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

Abstract\ud \ud \ud \ud Background\ud \ud 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.\ud \ud \ud \ud Results\ud \ud 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.\ud \ud \ud \ud Conclusions\ud \ud 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.We are deeply indebted to Alexsander Seixas de Souza (Departamento de Parasitologia, Instituto Butantan, Sao Paulo, Brazil) for use of Confocal facilities and helpful discussion. This work was supported by FAPESP, CNPq and Fundaçao Butantan, Brazil; RFD and MLV have fellowships from FAPESP

Canine leptospirosis in asymptomatic populations from the Southwest Region of São Paulo State, Brazil

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.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

Evaluation for leptospirosis vaccine. Relation between in vitro growh inibition test and potency test in hamsters

Foi investigada a existência da correlação entre o teste padrão de potência de bacterinas antileptospiras e o teste de inibição de crescimento de leptospiras in vitro. Os ensaios foram realizados, em hamsters machos, isoladamente segundo a bacterina comercial (A ou B) antileptospirose empregada. Foram comparados a proteção conferida pelas bacterinas e os níveis de anticorpos neutralizantes, respectivamente obtidos nos teste de desafio e inibição de crescimento de leptospiras in vitro (ICL). O protocolo de imunização adotou duas aplicações de 0,25 mL das bacterinas, puras e suas diluições, pela via subcutânea com o intervalo de 15 dias. Os desafios foram realizados após 15 dias da segunda dose com 0,2 mL de culturas vivas dos sorovares Canicola (bacterina A e B) ou Pomona (somente bacterina A), os óbitos por leptospirose foram registrados e, os animais sobreviventes foram submetidos a eutanásia no 21° dia de observação e a condição de portador renal foi investigada por cultivos de tecido renal em meio de Fletcher. Os animais destinados ao teste de inibição de crescimento de leptospiras in vitro foram sacrificados no mesmo dia em que se realizou o desafio dos animais submetidos ao teste de potência. As colheitas de sangue foram efetuadas assepticamente por punção intracardíaca. Constatada individualmente a esterilidade dos soros foram constituídos os pools com quantidades iguais de soro por animal e subgrupo (n=5). No teste de potência de bacterinas para ambas as estirpes a DL50 foi superior a diluição 10-9 do controle do inóculo de desafio, os animais foram desafiados com a diluição 10-6. O número de animais sobreviventes ao desafio e a proteção contra a infecção renal variou de acordo com a bacterina empregada e sua concentração. Os resultados do teste de desafio com as bacterinas antileptospirose A e B situaram-se dentro dos parâmetros exigidos, sendo as bacterinas aprovadas segundo o critério de avaliação internacional. A diluição 1:800 das duas bacterinas testadas recomendada pelas normas internacionais não foi capaz de proteger contra o estado de portador renal de leptospiras. Os hamsters imunizados com as bacterinas apresentaram anticorpos neutralizantes em níveis superiores ao de aglutininas. A comparação do desempenho das bacterinas testadas para os sorovares Canicola e/ou Pomona, segundo sua concentração, por meio das proporções de animais sobreviventes ao teste de desafio e a média dos títulos de anticorpos neutralizantes, estabeleceu que o título de anticorpos neutralizantes igual ou superior a 1,0log10 como correspondente ao nível de aprovação de bacterinas no teste de potência.It was investigated the existence in hamsters of correlation between the standard potency test of bacterins antileptospires and the in vitro leptospires growth inhibition test. The assays were performed, in male hamsters, separately according to the commercial (A and B) animal antileptospirosis bacterin used. The quality of the bacterins and the level of neutralizing antibodies were compared, respectively obtained in the challenge test and the in vitro leptospires growth inhibition test (ICL). The potency test with challenge, performed with serovars Pomona and Canicola, was modified from the protocol of The United States Agriculture Department. The immunization schedule employed two 0,25 mL of bacterins, pure and with dilutions, by subcutaneous route with 15-day interval. The challenge was performed after 15 days from the second dose with 0,2 mL of alive cultures from serovars Canicola (bacterins A and B) or Pomona (only bacterin A), the deaths by leptospirosis were registered and on the 21st observation day the survivors were sacrificed and the condition of renal carrier was investigated by culture of renal tissue for leptospires isolation in Fletcher medium. The animals destined to the in vitro leptospire growth inhibition test were sacrificed on the same day the potency assay with challenge was performed on the other group of hamsters. The blood samples were collected aseptically by intracardiac puncture. Once the sterility of each serum was verified the pools were formed with equal amounts of serum per animal and subgroup (n=5). The bacterins potency tests, for both strains, resulted in a higher DL50 for the dilution 10-9 of the challenge inoculum control, the animals were challenged with the dilution 10-6. The number of surviving animals to the challenge and the protection against renal infection varied according to the bacterin used and its concentration. The results of the potency tests with challenge using the bacterins antileptospirosis A and B were within the required standards, being therefore approved according to the international evaluation criteria. The dilution 1:800, for both bacterins tested, which is recommended by international criteria was not capable of protecting against the condition of leptospires renal carrier. Hamsters immunized with the bacterins showed neutralizing antibodies in higher titers in comparison to agglutinating antibodies. The comparison of the performance of the tested bacterins with the sorovars Canicola and/or Pomona, according to its concentration, by the proportions of surviving animals to the challenge assay and the average of the neutralizing antibodies titers, established a neutralizing antibodies titer equal or higher than 1,0log10 corresponding with the bacterins level of approval in the potency assay

Evaluation of the efficacy of a swine antileptospiral bacterin: relationship between the results of in vitro leptospiral growth inhibition test applied to swine sera with the ones in vivo potency test in hamsters

O controle da eficiência de bacterinas antileptospirose de uso animal é o teste de potência com desafio em hamsters, contudo, na atualidade, tem sido estimulada a busca de alternativas que dispensem o uso de animais de laboratório. O teste de inibição de crescimento de leptospiras in vitro (ICLIV) tem sido proposto como possível alternativa. O presente trabalho empregou os testes de ICLIV, soroaglutinação microscópica (SAM) e ELISA anti IgG para avaliar a intensidade e a duração da imunidade passiva em leitões em aleitamento e ativa em matrizes suínas e leitões desmamados imunizados com bacterina experimental antileptospirose aprovada no teste de potência em hamster. Foi produzida uma bacterina experimental antileptospirose com estirpe patogênica de Leptospira interrogans, sorovar Kennewicki, estirpe Pomona Fromm (LPF), padronizada para conter 109 leptospiras por mL e associada ao adjuvante de hidróxido de alumínio na proporção de 10% do volume da dose final. Para a avaliação da eficácia da concentração mínima de leptospiras a ser utilizada na bacterina, diluições seriadas de razão dez de cultivo de leptospiras variando de 105 a 109 leptospiras/mL, foram submetidas ao teste de potência com desafio em hamsters (Experimento A), apenas a bacterina produzida na concentração de 109 leptospiras/mL foi capaz de proteger os hamsters contra a infecção induzida pela estirpe LPF, quando a vacina foi testada na diluição de 1:800, critério internacional de aprovação. A bacterina na concentração de 109 leptospiras/mL foi submetida ao teste de potência em hamsters (Experimento B), imunizados com a vacina pura e em diluições seriadas de razão dois (200 a 25600). A bacterina foi aprovada no teste de desafio em hamster até a diluição de 1:6400. O controle do inóculo de desafio foi constituído por 100 DL50. Fêmeas suínas que nunca haviam sido vacinadas contra a leptospirose e que foram não reagentes no teste de soroaglutinação microscópica aplicado a leptospirose efetuado com 24 estirpes de referência e no teste ICLIV com a estirpe LPF (Experimento 1), receberam duas aplicações intervaladas de 30 dias e um reforço aos 210 da primeira dose da bacterina pura e em diluições seriadas de razão dois (400 a 3200). Estes animais foram 16 monitorados com colheitas de sangue efetuadas a cada 30 dias. Os picos máximos de anticorpos avaliados pelos testes de SAM e de ICLIV foram observados aos 30 dias da segunda aplicação da vacina, com maior magnitude para a vacina pura, contudo aos 120 dias da segunda aplicação da vacina houve um declínio acentuado nos níveis de anticorpos. Para encontrar um melhor intervalo entre as imunizações (Experimento 2), fêmeas suínas receberam duas aplicações da bacterina pura intervaladas de 30 dias e o reforço aos 150 da primeira dose. A redução do intervalo de revacinação após as duas doses iniciais determinou a persistência dos títulos de aglutininas e de anticorpos neutralizantes com níveis sempre superiores a 0,4 log. Nos leitões em aleitamento filhos das matrizes imunizadas com bacterina na concentração de 109 leptospiras/mL foi constatada a transferência de imunidade passiva, confirmada pelos títulos de anticorpos aglutinantes detectáveis no quinto dia de vida e de neutralizantes no quinto e décimo dia de vida. Nos ensaios realizados em leitões desmamados imunizados com uma série de diluições de razão dez variando de a 109 leptospiras/mL os picos máximos de anticorpos foram observados aos 30 dias da segunda imunização, com maior magnitude para a bacterina testada na concentração de 109 leptospiras/mL. Os parâmetros finalmente obtidos foram que matrizes suínas e leitões desmamados, primovacinados com duas aplicações intervaladas de 30 dias da bacterina aprovada no teste de potência com desafio em hamster, apresentaram no teste de ICLIV efetuado aos 60 dias da primo-vacinação, intervalos de títulos de anticorpos (95%) expressos em log variando, respectivamente de (0,87 a 1,35) e de (1,22 a 1,58). Os valores máximos (12.800) para o teste de ELISA anti IgG das matrizes suínas foram obtidos após o reforço efetuado aos 210 dias.The potency of antileptospirosis bacterins is controlled by in vivo experimental assay performed in hamsters; however, nowadays the search for in vitro methodologies that could replace the use of laboratory animals has been stimulated. For this purpose, the In vitro leptospiral growth inhibition test (IVLGIT) has been proposed as an alternative test. In this work, the intensity and duration of the passive immunity in suckling piglets and active immunity in weaned piglets and adult sows were evaluated, after vaccination with an experimental leptospirosis bacterin, which had been approved previously on the potency test in hamsters. The in vitro tests applied to swine sera were the growth inhibition test (IVLGIT), microscopic agglutination (MAT) and anti-IgG ELISA. The bacterin was produced with a pathogenic Leptospira interrogans serovar Kennewicki strain identified as Pomona Fromm (LPF), and added with aluminum hydroxide as adjuvant at a concentration of 10% of the final volume dose. In order to evaluate the efficacy of the minimum concentration of the leptospires to be used in the bacterin, ten-fold serial dilutions of a culture of leptospira varying from 105 to 109 leptospires/mL were submitted to potency-challenge test in hamsters (Trial A). Only the bacterin produced at the concentration of 109 leptospires/mL protected the hamsters against the infection by LPF strain, when the vaccine was tested at the dilution of 1:800, which is the international criterion for the approval. The bacterin at the concentration of 109 leptospires/mL was submitted to the potency test in hamsters (Trial B), which had been immunized with an undiluted and with two-fold serially diluted bacterin (from 1:200 to 1:25,600). In the challenge test in hamsters, the bacterin has passed till the dilution of 1:6,400. The control of the challenge inoculum presented a titer of 100 LD50. Adult sows, never been vaccinated against leptospirosis, and that showed negative MAT results using 24 reference serovars and with negative IVLGIT with the LPF strain (Trial 1) had administered two doses of bacterin with 30 day interval and a booster dose at 210th day after the first application of the undiluted bacterin and the two-fold diluted ones (1:400 to :3,200). The animals were 18 monitored with bleeding performed each 30 days. The levels of antibodies evaluated by MAT and IVLGIT showed the maximum peak at the 30th day after the second vaccination of the bacterin, with high magnitude found with the undiluted bacterin, however, at the 120th day after the second vaccination there were found a marked decline in antibodies levels. To find a better immunization scheme (Trial 2), the sows received two doses of undiluted bacterin with 30 day interval and the booster dose at the 150th day after the first dose. The reduction on the interval of revaccination after the two initial doses determined the persistence of a higher levels agglutinins and neutralizing antibodies with titers 0.4 log. In suckling piglets born from sows immunized with the bacterin at the concentration of 109 leptospires/mL, the passive transference of antibodies was confirmed by MAT titers detected on the fifth day, and by IVLGIT titers, at the fifth and tenth days after birth. In the study of weaned piglets immunized with the bacterin at the concentrations of 106, 107, 108 and 109 leptospires/mL (Trial 3), the highest antibodies levels were observed at the 30th day after the second immunization, with greater magnitude for the bacterin tested at the concentration of 109 leptospires/mL. The final results indicate that sows and weaned piglets, primo-vaccinated and revaccinated with 30 day interval with bacterin that had passed in the potency-challenge test in hamsters, presented the IVLGIT results varying (95% CI) from 0.87 log to 1.35 log for sows and 1.22 to 1.58 log for weaned piglets, at the 60th day after the first vaccination. In adult sows the highest anti-IgG ELISA titer reached 12,800, obtained after the booster vaccine dose performed at 210th day from the first vaccination

Refolding of the recombinant protein OmpA70 from Leptospira interrogans from inclusion bodies using high hydrostatic pressure and partial characterization of its immunological properties

Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects human populations worldwide. Available vaccines have demonstrated limited effectiveness, and therapeutic interventions are complicated by the difficulty of establishing an early diagnosis. The genome of Leptospira strains was sequenced, and bioinformatic analyses revealed potential vaccine and serodiagnosis candidates. The present work studied OmpA70, a putative outer membrane protein from Leptospira interrogans serovar Copenhageni that combines structural features of Loa22, the first genetically defined virulence factor in Leptospira, and Lp49, a protein that reacts with sera from early and convalescent patients. Recombinant OmpA was produced in Escherichia coli in an insoluble form. Considering the importance of the structural integrity of a protein to confer immune protection, high hydrostatic pressure (HHP) was used to refold OmpA70 aggregated as inclusion bodies. HHP was applied in association with redox-shuffling reagents (oxidized and reduced glutathione) and guanidine hydrochloride or l-arginine. About 40% of the protein was refolded by applying 200 MPa for 16 h in concentrations of l-arginine above 0.4 M. Circular dichroism revealed the presence of secondary structure. OmpA70 has immunogenic and antigenic properties as high antibody titers were seen after immunization with this protein, and sera from infected hamsters reacted with soluble OmpA70FAPESPCNPqFundação Butanta

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 (27°C and 37°C). 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 27°C 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 27°C, and no growth was obtained after 60-day storage at 37°C. 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 37°C). 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 27°C 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 27°C, e não houve crescimento a partir de 60 dias a 37°C. 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