars were autumnalis (20%), pomona (17,5%), grippotyphosa (10%) e patoc (10%).

Three leptospiral bacterins, produced with different serovars of Serogroup Sejroe, namely the hardjo (bacterin A), wolffi (bacterin B) and guaricura (bacterin C), were evaluated in male hamsters (Mesocricetus auratus) by comparing the agglutinating and neutralizing antibodies titers using microscopic agglutination (MAT) and in vitro growth inhibition (GIT) tests. The immunization schedule was based on two 1.0 mL doses of non-diluted formalininactivated whole culture bacterin given through subcutaneous route with 10-day interval. The challenge was performed ten days after the second vaccine dose, when the animals were inoculated with 0.2 mL of non-inactivated cultures of each serovar through intraperitoneal route. On the 21st post-challenge day (PCD), all animals were bled and their sera were joined in pools (n=8) and tested by MAT and GIT. All vaccinated and control animals presented no clinical signs of leptospirosis after the challenge, but the serovar guaricura was isolated from the kidneys of control animals on the 21st PCD. The MAT results showed cross agglutinins between serovars hardjo and wolffi, and between wolffi and guaricura. The GIT results revealed the presence of cross neutralizing antibodies between serovars wolffi or guaricura against hardjo, wolffi and guaricura. It was found that the tested strain of serovar hardjo did not produce detectable levels of neutralizing antibodies, indicating its poor immunogenicity.