RESUMO

Leptospirosis is a zoonosis that can infect buffaloes and other mammalians, and it's caused by Gram-negative bacteria of the genus Leptospira spp. The infection occurs through contact with urine or a contaminated environment with the mucous membranes of the animals, and the disease has two phases: leptospiremia and leptospiruria. The objective of this study was to determine the frequency of leptospirosis in buffaloes in the state of Paraná, Brazil, as well as the main serovars present in this area. A total of 276 blood samples were collected from buffaloes from 18 properties located in different cities and mesoregions of Paraná state. A microscopic agglutination test (MAT) was performed to detect the presence of anti-Leptospira spp. antibodies and to identify its serovars. The prevalence was 77.17% (213/276) of buffaloes seropositive for at least one serovar. The most frequent serovar was Icterohaemorrhagiae, followed by the Pomona serovar and serovar Hardjo. This study seems to be the first to determine the frequency of buffaloes with leptospirosis in the state of Paraná. Prevention and control measures are important to control the disease in herds.

Leptospirose é uma zoonose que infecta búfalos e outros mamíferos e é causada por bactérias Gram-negativas do gênero Leptospira spp. A infecção ocorre por meio do contato com a urina ou ambiente contaminado com as membranas mucosas dos animais e a doença possui duas fases: leptospiremia e leptospirúria. O objetivo deste trabalho foi determinar a frequência da leptospirose em bubalinos provenientes do estado do Paraná, assim como determinar os principais sorovares presentes no estado. Um total de 276 amostras de sangue foram coletados de búfalos provenientes de 18 propriedades localizadas em 14 municípios de diferentes mesorregiões do estado do Paraná. O teste de soroaglutinação microscópica (SAM) foi realizado para detecção da presença de anticorpos anti-Leptospira spp. e identificação dos respectivos sorovares. A prevalência de búfalos soropositivos para ao menos um sorovar foi de 77,17% (213/276). O sorovar mais frequente identificado nestes búfalos foi o Icterohaemorrhagiae, seguido pelos sorovares Pomona e Hardjo. Este é o primeiro estudo a determinar a frequência de búfalos com leptospirose no estado do Paraná. Medidas de controle e prevenção são importantes para o controle do agente nos rebanhos.

Assuntos

RESUMO

The first experimental infections with Leptospira in ruminants were conducted in the 1950s, primarily assessed the pathogenesis caused by serovar Pomona in cows. Throughout the decades, experimental infections have also demonstrated the clinical aspects of the infection by other strains, mainly Hardjo. Despite the important outcomes observed in experimental infections in ruminants, there is still a large discrepancy regarding the ideal dose, route, strain, model species or animal age that should be used to reproduce the acute and chronic leptospirosis in ruminants. In this context, the present study aimed to review the historical processes involved on the experimental leptospiral infection in ruminants. The inclusion criteria were papers that clearly described inoculation route, strain, dose, clinical signs and animal age. Overall, 37 experiments were noted. The most frequently reported clinical signs were fever, prostration, hematuria and death, with the majority of them occurring in young animals infected by incidental strains. Regarding reproductive problems, they occurred in the majority of the experiments and were also more related to incidental strains. In this context, abortions, retained placenta and weak fetuses were the most frequent symptoms. Noteworthy that although the mechanisms of the clinical acute disease either systemic or reproductive, is reasonably well understood, the physiopathology involved on reproductive problems due to the silent chronic infection is less discussed and remains to be elucidated. In this context, it is evident the need for studies focused on the genital infection and reproductive aspects of leptospiral infection in ruminants.

Assuntos

RESUMO

Leptospirosis is a zoonosis, caused by pathogenic spirochetes of the genus Leptospira. Although cattle are usually the maintenance hosts of serovar Hardjo, Pomona is the most frequent serovar circulating in Argentina. The understanding of bovine innate immune response and the virulence of this serovar is important for future control measures. This work compares infection of bovine macrophages with the virulent L. interrogans sv Pomona strain AKRFB (P1) and its attenuated counterpart (P19). First, we confirmed attenuation in the hamster model. Mortality and lung hemorrhages occurred after P1 inoculation, while the survival rate was 100% in P19-infected animals. Cells infected with both strains showed statistically upregulated gene expression of pro-inflammatory cytokines, IL-1ß, IL-6 and TNFα. The level of expression of anti-inflammatory cytokine IL-10 was statistically different between strains. Increased expression of IL-10 was observed only in P1-infected cells. For the first time, we describe macrophages extracellular traps induced by infection of bovine macrophages (bMETs) with both, the virulent and attenuated Leptospira interrogans Pomona strains. P1 was found higher internalized when the phagocytosis was inhibited, suggesting a cell entrance of this strain also by an independent-phagocytosis pathway. Furthermore, P1 was higher colocalized with acidic and late endosomal compartments compared with P19. This data emphasizes the importance to deepen in Leptospira bovine macrophages particular invasion mechanisms and, furthermore, underline the value of studying the main hosts.

Assuntos

RESUMO

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.

Assuntos

RESUMO

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.

Assuntos

RESUMO

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.

Assuntos

RESUMO

With the aim of isolating Leptospira spp., blood serum, kidney, liver and genital tract of 137 female swine (40 sows and 97 gilts) and also urine samples from 22 sows were collected in a slaughterhouse in the State of São Paulo, from April 2003 to August 2004. Four isolates were obtained from animals that presented microagglutination test (MAT) titers > 100 for the serovar Pomona and one was obtained from an animal negative by MAT in which Leptospira was isolated from the liver and reproductive tract. The presence of leptospiral DNA was investigated by PCR, and positive results were found in kidneys of 11 females, liver of two, genital tract of two and urine of one of them. Nephrosis, interstitial multifocal nephritis, moderate to severe changing, hyalines cylinders and hemorrhagic focuses, hepatic and uterine horns congestion were histological lesions observed in higher frequency in animals positive for leptospira. The silver impregnation (Warthin Starry) confirmed the presence of spirochetes in renal tubules of four females with positive leptospira cultures from kidneys. The serogroup of the five isolates was identified as Pomona by cross agglutination with reference polyclonal antibodies. Molecular characterization of the isolates was carried out by variable-number tandem-repeats analysis. All the isolates revealed a pattern distinct from the L. interrogans Pomona type strain, but identical to a previously identified pattern from strains isolated in Argentina belonging to serovar Pomona.

Amostras de soro sanguíneo, rim, fígado e trato genital de 137 fêmeas suínas (40 matrizes e 97 marrãs) e de urina de 22 matrizes foram colhidas em abatedouro no Estado de São Paulo, no período de abril de 2003 a agosto de 2004 tendo como objetivo o isolamento de Leptospira spp. Quatro estirpes foram isoladas de animais que apresentaram títulos, no teste de soroaglutinação microscópica (SAM) > 100, para o sorovar Pomona e de um animal, não reagente na SAM, em que houve isolamento de leptospiras do fígado e aparelho reprodutor. A presença do DNA de leptospira foi investigada pela técnica da PCR e foram observados resultados positivos nos rins de 11 fêmeas, no fígado de duas, no aparelho reprodutor de duas e na urina de uma delas. Nefrose, nefrite intersticial multifocal variando de moderada a severa, cilindros hialinos e focos hemorrágicos, congestão hepática e de cornos uterinos foram lesões histológicas evidenciadas com freqüência mais alta em animais positivos para leptospira. A impregnação argêntica (Warthin Starry) confirmou a presença de espiroquetas nos túbulos renais das quatro fêmeas onde houve cultura positiva para leptospiras dos rins. O sorogrupo dos cinco isolados foi identificado como Pomona pela técnica de aglutinação cruzada com anticorpos policlonais de referência. A caracterização molecular dos isolados foi realizada pela análise do número variável de repetições em tandem (VNTR). Os mesmos revelaram um padrão distinto da estirpe padrão de L. interrogans sorovar Pomona, porém idêntico a um padrão previamente identificado em estirpes isoladas na Argentina, pertencentes ao sorovar Pomona.

Assuntos

RESUMO

With the aim of isolating Leptospira spp., blood serum, kidney, liver and genital tract of 137 female swine (40 sows and 97 gilts) and also urine samples from 22 sows were collected in a slaughterhouse in the State of São Paulo, from April 2003 to August 2004. Four isolates were obtained from animals that presented microagglutination test (MAT) titers ≥ 100 for the serovar Pomona and one was obtained from an animal negative by MAT in which Leptospira was isolated from the liver and reproductive tract. The presence of leptospiral DNA was investigated by PCR, and positive results were found in kidneys of 11 females, liver of two, genital tract of two and urine of one of them. Nephrosis, interstitial multifocal nephritis, moderate to severe changing, hyalines cylinders and hemorrhagic focuses, hepatic and uterine horns congestion were histological lesions observed in higher frequency in animals positive for leptospira. The silver impregnation (Warthin Starry) confirmed the presence of spirochetes in renal tubules of four females with positive leptospira cultures from kidneys. The serogroup of the five isolates was identified as Pomona by cross agglutination with reference polyclonal antibodies. Molecular characterization of the isolates was carried out by variable-number tandem-repeats analysis. All the isolates revealed a pattern distinct from the L. interrogans Pomona type strain, but identical to a previously identified pattern from strains isolated in Argentina belonging to serovar Pomona.

RESUMO

With the aim of isolating Leptospira spp., blood serum, kidney, liver and genital tract of 137 female swine (40 sows and 97 gilts) and also urine samples from 22 sows were collected in a slaughterhouse in the State of São Paulo, from April 2003 to August 2004. Four isolates were obtained from animals that presented microagglutination test (MAT) titers > 100 for the serovar Pomona and one was obtained from an animal negative by MAT in which Leptospira was isolated from the liver and reproductive tract. The presence of leptospiral DNA was investigated by PCR, and positive results were found in kidneys of 11 females, liver of two, genital tract of two and urine of one of them. Nephrosis, interstitial multifocal nephritis, moderate to severe changing, hyalines cylinders and hemorrhagic focuses, hepatic and uterine horns congestion were histological lesions observed in higher frequency in animals positive for leptospira. The silver impregnation (Warthin Starry) confirmed the presence of spirochetes in renal tubules of four females with positive leptospira cultures from kidneys. The serogroup of the five isolates was identified as Pomona by cross agglutination with reference polyclonal antibodies. Molecular characterization of the isolates was carried out by variable-number tandem-repeats analysis. All the isolates revealed a pattern distinct from the L. interrogans Pomona type strain, but identical to a previously identified pattern from strains isolated in Argentina belonging to serovar Pomona.

Amostras de soro sanguíneo, rim, fígado e trato genital de 137 fêmeas suínas (40 matrizes e 97 marrãs) e de urina de 22 matrizes foram colhidas em abatedouro no Estado de São Paulo, no período de abril de 2003 a agosto de 2004 tendo como objetivo o isolamento de Leptospira spp. Quatro estirpes foram isoladas de animais que apresentaram títulos, no teste de soroaglutinação microscópica (SAM) > 100, para o sorovar Pomona e de um animal, não reagente na SAM, em que houve isolamento de leptospiras do fígado e aparelho reprodutor. A presença do DNA de leptospira foi investigada pela técnica da PCR e foram observados resultados positivos nos rins de 11 fêmeas, no fígado de duas, no aparelho reprodutor de duas e na urina de uma delas. Nefrose, nefrite intersticial multifocal variando de moderada a severa, cilindros hialinos e focos hemorrágicos, congestão hepática e de cornos uterinos foram lesões histológicas evidenciadas com freqüência mais alta em animais positivos para leptospira. A impregnação argêntica (Warthin Starry) confirmou a presença de espiroquetas nos túbulos renais das quatro fêmeas onde houve cultura positiva para leptospiras dos rins. O sorogrupo dos cinco isolados foi identificado como Pomona pela técnica de aglutinação cruzada com anticorpos policlonais de referência. A caracterização molecular dos isolados foi realizada pela análise do número variável de repetições em tandem (VNTR). Os mesmos revelaram um padrão distinto da estirpe padrão de L. interrogans sorovar Pomona, porém idêntico a um padrão previamente identificado em estirpes isoladas na Argentina, pertencentes ao sorovar Pomona.

RESUMO

O presente trabalho teve por objetivo identificar a presença da Leptospira interrogans sorovar pomona em camundongos geneticamente selecionados para a alta e baixa resposta a anticorpos. Todos os animais foram submetidos ao isolamento bacteriano, imunohistoquímica (imunoperoxidase) em cortes de tecido renal e coloração através da hematoxilina-eosina. A técnica de imunoperoxidase apresentou-se pouco mais sensível em relação ao cultivo, entretanto, ambas foram bons parâmetros de identificação do agente. Presença de lesões renais mais intensas ocorreram em períodos em que houve maior número de bactérias isoladas em meio de cultivo. Camundongos da linhagem HIV-A conseguiram eliminar as leptospiras com maior eficiência e rapidez em relação as linhagem LIV-A, entretanto o estudo demonstrou que ambas linhagens da seleção IV-A foram eficientes em controlar o processo infeccioso.

The present work had the objective of identifying the presence of Leptospira interrogans serovar pomona in mice that had been genetically selected for high and low response to antibodies. All the animals were subjected to bacterial isolation, immunohistochemical analysis (immunoperoxidase) in renal tissue sections and hematoxylin-eosin staining. The immunoperoxidase technique was little more sensitive than culturing, but both were good parameters for agent identification. More severe renal lesions were present at times when there were greater numbers of bacteria isolated in culture medium. Mice of the lineage HIV-A were able to eliminate the Leptospira more efficiently and faster than the lineage LIV-A could. However, the study demonstrated that both lineages of the IV-A selection were efficient in controlling the infectious process.

Assuntos

RESUMO

Foi investigada a influência da via de inoculação sobre o estabelecimento e a evolução da leptospirose em hamsters (Mesocricetus auratus) experimentalmente infectados com Leptospira interrogans sorovar pomona. As vias de inoculação ensaiadas foram: intraperitoneal, subcutânea, oral, conjuntival e escarificação cutânea. O inóculo infeccioso foi constituído por uma cultura em meio de Fletcher, com 20 a 30 leptospiras por campo microscópio no aumento de 200 vezes. Os animais controle foram inoculados apenas com meio de Fletcher. Foram colhidas amostras de soro sanguíneo e fragmentos de rins na fase agônica da doença ou no 21º dia pós-infecção, quando todos os animais foram sacrificados. Para a pesquisa de leptospiras, foi feito o exame direto com microscopia óptica em campo escuro e cultivo em meio de Fletcher, pela técnica das diluições seriadas. A detecção de aglutininas anti-leptospiras foi realizada pela técnica de soroaglutinação microscópica. A instalação e evolução da leptospirose foram afetadas pela via de inoculação. A via oral foi a menos efetiva em estabelecer a infecção. Não foi observada associação estatística entre a freqüência de portadores e a via de inoculação. (AU)

The influence of the route of inoculation on the development of the leptospiral infection was investigated in 60 hamsters (Mesocricetus auratos) experimentally infected with Leptospira interrogans serovar pomona. The routes of inoculation were intraperitoneal, subcutaneous, oral, conjuntival and scrapped skin. All animals received the leptospiral inoculum cultivated in Fletcher's medium, with 20 to 30 leptospires per field under 200x magnification. The controls were inoculated only with Fletcher's medium. Samples of blood sera and the kidney fragments were collected in the agonic stage of the disease or at 21st experimental day, when all survivors were killed. The antibodies detection was done by the microscopic agglutination test and the bacteria detection by the dark field microscopic examination and culture in Fletcher's medium, by the tenfold dilutions technique. The presence and development of disease was affected by the route of infection. The oral route was the less effective for promoting the disease. There was no association between the frequency of carrier state and the route of infection. (AU)

Assuntos

RESUMO

In order to comparatively study the methods of in vitro" isolation of leptospires, the Pasteur"s pipette and the serial dilution techniques were performed on the renal tissue samples of 60 hamsters experimentally infected either by two strains of Leptospira interrogans serovar pomona, using the Fletcher"s medium added with 100 micrograms /mL of 5-fluor-uracil or 2.5 micrograms /mL of neomycin sulfate. The hamsters were divided into groups A and B, the 30 animals of group A were inoculated with a strain of Leptospira that was passed 39 times in hamsters, and the remaining 30 animals, inoculated with a field isolate of Leptospira with only 2 passages in hamsters. The hamsters died three to seven days after the inoculation, or sacrificed in agonic state, and the presence of leptospires in the renal tissue was confirmed by at least one of the three microscopic examination procedures: direct examination of the renal tissue suspension with dark field microscopy, Levaditis histologic staining and the direct fluorescent  antibody technique. The specimens taken from the group A were found to be richer in the number of leptospires than the materials of group B. The results of "in vitro" cultivation of leptospires varied according to the strain of leptospires tested. For the group A, the serial dilution technique was the most sensitive, combined with the use of 5-fluor-uracil. For the g

Com o objetivo de estudar comparativamente os métodos de isolamento de leptospiras, através das técnicas da pipeta Pasteur e a das diluições seriadas, em meio de cultura de Fletcher tratado com os antimicrobianos 5-fluor-uracil (5-FU) na concentração de 100 microgramas por mililitro ou o sulfato de neomicina na concentração de 2,5 microgramas por mililitro, foram examinados 60 pares de rins obtidos de hamsters jovens, experimentalmente inocinoculados com estirpes virulentas de Leptospira interrogans sorotipo pomona. Os trinta animais do grupo A" foram inoculados com uma estirpe que apresentava 33 passagens em hamsters. 0 grupo " B foi constituído por outrs trinta animais inoculados com a estirpe que contemplava apenas duas passagens em hamsters. Decorridos três a sete dias da inoculação, todos os animais vieram a óbito ou foram sacrificados em estado agânico. Nesta oportunidade, a existência de leptospiras em todos os rins processados foi confirmada por pelo menos um de três métodos utilizados para a visualização do agente: exame direto em microscopia de campo escuro, coloração de Levaditi, e a reação de imunofluorescência direta. Segundo estas técnicas, os materiais provenientes do grupo "A" foram caracterizados como apresentando maior riqueza em leptospiras do que aqueles originários do grup o B". Os resultados das provas culturais variaram conforme a estirpe de Leptospira

RESUMO

In order to comparatively study the methods of in vitro" isolation of leptospires, the Pasteur"s pipette and the serial dilution techniques were performed on the renal tissue samples of 60 hamsters experimentally infected either by two strains of Leptospira interrogans serovar pomona, using the Fletcher"s medium added with 100 micrograms /mL of 5-fluor-uracil or 2.5 micrograms /mL of neomycin sulfate. The hamsters were divided into groups A and B, the 30 animals of group A were inoculated with a strain of Leptospira that was passed 39 times in hamsters, and the remaining 30 animals, inoculated with a field isolate of Leptospira with only 2 passages in hamsters. The hamsters died three to seven days after the inoculation, or sacrificed in agonic state, and the presence of leptospires in the renal tissue was confirmed by at least one of the three microscopic examination procedures: direct examination of the renal tissue suspension with dark field microscopy, Levaditis histologic staining and the direct fluorescent  antibody technique. The specimens taken from the group A were found to be richer in the number of leptospires than the materials of group B. The results of "in vitro" cultivation of leptospires varied according to the strain of leptospires tested. For the group A, the serial dilution technique was the most sensitive, combined with the use of 5-fluor-uracil. For the g

Com o objetivo de estudar comparativamente os métodos de isolamento de leptospiras, através das técnicas da pipeta Pasteur e a das diluições seriadas, em meio de cultura de Fletcher tratado com os antimicrobianos 5-fluor-uracil (5-FU) na concentração de 100 microgramas por mililitro ou o sulfato de neomicina na concentração de 2,5 microgramas por mililitro, foram examinados 60 pares de rins obtidos de hamsters jovens, experimentalmente inocinoculados com estirpes virulentas de Leptospira interrogans sorotipo pomona. Os trinta animais do grupo A" foram inoculados com uma estirpe que apresentava 33 passagens em hamsters. 0 grupo " B foi constituído por outrs trinta animais inoculados com a estirpe que contemplava apenas duas passagens em hamsters. Decorridos três a sete dias da inoculação, todos os animais vieram a óbito ou foram sacrificados em estado agânico. Nesta oportunidade, a existência de leptospiras em todos os rins processados foi confirmada por pelo menos um de três métodos utilizados para a visualização do agente: exame direto em microscopia de campo escuro, coloração de Levaditi, e a reação de imunofluorescência direta. Segundo estas técnicas, os materiais provenientes do grupo "A" foram caracterizados como apresentando maior riqueza em leptospiras do que aqueles originários do grup o B". Os resultados das provas culturais variaram conforme a estirpe de Leptospira