Molecular and serological characterization of pathogenic Leptospira spp. isolated from symptomatic dogs in a highly endemic area, Brazil.

BACKGROUND: Leptospirosis is an endemic zoonosis in Brazil, with a great impact on human and animal health. Although dogs are frequently infected by pathogenic Leptospira, the current epidemiological understanding of canine leptospirosis is mainly based on serological tests that predict the infecting serogroup/serovar. Thus, the present study aimed at identifying the causative agent for severe cases of canine leptospirosis in a highly endemic area through the isolation and characterization of the isolated strains. RESULTS: Urine, serum and blood samples were collected from 31 dogs with suspected acute leptospirosis treated at the Veterinary Hospital Service of Santo Amaro University between 2018 and 2019. Acute infection was confirmed in 17 dogs (54.8%) by the associated use of Polymerase Chain Reaction (PCR), Microscopic Agglutination (MAT) and bacteriological culture. Eleven dogs (35.5%) had titers ≥800, with the most frequent serogroups being Autumnalis and Icterohaemorrhagiae (n = 4 each) and Canicola (n = 2). Leptospires were recovered from four dogs, and Multilocus Sequence Analysis (MLSA) revealed infection caused by L. interrogans, which were further characterized as serogroups Canicola (n = 1) and Icterohaemorrhagiae (n = 3). CONCLUSION: The identity of the isolates and serological pattern of MAT suggest that dogs are highly exposed to the serogroup Icterohaemorrhagiae and Canicola, also indicating possible circulation of serogroups not yet isolated in Brazil, notably serogroup Autumnalis. Our findings also reinforce the usefulness of using multiple diagnostic approaches to confirm acute canine leptospirosis.

Canine leptospirosis in asymptomatic populations from the Southwest Region of São Paulo State, Brazil / Leptospirose canina em uma população assintomática da região sudoeste do estado de São Paulo, Brasil

Leptospirosis is an infectious disease that can affect animals and humans. Distributed worldwide, the disease is more prevalent in tropical regions due to socioenvironmental characteristics. Dogs can serve as sentinels for environmental contamination due to their frequent contact with humans. This study investigated the frequency of occurrence of canine leptospirosis in asymptomatic populations from the Southwest Region of the State of São Paulo, Brazil. Thus, blood samples collected from 572 asymptomatic dogs from the cities of Apiaí, Cananeia, Itapeva, and Itu were tested with a microscopic agglutination test (MAT). A total of 40.5% of animals in Apiaí reacted to Leptospira spp., 42.6% in Itapeva, 7.1% in Cananeia, and 5.1% in Itu. The data from the present study demonstrate that at least one animal from the municipalities of Itapeva, Apiaí, and Cananeia had a titer equal to or higher than 800, indicating that Leptospira is circulating in these municipalities and that the teams working on castration campaigns need to be educated on the correct use of personal protective equipment, especially when mechanically emptying the bladder of these animals. This study also suggests that castration campaigns can strategically monitor zoonotic diseases and assist in establishing preventive strategies for human and animal health.(AU)

A leptospirose é uma enfermidade infectocontagiosa que pode acometer os animais e o homem. Nos países tropicais e em desenvolvimento ocorrem 70% dos casos humanos, com mortalidade variando entre 10 a 70%. Os cães podem se tornar portadores assintomáticos por um longo período, podendo transmitir a Leptospira para humanos. Devido ao intenso convívio com o ser humano, os cães podem servir como sentinelas da contaminação ambiental. Esse trabalho investigou a frequência de ocorrência da leptospirose canina em populações assintomáticas da região sudoeste do estado de São Paulo. Para isso foram examinadas pela técnica de soroaglutinação microscópica (MAT), amostras de sangue provenientes de 572 cães assintomáticos dos municípios de Apiaí, Cananeia, Itapeva e Itu por amostragem de conveniência, oriundos de campanhas de castração. Em Apiaí, foram encontrados 40,5% dos animais reagentes para Leptospira spp.; em Itapeva, 42,6%; em Cananeia, 7,7% e em Itu, 5,1%. Os dados encontrados demonstram que, pelo menos, um animal dos municípios de Itapeva, Apiaí e Cananeia apresentaram título igual ou maior que 800, indicando a circulação da bactéria nessas localidades e que a equipe envolvida nas campanhas de castração precisam ser alertadas sobre o correto uso de equipamento de proteção individual, principalmente no esvaziamento mecânico da bexiga antes do procedimento cirúrgico. O estudo também sugere que as campanhas de castração podem ser estratégicas no monitoramento de doenças zoonóticas e poderiam auxiliar no estabelecimento de ações preventivas para a saúde humana e animal.(AU)

Canine leptospirosis in asymptomatic populations from the Southwest Region of São Paulo State, Brazil / Leptospirose canina em uma população assintomática da região sudoeste do estado de São Paulo, Brasil

Leptospirosis is an infectious disease that can affect animals and humans. Distributed worldwide, the disease is more prevalent in tropical regions due to socioenvironmental characteristics. Dogs can serve as sentinels for environmental contamination due to their frequent contact with humans. This study investigated the frequency of occurrence of canine leptospirosis in asymptomatic populations from the Southwest Region of the State of São Paulo, Brazil. Thus, blood samples collected from 572 asymptomatic dogs from the cities of Apiaí, Cananeia, Itapeva, and Itu were tested with a microscopic agglutination test (MAT). A total of 40.5% of animals in Apiaí reacted to Leptospira spp., 42.6% in Itapeva, 7.1% in Cananeia, and 5.1% in Itu. The data from the present study demonstrate that at least one animal from the municipalities of Itapeva, Apiaí, and Cananeia had a titer equal to or higher than 800, indicating that Leptospira is circulating in these municipalities and that the teams working on castration campaigns need to be educated on the correct use of personal protective equipment, especially when mechanically emptying the bladder of these animals. This study also suggests that castration campaigns can strategically monitor zoonotic diseases and assist in establishing preventive strategies for human and animal health.

A leptospirose é uma enfermidade infectocontagiosa que pode acometer os animais e o homem. Nos países tropicais e em desenvolvimento ocorrem 70% dos casos humanos, com mortalidade variando entre 10 a 70%. Os cães podem se tornar portadores assintomáticos por um longo período, podendo transmitir a Leptospira para humanos. Devido ao intenso convívio com o ser humano, os cães podem servir como sentinelas da contaminação ambiental. Esse trabalho investigou a frequência de ocorrência da leptospirose canina em populações assintomáticas da região sudoeste do estado de São Paulo. Para isso foram examinadas pela técnica de soroaglutinação microscópica (MAT), amostras de sangue provenientes de 572 cães assintomáticos dos municípios de Apiaí, Cananeia, Itapeva e Itu por amostragem de conveniência, oriundos de campanhas de castração. Em Apiaí, foram encontrados 40,5% dos animais reagentes para Leptospira spp.; em Itapeva, 42,6%; em Cananeia, 7,7% e em Itu, 5,1%. Os dados encontrados demonstram que, pelo menos, um animal dos municípios de Itapeva, Apiaí e Cananeia apresentaram título igual ou maior que 800, indicando a circulação da bactéria nessas localidades e que a equipe envolvida nas campanhas de castração precisam ser alertadas sobre o correto uso de equipamento de proteção individual, principalmente no esvaziamento mecânico da bexiga antes do procedimento cirúrgico. O estudo também s

Canine leptospirosis in asymptomatic populations from the Southwest Region of São Paulo State, Brazil / Leptospirose canina em uma população assintomática da região sudoeste do estado de São Paulo, Brasil

Leptospirosis is an infectious disease that can affect animals and humans. Distributed worldwide, the disease is more prevalent in tropical regions due to socioenvironmental characteristics. Dogs can serve as sentinels for environmental contamination due to their frequent contact with humans. This study investigated the frequency of occurrence of canine leptospirosis in asymptomatic populations from the Southwest Region of the State of São Paulo, Brazil. Thus, blood samples collected from 572 asymptomatic dogs from the cities of Apiaí, Cananeia, Itapeva, and Itu were tested with a microscopic agglutination test (MAT). A total of 40.5% of animals in Apiaí reacted to Leptospira spp., 42.6% in Itapeva, 7.1% in Cananeia, and 5.1% in Itu. The data from the present study demonstrate that at least one animal from the municipalities of Itapeva, Apiaí, and Cananeia had a titer equal to or higher than 800, indicating that Leptospira is circulating in these municipalities and that the teams working on castration campaigns need to be educated on the correct use of personal protective equipment, especially when mechanically emptying the bladder of these animals. This study also suggests that castration campaigns can strategically monitor zoonotic diseases and assist in establishing preventive strategies for human and animal health.

A leptospirose é uma enfermidade infectocontagiosa que pode acometer os animais e o homem. Nos países tropicais e em desenvolvimento ocorrem 70% dos casos humanos, com mortalidade variando entre 10 a 70%. Os cães podem se tornar portadores assintomáticos por um longo período, podendo transmitir a Leptospira para humanos. Devido ao intenso convívio com o ser humano, os cães podem servir como sentinelas da contaminação ambiental. Esse trabalho investigou a frequência de ocorrência da leptospirose canina em populações assintomáticas da região sudoeste do estado de São Paulo. Para isso foram examinadas pela técnica de soroaglutinação microscópica (MAT), amostras de sangue provenientes de 572 cães assintomáticos dos municípios de Apiaí, Cananeia, Itapeva e Itu por amostragem de conveniência, oriundos de campanhas de castração. Em Apiaí, foram encontrados 40,5% dos animais reagentes para Leptospira spp.; em Itapeva, 42,6%; em Cananeia, 7,7% e em Itu, 5,1%. Os dados encontrados demonstram que, pelo menos, um animal dos municípios de Itapeva, Apiaí e Cananeia apresentaram título igual ou maior que 800, indicando a circulação da bactéria nessas localidades e que a equipe envolvida nas campanhas de castração precisam ser alertadas sobre o correto uso de equipamento de proteção individual, principalmente no esvaziamento mecânico da bexiga antes do procedimento cirúrgico. O estudo também s

Canine leptospirosis in asymptomatic populations from the Southwest Region of São Paulo State, Brazil / Leptospirose canina em uma população assintomática da região sudoeste do estado de São Paulo, Brasil

Leptospirosis is an infectious disease that can affect animals and humans. Distributed worldwide, the disease is more prevalent in tropical regions due to socioenvironmental characteristics. Dogs can serve as sentinels for environmental contamination due to their frequent contact with humans. This study investigated the frequency of occurrence of canine leptospirosis in asymptomatic populations from the Southwest Region of the State of São Paulo, Brazil. Thus, blood samples collected from 572 asymptomatic dogs from the cities of Apiaí, Cananeia, Itapeva, and Itu were tested with a microscopic agglutination test (MAT). A total of 40.5% of animals in Apiaí reacted to Leptospira spp., 42.6% in Itapeva, 7.1% in Cananeia, and 5.1% in Itu. The data from the present study demonstrate that at least one animal from the municipalities of Itapeva, Apiaí, and Cananeia had a titer equal to or higher than 800, indicating that Leptospira is circulating in these municipalities and that the teams working on castration campaigns need to be educated on the correct use of personal protective equipment, especially when mechanically emptying the bladder of these animals. This study also suggests that castration campaigns can strategically monitor zoonotic diseases and assist in establishing preventive strategies for human and animal health.

A leptospirose é uma enfermidade infectocontagiosa que pode acometer os animais e o homem. Nos países tropicais e em desenvolvimento ocorrem 70% dos casos humanos, com mortalidade variando entre 10 a 70%. Os cães podem se tornar portadores assintomáticos por um longo período, podendo transmitir a Leptospira para humanos. Devido ao intenso convívio com o ser humano, os cães podem servir como sentinelas da contaminação ambiental. Esse trabalho investigou a frequência de ocorrência da leptospirose canina em populações assintomáticas da região sudoeste do estado de São Paulo. Para isso foram examinadas pela técnica de soroaglutinação microscópica (MAT), amostras de sangue provenientes de 572 cães assintomáticos dos municípios de Apiaí, Cananeia, Itapeva e Itu por amostragem de conveniência, oriundos de campanhas de castração. Em Apiaí, foram encontrados 40,5% dos animais reagentes para Leptospira spp.; em Itapeva, 42,6%; em Cananeia, 7,7% e em Itu, 5,1%. Os dados encontrados demonstram que, pelo menos, um animal dos municípios de Itapeva, Apiaí e Cananeia apresentaram título igual ou maior que 800, indicando a circulação da bactéria nessas localidades e que a equipe envolvida nas campanhas de castração precisam ser alertadas sobre o correto uso de equipamento de proteção individual, principalmente no esvaziamento mecânico da bexiga antes do procedimento cirúrgico. O estudo também s

Canine leptospirosis in asymptomatic populations from the Southwest Region of São Paulo State, Brazil / Leptospirose canina em uma população assintomática da região sudoeste do estado de São Paulo, Brasil

Leptospirosis is an infectious disease that can affect animals and humans. Distributed worldwide, the disease is more prevalent in tropical regions due to socioenvironmental characteristics. Dogs can serve as sentinels for environmental contamination due to their frequent contact with humans. This study investigated the frequency of occurrence of canine leptospirosis in asymptomatic populations from the Southwest Region of the State of São Paulo, Brazil. Thus, blood samples collected from 572 asymptomatic dogs from the cities of Apiaí, Cananeia, Itapeva, and Itu were tested with a microscopic agglutination test (MAT). A total of 40.5% of animals in Apiaí reacted to Leptospira spp., 42.6% in Itapeva, 7.1% in Cananeia, and 5.1% in Itu. The data from the present study demonstrate that at least one animal from the municipalities of Itapeva, Apiaí, and Cananeia had a titer equal to or higher than 800, indicating that Leptospira is circulating in these municipalities and that the teams working on castration campaigns need to be educated on the correct use of personal protective equipment, especially when mechanically emptying the bladder of these animals. This study also suggests that castration campaigns can strategically monitor zoonotic diseases and assist in establishing preventive strategies for human and animal health.

A leptospirose é uma enfermidade infectocontagiosa que pode acometer os animais e o homem. Nos países tropicais e em desenvolvimento ocorrem 70% dos casos humanos, com mortalidade variando entre 10 a 70%. Os cães podem se tornar portadores assintomáticos por um longo período, podendo transmitir a Leptospira para humanos. Devido ao intenso convívio com o ser humano, os cães podem servir como sentinelas da contaminação ambiental. Esse trabalho investigou a frequência de ocorrência da leptospirose canina em populações assintomáticas da região sudoeste do estado de São Paulo. Para isso foram examinadas pela técnica de soroaglutinação microscópica (MAT), amostras de sangue provenientes de 572 cães assintomáticos dos municípios de Apiaí, Cananeia, Itapeva e Itu por amostragem de conveniência, oriundos de campanhas de castração. Em Apiaí, foram encontrados 40,5% dos animais reagentes para Leptospira spp.; em Itapeva, 42,6%; em Cananeia, 7,7% e em Itu, 5,1%. Os dados encontrados demonstram que, pelo menos, um animal dos municípios de Itapeva, Apiaí e Cananeia apresentaram título igual ou maior que 800, indicando a circulação da bactéria nessas localidades e que a equipe envolvida nas campanhas de castração precisam ser alertadas sobre o correto uso de equipamento de proteção individual, principalmente no esvaziamento mecânico da bexiga antes do procedimento cirúrgico. O estudo também s

Canine leptospirosis in asymptomatic populations from the Southwest Region of São Paulo State, Brazil / Leptospirose canina em uma população assintomática da região sudoeste do estado de São Paulo, Brasil

Leptospirosis is an infectious disease that can affect animals and humans. Distributed worldwide, the disease is more prevalent in tropical regions due to socioenvironmental characteristics. Dogs can serve as sentinels for environmental contamination due to their frequent contact with humans. This study investigated the frequency of occurrence of canine leptospirosis in asymptomatic populations from the Southwest Region of the State of São Paulo, Brazil. Thus, blood samples collected from 572 asymptomatic dogs from the cities of Apiaí, Cananeia, Itapeva, and Itu were tested with a microscopic agglutination test (MAT). A total of 40.5% of animals in Apiaí reacted to Leptospira spp., 42.6% in Itapeva, 7.1% in Cananeia, and 5.1% in Itu. The data from the present study demonstrate that at least one animal from the municipalities of Itapeva, Apiaí, and Cananeia had a titer equal to or higher than 800, indicating that Leptospira is circulating in these municipalities and that the teams working on castration campaigns need to be educated on the correct use of personal protective equipment, especially when mechanically emptying the bladder of these animals. This study also suggests that castration campaigns can strategically monitor zoonotic diseases and assist in establishing preventive strategies for human and animal health.(AU)

A leptospirose é uma enfermidade infectocontagiosa que pode acometer os animais e o homem. Nos países tropicais e em desenvolvimento ocorrem 70% dos casos humanos, com mortalidade variando entre 10 a 70%. Os cães podem se tornar portadores assintomáticos por um longo período, podendo transmitir a Leptospira para humanos. Devido ao intenso convívio com o ser humano, os cães podem servir como sentinelas da contaminação ambiental. Esse trabalho investigou a frequência de ocorrência da leptospirose canina em populações assintomáticas da região sudoeste do estado de São Paulo. Para isso foram examinadas pela técnica de soroaglutinação microscópica (MAT), amostras de sangue provenientes de 572 cães assintomáticos dos municípios de Apiaí, Cananeia, Itapeva e Itu por amostragem de conveniência, oriundos de campanhas de castração. Em Apiaí, foram encontrados 40,5% dos animais reagentes para Leptospira spp.; em Itapeva, 42,6%; em Cananeia, 7,7% e em Itu, 5,1%. Os dados encontrados demonstram que, pelo menos, um animal dos municípios de Itapeva, Apiaí e Cananeia apresentaram título igual ou maior que 800, indicando a circulação da bactéria nessas localidades e que a equipe envolvida nas campanhas de castração precisam ser alertadas sobre o correto uso de equipamento de proteção individual, principalmente no esvaziamento mecânico da bexiga antes do procedimento cirúrgico. O estudo também sugere que as campanhas de castração podem ser estratégicas no monitoramento de doenças zoonóticas e poderiam auxiliar no estabelecimento de ações preventivas para a saúde humana e animal.(AU)

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.

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

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.(AU)

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

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.

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.

Evaluation of optimal storage time of tuberculous lesions stored in sodium borate / Avaliação do tempo de armazenamento do borato de sódio na conservação de lesões tuberculosas

Preservation of specimens during transportation between abattoirs and diagnostic laboratories defines a critical stage in the definitive diagnosis of bovine tuberculosis by the isolation of Mycobacterium bovis. A 2-step study was designed to verify the maximum time that tissue samples can be stored in saturated sodium borate solution (SSB) with the highest detection of M. bovis isolates. Ninety hamsters were inoculated intraperitoneally with a suspension of M. bovis strain AN5 and were humanely euthanized after 40 days. Their spleens were collected and stored in SSB during four distinct periods (30, 60, 90 and 120 days) with incubation at two temperatures (27C and 37C). The control group was cultured on the day of euthanasia. Sixty-nine suspected tuberculous lesions samples were collected in the abattoir and were stored in SSB for three periods (30, 60 and 90 days) at 27C in the laboratory. The bovine control group was cultured on the day of entry in the laboratory. Both experiments were analyzed separately based on the growth proportion of isolates and the number of colonies. SSB was able to maintain the viability of most M. bovis at high temperatures for up to 30 days. A progressive decline was observed with other storage periods at 27C, and no growth was obtained after 60-day storage at 37C. Despite the loss in viability of M. bovis, SSB is the most favorable choice to preserve specimens during transportation across a large country with high variation in environmental temperature. The sensitivity of M. bovis detection by bacteriological examination is inversely proportional to storage time. Therefore, the storage of tuberculous lesion specimens in SSB is recommended to not exceed 30 days at 27°C before cultivation.(AU)

A conservação de espécimes durante o transporte entre abatedouros e laboratórios de diagnóstico define uma etapa crítica no diagnóstico definitivo da tuberculose bovina pelo isolamento de Mycobacterium bovis. Um estudo de duas fases foi delineado para verificar o tempo máximo de armazenamento que amostras teciduais podem ser mantidas em solução saturada de borato de sódio (SSB) com a mais alta detecção de isolados de M. bovis. Noventa hamsters foram inoculados com suspensão de M. bovis cepa AN5 por via intraperitoneal e, após 40 dias, submetidos à eutanásia humanitária. Os baços foram coletados, armazenados em SSB por quatro períodos distintos (30, 60, 90 e 120 dias) e incubados a duas temperaturas (27 e 37C). O grupo controle foi cultivado no mesmo dia da eutanásia. Sessenta e nove amostras de lesões suspeitas de tuberculose foram coletadas em abatedouro e armazenadas em SSB por três períodos (30, 60 e 90 dias) a 27C no laboratório. O grupo controle bovino foi cultivado no dia da entrada no laboratório. Ambos os experimentos foram analisados separadamente baseados na proporção de crescimento de isolados e no número de colônias. A SSB foi capaz de manter a maioria de M. bovis viáveis em altas temperaturas por até 30 dias. Houve um declínio progressivo nos outros períodos de armazenamento a 27C, e não houve crescimento a partir de 60 dias a 37C. Apesar da perda de viabilidade de M. bovis, a SSB é a escolha mais favorável para preservar as amostras durante o transporte em um grande país com alta variação de temperatura ambiente. A sensibilidade de detecção de M. bovis por exames bacteriológicos é inversamente proporcional ao tempo de armazenamento. Portanto, é recomendado que o armazenamento de amostras de lesões tuberculosas em SSB não exceda 30 dias a 27°C antes do cultivo.(AU)

Evaluation of optimal storage time of tuberculous lesions stored in sodium borate / Avaliação do tempo de armazenamento do borato de sódio na conservação de lesões tuberculosas

Preservation of specimens during transportation between abattoirs and diagnostic laboratories defines a critical stage in the definitive diagnosis of bovine tuberculosis by the isolation of Mycobacterium bovis. A 2-step study was designed to verify the maximum time that tissue samples can be stored in saturated sodium borate solution (SSB) with the highest detection of M. bovis isolates. Ninety hamsters were inoculated intraperitoneally with a suspension of M. bovis strain AN5 and were humanely euthanized after 40 days. Their spleens were collected and stored in SSB during four distinct periods (30, 60, 90 and 120 days) with incubation at two temperatures (27C and 37C). The control group was cultured on the day of euthanasia. Sixty-nine suspected tuberculous lesions samples were collected in the abattoir and were stored in SSB for three periods (30, 60 and 90 days) at 27C in the laboratory. The bovine control group was cultured on the day of entry in the laboratory. Both experiments were analyzed separately based on the growth proportion of isolates and the number of colonies. SSB was able to maintain the viability of most M. bovis at high temperatures for up to 30 days. A progressive decline was observed with other storage periods at 27C, and no growth was obtained after 60-day storage at 37C. Despite the loss in viability of M. bovis, SSB is the most favorable choice to preserve specimens during transportation across a large country with high variation in environmental temperature. The sensitivity of M. bovis detection by bacteriological examination is inversely proportional to storage time. Therefore, the storage of tuberculous lesion specimens in SSB is recommended to not exceed 30 days at 27°C before cultivation.

A conservação de espécimes durante o transporte entre abatedouros e laboratórios de diagnóstico define uma etapa crítica no diagnóstico definitivo da tuberculose bovina pelo isolamento de Mycobacterium bovis. Um estudo de duas fases foi delineado para verificar o tempo máximo de armazenamento que amostras teciduais podem ser mantidas em solução saturada de borato de sódio (SSB) com a mais alta detecção de isolados de M. bovis. Noventa hamsters foram inoculados com suspensão de M. bovis cepa AN5 por via intraperitoneal e, após 40 dias, submetidos à eutanásia humanitária. Os baços foram coletados, armazenados em SSB por quatro períodos distintos (30, 60, 90 e 120 dias) e incubados a duas temperaturas (27 e 37C). O grupo controle foi cultivado no mesmo dia da eutanásia. Sessenta e nove amostras de lesões suspeitas de tuberculose foram coletadas em abatedouro e armazenadas em SSB por três períodos (30, 60 e 90 dias) a 27C no laboratório. O grupo controle bovino foi cultivado no dia da entrada no laboratório. Ambos os experimentos foram analisados separadamente baseados na proporção de crescimento de isolados e no número de colônias. A SSB foi capaz de manter a maioria de M. bovis viáveis em altas temperaturas por até 30 dias. Houve um declínio progressivo nos outros períodos de armazenamento a 27C, e não houve crescimento a partir de 60 dias a 37C. Apesar da perda de viabilidade de M. bovis, a SSB é a escolha mais favorável para preservar as amostras durante o transporte em um grande país com alta variação de temperatura ambiente. A sensibilidade de detecção de M. bovis por exames bacteriológicos é inversamente proporcional ao tempo de armazenamento. Portanto, é recomendado que o armazenamento de amostras de lesões tuberculosas em SSB não exceda 30 dias a 27°C antes do cultivo.

Cross-protection between experimental anti-leptospirosis bacterins.

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.

Cross-protection between experimental anti-leptospirosis bacterins

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.

Cross-protection between experimental anti-leptospirosis bacterins

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.

Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions.

BACKGROUND: Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. RESULTS: We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (KD) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a KD of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. CONCLUSIONS: We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

The novel leptospiral surface adhesin Lsa20 binds laminin and human plasminogen and is probably expressed during infection.

Leptospirosis is an emerging infectious disease caused by pathogenic species of Leptospira. In this work, we report the cloning, expression, purification, and characterization of two predicted leptospiral outer membrane proteins, LIC11469 and LIC11030. The LIC11469 protein is well conserved among leptospiral strains, while LIC11030 was identified only in Leptospira interrogans. We confirmed by surface proteolysis of intact leptospires with proteinase K that these proteins are most likely new surface leptospiral proteins. The recombinant proteins were evaluated for their capacity to attach to extracellular matrix (ECM) components and to plasminogen. The leptospiral protein encoded by LIC11469, named Lsa20 (leptospiral surface adhesin of 20 kDa), binds to laminin and to plasminogen. The binding with both components was not detected when Lsa20 was previously denatured or blocked with anti-Lsa20 antibodies. Moreover, Lsa20 binding to laminin was also confirmed by surface plasmon resonance (SPR). Laminin competes with plasminogen for binding to Lsa20, suggesting the same ligand-binding site. Lsa20-bound plasminogen could be converted to enzymatically active plasmin, capable of cleaving plasmin substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Lsa20 was recognized by antibodies in confirmed-leptospirosis serum samples, suggesting that this protein is expressed during infection. Taken together, our results indicate that Lsa20 is a novel leptospiral adhesin that in concert with the host-derived plasmin may help the bacteria to adhere and to spread through the hosts.

Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D) of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

Characterization of Novel OmpA-Like Protein of Leptospira interrogans That Binds Extracellular Matrix Molecules and Plasminogen

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (KD, 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a KD of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.