Biofilm production of Leptospira spp. strains

Background: Leptospirosis is a zoonosis that affects many species of mammals and occurs endemically in Brazil. The biofilm matrix provides structure and protection to the biofilm cells working as a physical barrier to antibiotic agents, which are attached or consumed by the matrix components. However, this attribute varies according to the matrix, antimicrobial agent and biofilm age. Leptospira may change morphologically according to environmental conditions, including cell aggregation and biofilm formation. Leptospira can colonize the ducts of kidney from hosts for a long time, forming a biofilm, which is believed to be an important factor for their maintenance in animals and in the environment. Thus, the objective of this research was to determine the biofilm formation capacity of four strains of Leptospira interrogans.Materials, Methods & Results: The strains were typified by WHO/FAO/OIE and National Collaborating Center for Reference and Research on Leptospirosis (Kit Biomedical Research, Amsterdam, Netherlands). Leptospira interrogans strains, two isolated from cattle and two isolated from dogs were biofilms tested for adhesion on polystyrene plates, extracellular matrix composition and confocal microscopy. In the plating adhesion test, the suspension was inoculated into 96-well sterile polystyrene microplates with flat bottom at a ratio of 1:200 in EMJH medium, followed by 24 h incubation at 28°C, with medium renewal after 12 h. After this period the wells were washed three times with sterile PBS and following incubation; the plates were dried in the oven at 60°C for 30 min and added 200 μL of 1% violet crystal for five min. Subsequently, the plates were washed with distilled water, after complete removal, 200 μL of acetic acid 33% was added and the readings were performed at 570 nm in the ELISA reader.[...]

Biofilm production of Leptospira spp. strains

Background: Leptospirosis is a zoonosis that affects many species of mammals and occurs endemically in Brazil. The biofilm matrix provides structure and protection to the biofilm cells working as a physical barrier to antibiotic agents, which are attached or consumed by the matrix components. However, this attribute varies according to the matrix, antimicrobial agent and biofilm age. Leptospira may change morphologically according to environmental conditions, including cell aggregation and biofilm formation. Leptospira can colonize the ducts of kidney from hosts for a long time, forming a biofilm, which is believed to be an important factor for their maintenance in animals and in the environment. Thus, the objective of this research was to determine the biofilm formation capacity of four strains of Leptospira interrogans.Materials, Methods & Results: The strains were typified by WHO/FAO/OIE and National Collaborating Center for Reference and Research on Leptospirosis (Kit Biomedical Research, Amsterdam, Netherlands). Leptospira interrogans strains, two isolated from cattle and two isolated from dogs were biofilms tested for adhesion on polystyrene plates, extracellular matrix composition and confocal microscopy. In the plating adhesion test, the suspension was inoculated into 96-well sterile polystyrene microplates with flat bottom at a ratio of 1:200 in EMJH medium, followed by 24 h incubation at 28°C, with medium renewal after 12 h. After this period the wells were washed three times with sterile PBS and following incubation; the plates were dried in the oven at 60°C for 30 min and added 200 μL of 1% violet crystal for five min. Subsequently, the plates were washed with distilled water, after complete removal, 200 μL of acetic acid 33% was added and the readings were performed at 570 nm in the ELISA reader.[...](AU)

Intercurrence between enzootic leukosis and tuberculosis in slaughtered bovine in slaughterhouses / Intercorrência entre leucose enzoótica e tuberculose em bovinos abatidos em frigorífico

Leukosis and tuberculosis are diseases that affect cattle and entail considerable economic losses, due to theim pact on productivity, but there are few reports that they occur simultaneously. Therefore, aimed to evaluate the complications of these diseases in cattles laughtered in São Gotardo, MG, these animals were positive to the tuberculin and showed lesions characteristic of the disease during the stamping. One hundred and thirty three samples of blood serum, all from positive to bovine tuberculosis, were analyzed by immuno diffusion technique in agar gel (AGID), and the prevalence of cattle that were positive by AGID test was 55.64% (74/133). The results of this research showed that tuberculos is and leukosis are diseases present in the studied regions and are disseminated in this population, with significant levels, causing risk to the health of animals and people, as the zoonotic bovine tuberculosis.

Leucose e tuberculose são doenças que acometem o rebanho bovino e acarretam consideráveis prejuízos econômicos, em virtude do impacto na produtividade, porém há poucos relatos de que elas ocorram concomitantemente. Diante disso, objetivouse avaliar a intercorrência entre essas doenças em bovinos abatidos no município de São Gotardo, MG, sendo que esses animais foram reagentes à tuberculinização e apresentaram lesões características da doença durante o abate sanitário. Cento e trinta e três amostras de soro sanguíneo, todas oriundas de bovinos positivos para tuberculose, foram analisadas pela técnica de imunodifusão em gel de ágar (IDGA), e a prevalência de bovinos que apresentaram positividade ao teste de IDGA foi de 55,64% (74/133). Os resultados obtidos nesta pesquisa permitiram concluir que a tuberculose e a leucose são doenças presentes nas regiões estudadas e encontramse disseminadas na população avaliada, com níveis significativos, causando risco à saúde dos animais e das pessoas, dado o caráter zoonótico da tuberculose bovina.

Intercorrência entre leucose enzoótica e tuberculose em bovinos abatidos em frigorífico / Intercurrence between enzootic leukosis and tuberculosis in slaughtered bovine in slaughterhouses

Leucose e tuberculose são doenças que acometem o rebanho bovino e acarretam consideráveis prejuízos econômicos, em virtude do impacto na produtividade, porém há poucos relatos de que elas ocorram concomitantemente. Diante disso, objetivou-se avaliar a intercorrência entre essas doenças em bovinos abatidos no município de São Gotardo, MG, sendo que esses animais foram reagentes à tuberculinização e apresentaram lesões características da doença durante o abate sanitário. Cento e trinta e três amostras de soro sanguíneo, todas oriundas de bovinos positivos para tuberculose, foram analisadas pela técnica de imunodifusão em gel de ágar (IDGA), e a prevalência de bovinos que apresentaram positividade ao teste de IDGA foi de 55,64% (74/133). Os resultados obtidos nesta pesquisa permitiram concluir que a tuberculose e a leucose são doenças presentes nas regiões estudadas e encontram-se disseminadas na população avaliada, com níveis significativos, causando risco à saúde dos animais e das pessoas, dado o caráter zoonótico da tuberculose bovina.(AU)

Leukosis and tuberculosis are diseases that affect cattle and entail considerable economic losses, due to theim pact on productivity, but there are few reports that they occur simultaneously. Therefore, aimed to evaluate the complications of these diseases in cattles laughtered in São Gotardo, MG, these animals were positive to the tuberculin and showed lesions characteristic of the disease during the stamping. One hundred and thirty three samples of blood serum, all from positive to bovine tuberculosis, were analyzed by immuno diffusion technique in agar gel (AGID), and the prevalence of cattle that were positive by AGID test was 55.64% (74/133). The results of this research showed that tuberculos is and leukosis are diseases present in the studied regions and are disseminated in this population, with significant levels, causing risk to the health of animals and people, as the zoonotic bovine tuberculosis.(AU)

Intercurrence between enzootic leukosis and tuberculosis in slaughtered bovine in slaughterhouses / Intercorrência entre leucose enzoótica e tuberculose em bovinos abatidos em frigorífico

Leukosis and tuberculosis are diseases that affect cattle and entail considerable economic losses, due to theim pact on productivity, but there are few reports that they occur simultaneously. Therefore, aimed to evaluate the complications of these diseases in cattles laughtered in São Gotardo, MG, these animals were positive to the tuberculin and showed lesions characteristic of the disease during the stamping. One hundred and thirty three samples of blood serum, all from positive to bovine tuberculosis, were analyzed by immuno diffusion technique in agar gel (AGID), and the prevalence of cattle that were positive by AGID test was 55.64% (74/133). The results of this research showed that tuberculos is and leukosis are diseases present in the studied regions and are disseminated in this population, with significant levels, causing risk to the health of animals and people, as the zoonotic bovine tuberculosis.(AU)

Leucose e tuberculose são doenças que acometem o rebanho bovino e acarretam consideráveis prejuízos econômicos, em virtude do impacto na produtividade, porém há poucos relatos de que elas ocorram concomitantemente. Diante disso, objetivouse avaliar a intercorrência entre essas doenças em bovinos abatidos no município de São Gotardo, MG, sendo que esses animais foram reagentes à tuberculinização e apresentaram lesões características da doença durante o abate sanitário. Cento e trinta e três amostras de soro sanguíneo, todas oriundas de bovinos positivos para tuberculose, foram analisadas pela técnica de imunodifusão em gel de ágar (IDGA), e a prevalência de bovinos que apresentaram positividade ao teste de IDGA foi de 55,64% (74/133). Os resultados obtidos nesta pesquisa permitiram concluir que a tuberculose e a leucose são doenças presentes nas regiões estudadas e encontramse disseminadas na população avaliada, com níveis significativos, causando risco à saúde dos animais e das pessoas, dado o caráter zoonótico da tuberculose bovina.(AU)

Comparison of complementary diagnostic methods of bovine tuberculosis on skintest reactive cattle / Exames complementares no diagnóstico da tuberculose em bovinos reagentes à tuberculinização comparada

The aim of this study was to compare different complementary diagnostic methods (macroscopic, histological, serological and molecular) of tuberculosis in naturally infected cattle. The study was conducted from samples taken from 40 reagents cattle in cervical comparative intradermal tuberculin test  (ITT). The macroscopic post mortem inspection of carcasses was followed by the collection of nasal swabs, blood and tissue samples (liver, lung and mediastinal lymph node) for polymerase chain reaction (PCR) test, EnzymeLinked Immunosorbent Assay (ELISA) and histopathologic test with hematoxylineosin (HE) and ZiehlNeelsen (ZN), respectively. Of the 40 cattle slaughtered, 22 (55%) carcasses had macroscopic lesions suggestive of tuberculosis. The histopathology HE identified 13 (32.5%) carcasses with histological changes: 6 (15%) in the mediastinal lymph nodes, 5 (12.5%) in the liver and 3 (7.5%) in the lung. At  ZN, the presence of acidfast bacilli was not detected in any of the samples tested. The ELISA/IDEXX® identified one (2.5%) reagent animal, and the PCR test detected DNA of Mycobacterium bovis in one (2.5%) cow. It is concluded that among the diagnostic methods reviews none was able to detect all animals that were positive in tuberculin test, but the association of different methods can ensure diagnostic accuracy.

Objetivou-se com este estudo comparar diferentes métodos complementares de diagnóstico (macroscópico, histopatológico, sorológico e molecular) da tuberculose, em bovinos naturalmente infectados. O trabalho deu-se por meio de amostras colhidas em abate sanitário de 40 bovinos reagentes no teste cervical comparativo (TCC) para tuberculose. A inspeção macroscópica post mor tem das carcaças foi acompanhada de colheita de amostras de muco nasal, sangue e tecido (fígado, pulmão e linfonodo mediastínico) para realização do exame de reação em cadeia pela polimerase (PCR), de EnzymeLinked Immunosorbent Assay (ELISA) e histopatológico com colorações de hematoxilinaeosina (HE) e ZiehlNeelsen (ZN), respectivamente. Dos 40 bovinos inspecionados no abate, 22 (55%) animais apresentaram algum tipo de lesão macroscópica sugestiva de tuberculose. Nos achados histopatológicos visualizados em HE, 13 (32,5%) carcaças apontaram alterações histológicas, sendo 6 (15%) nos linfonodos mediastínicos, 5 (12,5%) no fígado e 3 (7,5%) no pulmão. Não foi observada a presença de bacilos álcool-ácido resistentes em nenhuma das amostras avaliadas. O ensaio sorológico de ELISA/IDEXX® identificou um (2,5%) animal reagente, e o teste de PCR detectou DNA de Mycobacterium bovis em uma (2,5%) amostra. Concluiu-se que entre os exames complementares de diagnóstico avaliados nenhum foi capaz de detectar todos os animais que estavam positivos na tuberculinização, porém a associação de diferentes métodos pode garantir a confiabilidade do diagnóstico.

Exames complementares no diagnóstico da tuberculose em bovinos reagentes à tuberculinização comparada / Comparison of complementary diagnostic methods of bovine tuberculosis on skin-test reactive cattle

Objetivou-se com este estudo comparar diferentes métodos complementares de diagnóstico (macroscópico, histopatológico, sorológico e molecular) da tuberculose, em bovinos naturalmente infectados. O trabalho deu-se por meio de amostras colhidas em abate sanitário de 40 bovinos reagentes no teste cervical comparativo (TCC) para tuberculose. A inspeção macroscópica post mortem das carcaças foi acompanhada de colheita de amostras de muco nasal, sangue e tecido (fígado, pulmão e linfonodo mediastínico) para realização do exame de reação em cadeia pela polimerase (PCR), de Enzyme-Linked Immunosorbent Assay (ELISA) e histopatológico com colorações de hematoxilina-eosina (HE) e Ziehl-Neelsen (ZN), respectivamente. Dos 40 bovinos inspecionados no abate, 22 (55%) animais apresentaram algum tipo de lesão macroscópica sugestiva de tuberculose. Nos achados histopatológicos visualizados em HE, 13 (32,5%) carcaças apontaram alterações histológicas, sendo 6 (15%) nos linfonodos mediastínicos, 5 (12,5%) no fígado e 3 (7,5%) no pulmão. Não foi observada a presença de bacilos álcool-ácido resistentes em nenhuma das amostras avaliadas. O ensaio sorológico de ELISA/IDEXX(r) identificou um (2,5%) animal reagente, e o teste de PCR detectou DNA de Mycobacterium bovis em uma (2,5%) amostra. Concluiu-se que entre os exames complementares de diagnóstico avaliados nenhum foi capaz de detectar todos os animais que estavam positivos na tuberculinização, porém a associação de diferentes métodos pode garantir a confiabilidade do diagnóstico.(AU)

The aim of this study was to compare different complementary diagnostic methods (macroscopic, histological, serological and molecular) of tuberculosis in naturally infected cattle. The study was conducted from samples taken from 40 reagents cattle in cervical comparative intradermal tuberculin test (ITT). The macroscopic post mortem inspection of carcasses was followed by the collection of nasal swabs, blood and tissue samples (liver, lung and mediastinal lymph node) for polymerase chain reaction (PCR) test, Enzyme-Linked Immunosorbent Assay (ELISA) and histopathologic test with hematoxylin-eosin (HE) and Ziehl-Neelsen (ZN), respectively. Of the 40 cattle slaughtered, 22 (55%) carcasses had macroscopic lesions suggestive of tuberculosis. The histopathology HE identified 13 (32.5%) carcasses with histological changes: 6 (15%) in the mediastinal lymph nodes, 5 (12.5%) in the liver and 3 (7.5%) in the lung. At ZN, the presence of acid-fast bacilli was not detected in any of the samples tested. The ELISA/IDEXX(r) identified one (2.5%) reagent animal, and the PCR test detected DNA of Mycobacterium bovis in one (2.5%) cow. It is concluded that among the diagnostic methods reviews none was able to detect all animals that were positive in tuberculin test, but the association of different methods can ensure diagnostic accuracy.(AU)

Comparison of complementary diagnostic methods of bovine tuberculosis on skintest reactive cattle / Exames complementares no diagnóstico da tuberculose em bovinos reagentes à tuberculinização comparada

The aim of this study was to compare different complementary diagnostic methods (macroscopic, histological, serological and molecular) of tuberculosis in naturally infected cattle. The study was conducted from samples taken from 40 reagents cattle in cervical comparative intradermal tuberculin test  (ITT). The macroscopic post mortem inspection of carcasses was followed by the collection of nasal swabs, blood and tissue samples (liver, lung and mediastinal lymph node) for polymerase chain reaction (PCR) test, EnzymeLinked Immunosorbent Assay (ELISA) and histopathologic test with hematoxylineosin (HE) and ZiehlNeelsen (ZN), respectively. Of the 40 cattle slaughtered, 22 (55%) carcasses had macroscopic lesions suggestive of tuberculosis. The histopathology HE identified 13 (32.5%) carcasses with histological changes: 6 (15%) in the mediastinal lymph nodes, 5 (12.5%) in the liver and 3 (7.5%) in the lung. At  ZN, the presence of acidfast bacilli was not detected in any of the samples tested. The ELISA/IDEXX® identified one (2.5%) reagent animal, and the PCR test detected DNA of Mycobacterium bovis in one (2.5%) cow. It is concluded that among the diagnostic methods reviews none was able to detect all animals that were positive in tuberculin test, but the association of different methods can ensure diagnostic accuracy.(AU)

Objetivou-se com este estudo comparar diferentes métodos complementares de diagnóstico (macroscópico, histopatológico, sorológico e molecular) da tuberculose, em bovinos naturalmente infectados. O trabalho deu-se por meio de amostras colhidas em abate sanitário de 40 bovinos reagentes no teste cervical comparativo (TCC) para tuberculose. A inspeção macroscópica post mor tem das carcaças foi acompanhada de colheita de amostras de muco nasal, sangue e tecido (fígado, pulmão e linfonodo mediastínico) para realização do exame de reação em cadeia pela polimerase (PCR), de EnzymeLinked Immunosorbent Assay (ELISA) e histopatológico com colorações de hematoxilinaeosina (HE) e ZiehlNeelsen (ZN), respectivamente. Dos 40 bovinos inspecionados no abate, 22 (55%) animais apresentaram algum tipo de lesão macroscópica sugestiva de tuberculose. Nos achados histopatológicos visualizados em HE, 13 (32,5%) carcaças apontaram alterações histológicas, sendo 6 (15%) nos linfonodos mediastínicos, 5 (12,5%) no fígado e 3 (7,5%) no pulmão. Não foi observada a presença de bacilos álcool-ácido resistentes em nenhuma das amostras avaliadas. O ensaio sorológico de ELISA/IDEXX® identificou um (2,5%) animal reagente, e o teste de PCR detectou DNA de Mycobacterium bovis em uma (2,5%) amostra. Concluiu-se que entre os exames complementares de diagnóstico avaliados nenhum foi capaz de detectar todos os animais que estavam positivos na tuberculinização, porém a associação de diferentes métodos pode garantir a confiabilidade do diagnóstico.(AU)

Frequência de lesões macroscópicas em carcaças de bovinos reagentes ao teste tuberculínico / Frequency of macroscopic lesions in cattle positive to the tuberculin skin test

O objetivo deste trabalho foi analisar a frequência de lesões macroscópicas sugestivas de tuberculose em carcaças de bovinos reagentes ao teste intradérmico de tuberculose. Os animais do estudo foram provenientes de rebanhos de exploração leiteira, localizados nos municípios de Perdizes, Patos de Minas, Lagoa Formosa e Uberlândia, Minas Gerais. Foram avaliadas 140 carcaças de bovinos positivos no teste cervical comparativo. Durante o abate sanitário foi realizada inspeção post mortem das carcaças, e as lesões macroscópicas sugestivas de tuberculose foram registradas. Foram avaliados os linfonodos mediastínicos, fígado, pulmão e carcaça. Dos 140 bovinos examinados, 78 (55%) apresentaram algum tipo de lesão macroscópica sugestiva de tuberculose, 38 (49%) ocorreram exclusivamente nos linfonodos mediastínicos, 22 (28%) no fígado e 11 (14%) no pulmão; 5 (6%) carcaças apresentaram lesões no fígado, pulmão e linfonodo mediastínico, e 2 (4%) tiveram lesões no pulmão e linfonodo. Concluem-se que as lesões não visualizadas em bovinos reagentes ao teste tuberculínico podem ocorrer e estariam relacionadas principalmente ao estágio de evolução da doença, tempo insuficiente na inspeção post mortempara detecção, bem como reações inespecíficas para outras micobactérias.(AU)

The aim of this study was to analyze the frequency of macroscopic lesions suggestive of tuberculosis in cattle reacting to the tuberculin test. Cattle of this study were from dairy herds located in the cities of Perdizes, Patos de Minas, Lagoa Formosa and Uberlândia, Minas Gerais, Brazil. We evaluated 140 cattle carcasses positive to the cervical comparative intradermal tuberculin test. During the slaughter a post-mortem inspection of carcasses was done, and the lesions suggestive of tuberculosis were recorded. We evaluated the mediastinal lymph nodes, liver, lung and carcass. Of the 140 cattle examined, 78 (55%) carcasses had some kind of macroscopic lesions suggestive of tuberculosis, 38 (49%) occurred only in the mediastinal lymph nodes, 22 (28%) in the liver and 11 (14%) in the lungs; 5 (6%) carcasses showed lesions in liver, lungs and lymph node, and 2 (4%) showed lesions in lung and lymph nodes. We concluded that lesions that were not visualized in bovine tuberculin skin test reagents may occur and are related mainly to the stage of the disease, insufficient time to detect the lesions, and nonspecific reactions to other mycobacteria.(AU)

Frequência de lesões macroscópicas em carcaças de bovinos reagentes ao teste tuberculínico / Frequency of macroscopic lesions in cattle positive to the tuberculin skin test

O objetivo deste trabalho foi analisar a frequência de lesões macroscópicas sugestivas de tuberculose em carcaças de bovinos reagentes ao teste intradérmico de tuberculose. Os animais do estudo foram provenientes de rebanhos de exploração leiteira, localizados nos municípios de Perdizes, Patos de Minas, Lagoa Formosa e Uberlândia, Minas Gerais. Foram avaliadas 140 carcaças de bovinos positivos no teste cervical comparativo. Durante o abate sanitário foi realizada inspeção post mortem das carcaças, e as lesões macroscópicas sugestivas de tuberculose foram registradas. Foram avaliados os linfonodos mediastínicos, fígado, pulmão e carcaça. Dos 140 bovinos examinados, 78 (55%) apresentaram algum tipo de lesão macroscópica sugestiva de tuberculose, 38 (49%) ocorreram exclusivamente nos linfonodos mediastínicos, 22 (28%) no fígado e 11 (14%) no pulmão; 5 (6%) carcaças apresentaram lesões no fígado, pulmão e linfonodo mediastínico, e 2 (4%) tiveram lesões no pulmão e linfonodo. Concluem-se que as lesões não visualizadas em bovinos reagentes ao teste tuberculínico podem ocorrer e estariam relacionadas principalmente ao estágio de evolução da doença, tempo insuficiente na inspeção post mortempara detecção, bem como reações inespecíficas para outras micobactérias.(AU)

The aim of this study was to analyze the frequency of macroscopic lesions suggestive of tuberculosis in cattle reacting to the tuberculin test. Cattle of this study were from dairy herds located in the cities of Perdizes, Patos de Minas, Lagoa Formosa and Uberlândia, Minas Gerais, Brazil. We evaluated 140 cattle carcasses positive to the cervical comparative intradermal tuberculin test. During the slaughter a post-mortem inspection of carcasses was done, and the lesions suggestive of tuberculosis were recorded. We evaluated the mediastinal lymph nodes, liver, lung and carcass. Of the 140 cattle examined, 78 (55%) carcasses had some kind of macroscopic lesions suggestive of tuberculosis, 38 (49%) occurred only in the mediastinal lymph nodes, 22 (28%) in the liver and 11 (14%) in the lungs; 5 (6%) carcasses showed lesions in liver, lungs and lymph node, and 2 (4%) showed lesions in lung and lymph nodes. We concluded that lesions that were not visualized in bovine tuberculin skin test reagents may occur and are related mainly to the stage of the disease, insufficient time to detect the lesions, and nonspecific reactions to other mycobacteria.(AU)