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

Serological survey of toxoplasmosis and leptospirosis in neotropical wild mammals from Aracaju Zoo, Sergipe, Brazil

Os zoológicos modernos são instituições destinadas à manutenção da fauna selvagem com o objetivo de promover a conservação, pesquisa científica, lazer, recreação e educação ambiental. A ampla variedade de espécies selvagens, vivendo em condições diferentes do seu habitat natural, representa um ambiente propício à disseminação de doenças, muitas delas zoonóticas. Devido à escassez de dados e à relevância dos mamíferos selvagens neste contexto epidemiológico, tanto na toxoplasmose, quanto na leptospirose, foi efetuado o inquérito sorológico para toxoplasmose e leptospirose em mamíferos selvagens neotropicais do Zoológico de Aracaju, Sergipe, Brasil. Para tanto foram colhidas amostras sanguíneas de 32 animais, adultos, de ambos os sexos incluindo: 14 macacos-prego (Cebus libidinosus), quatro macacos-prego-do-peito-amarelo (Cebus xanthosternus), três onças-suçuaranas (Puma concolor), uma onça-pintada (Pantheraonca), uma raposa (Cerdocyon thous), seis guaxinins (Procyon cancrivorus), dois quatis (Nasua nasua) e um papa-mel (Eira barbara). Para a pesquisa de anticorpos anti-Toxoplasma gondii foi utilizado o Teste de Aglutinação Modificada (MAT ³"1:25) e para pesquisa de anticorpos anti-Leptospira spp. foi utilizado o teste de Soroaglutinação Microscópica (ponto de corte ³1:100) com uma coleção de antígenos vivos que incluiu 24 variantes sorológicas de leptospiras patogênicas e duas leptospiras saprófitas. Dentre os 32 mamíferos, 17 (53,1%) apresentaram anticorpos anti-T. gondii e quatro (12,5%) foram positivos para anticorpos anti-Leptospira spp. De acordo com o sexo, 60% (9/15) dos machos e 47,1% (8/17) das fêmeas foram soropositivos para T. gondii e 26,7% (4/15) dos machos apresentaram anticorpos anti-Leptospira spp. Dos mamíferos que apresentaram anticorpos anti-T. gondii, 47% (8/17) nasceram no zoológico, 41,2% (7/17) foram oriundos de outras instituições e dois (11,8%) foram ) provenientes da natureza. Em relação aos quatro mamíferos soropositivos para Leptospira spp., três (75%) foram procedentes da natureza e um (25%) nasceu no zoológico. Este foi o primeiro inquérito sorológico de anticorpos anti-Leptospira spp. em primatas e carnívoros neotropicais em um zoológico do Nordeste do Brasil e descreveu pela primeira vez a ocorrência de anticorpos anti-T. gondii e anti-Leptospira spp. com sorovar mais provável Copenhageni no primata ameaçado de extinção macaco-prego-de-peito-amarelo (C. xanthosternus) em Aracaju, SEModern zoological gardens are institutions to wildlife maintenance, aiming its conservation, the performance of scientific research and leisure, recreation and environ-mental education activities. The variety of wild species living in conditions different from the one found in its natural habitats represents a propitious environment for diseases spread, specially the zoonotic ones. Due to data shortage and wild mammals' epidemio-logical relevance both for toxoplasmosis as for leptospirosis, this study aimed to determine the serological survey of toxoplasmosis and leptospirosis in Neotropical wild mammals, from Zoo of Aracaju, Sergipe, Brazil. Blood samples were collected from 32 wild mammals, adults and from both sexes: 14 wild bearded capuchins (Cebus libidinosus), four golden-bellied capuchins (Cebus xanthosternus), three pumas (Puma concolor), one jaguar (Pantheraonca), one crab-eating fox (Cerdocyon thous), six crab-eating raccoons (Procyon cancrivorus), two South American coatis (Nasua nasua), and one tayra (Eira barbara). Sera were tested to Toxoplasma gondii antibodies by the modified agglutination test (MAT e"1:25), and by the microscopic seroaglutination test (cut-off e"1:100) using 24 serovar of pathogenic leptospiras and two serovar of saprophyte leptospiras. Antibodies to T. gondii were found in 17 of 32 (53.1%) wild mammals, and antibodies to Leptospira spp. were found in 4 of 32 (12.5%) wild mammals. In relation to gender, 9 of 15 (60%) males, and 8 of 17 (47.1%) females had antibodies to Toxoplasma gondii, and 4 of 15 (26.7%) males had antibodies to Leptospira spp. Considering the origin of the seropositive animals to T. gondii, 8 of 17 (47%) were born in the zoo, 7 of 17 (41.2%) were from other zoos, and 2 (11.8%) were from the wild. Three of 4 (75%) mammals seropositive to Leptospira spp. were born in zoo, and 1 of 4 (25%) was from the wild. This is the first serological survey of Leptospira spp. in Neotropical and carnivores from Northeast zoo of Brazil, and the first time that antibodies to T. gondii and Leptospira spp. (serovar Copenhageni) were observed in the threaten specie of primate golden-bellied capuchins (Cebus xanthosternus) in Aracaju, S

Serosurvey of Smooth Brucella, Leptospira spp. and Toxoplasma gondii in Free-Ranging Jaguars (Panthera onca) and Domestic Animals from Brazil.

This study investigated the exposure of jaguar populations and domestic animals to smooth Brucella, Leptospira spp. and Toxoplasma gondii in the Cerrado, Pantanal and Amazon biomes of Brazil. Between February 2000 and January 2010, serum samples from 31 jaguars (Panthera onca), 1,245 cattle (Bos taurus), 168 domestic dogs (Canis lupus familiaris) and 29 domestic cats (Felis catus) were collected and analysed by rose bengal test for smooth Brucella, microscopic agglutination test for Leptospira spp. and modified agglutination test for T. gondii. Cattle populations from all sites (9.88%) were exposed to smooth Brucella, but only one jaguar from Cerrado was exposed to this agent. Jaguars captured in the Cerrado (60.0%) and in the Pantanal (45.5%) were seropositive for different serovars of Leptospira spp., cattle (72.18%) and domestic dogs (13.1%) from the three sites and one domestic cat from Pantanal were also seropositive for the agent. The most prevalent serotype of Leptospira spp. identified in jaguars from the Cerrado (Grippotyphosa) and the Pantanal (Pomona) biomes were distinct from those found in the domestic animals sampled. Jaguars (100%), domestic dogs (38.28%) and domestic cats (82.76%) from the three areas were exposed to T. gondii. Our results show that brucellosis and leptospirosis could have been transmitted to jaguars by domestic animals; and jaguars probably play an important role in the maintenance of T. gondii in nature

Ocorrência de anticorpos anti-Toxoplasma gondii, anti-Neospora caninum e anti-Leptospira spp. em gambás (Didelphis spp.) no estado de São Paulo, Brasil

Opossum (Didelphis spp.) is an omnivorous marsupial native to the Americas that shows synanthropic behavior in urban areas. Despite its proximity to domestic animals and humans, knowledge of its participation in the epidemiology of some zoonotic agents is substantial. This study aimed to determine the presence of antibodies against Toxoplasma gondii, Neospora spp. and Leptospira spp. in blood samples collected from opossums in 18 municipalities in the state of São Paulo, Brazil, between 2003 and 2008. Blood samples from 343 opossums: Didelphis aurita (n = 256) and Didelphis albiventris (n = 87) were obtained. These were tested to detect antibodies against T. gondii, using the modified agglutination test (MAT-Toto; cutoff ≥ 25); Neospora spp., using the indirect fluorescent antibody test (IFAT; cutoff ≥ 25); and Leptospira spp., using the microscopic agglutination test (MAT-Lepto; cutoff ≥ 100). Frequency of anti-T. gondii, Neospora spp. and Leptospira spp. antibodies were in 22.7%, 1.5% and 3.5%, respectively. The serogroups-serovars of Leptospira spp. presenting positive MAT-Lepto reactions were: Autumnalis-Butembo; Mini-Mini; Ballum-Castellonis; Icterohaemorrhagiae-Icterohaemorrhagiae; Icterohaemorrhagiae-Copenhageni and Grippotyphosa-Grippotyphosa or Bananal. This study demonstrated that these zoonotic agents are circulating in opossum populations in the state of São Paulo. Therefore, investigations regarding the role of marsupials in the epidemiology of each of these diseases should be conducted, especially to understand the behavior of these animals as zoonosis maintenance hosts. O gambá (Didelphis spp.) é um marsupial onívoro nativo das Américas que apresenta comportamento sinantrópico em áreas urbanas. Apesar da sua proximidade com animais domésticos e o homem, o conhecimento da sua participação na epidemiologia de alguns agentes zoonóticos é fundamental. Este estudo objetivou determinar a presença de anticorpos contra Toxoplasma gondii, Neospora spp. e Leptospira spp. em amostras de sangue colhidas de gambás de 18 municípios do estado de São Paulo, Brasil, entre 2003 e 2008. Foram obtidas amostras sanguíneas de 343 gambás: Didelphis aurita (n = 256) e Didelphis albiventris (n = 87). As amostras foram testadas para detecção de anticorpos contra T. gondii, utilizando o teste de aglutinação modificado (TAM-Toxo; ponto de corte ≥ 25); Neospora spp., utilizando a reação de imunofluorescência indireta (RIFI; ponto de corte ≥ 25); e Leptospira spp., utilizando a soroaglutinação microscópica (SAM-Lepto; ponto de corte ≥ 100). As frequências de anticorpos contra T. gondii, Neospora spp. e Leptospira spp. foram 22,7%, 1,5% e 3,5%, respectivamente. Os sorogrupos-sorovares de Leptospira spp. que apresentaram soropositividade foram: Autumnalis-Butembo; Mini-Mini; Ballum-Castellonis; Icterohaemorrhagiae-Icterohaemorrhagiae; Icterohaemorrhagiae-Copenhageni e Grippotyphosa-Grippotyphosa ou Bananal. Esse estudo demonstrou que esses agentes estão circulando em populações de gambás no estado de São Paulo. Desta forma, investigações que visam determinar o papel dos marsupiais na epidemiologia de cada doença devem ser conduzidas, especialmente visando o entendimento do comportamento desses animais como hospedeiros dessas zoonoses

Exposure of free-ranging wild carnivores, horses and domestic dogs to Leptospira spp in the northern Pantanal, Brazil

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

Influência da matéria orgânica na atividade micobactericida de cinco desinfetantes de uso pecuário

Tendo em vista que a maioria dos desinfetantes químicos tem a sua atuação antimicrobiana prejudicada pela presença de matéria orgânica, o objetivo do presente trabalho foi o de investigar, comparativamente, a influência da presença de fezes ou de soro de bovinos sobre a atividade micobactericida de desinfetantes químicos de uso pecuário. Foram ensaiados cinco desinfetantes: A - hipoclorito de sódio; B - combinação de fenóis e cresóis; C - combinação de aldeídos e amorno quaternário; D - iodóforo; E - composto fenólico. A suspensão microbiana foi representada por uma cultura de Mycobactenum fortuitum na concentração de 0,0005% (peso úmido). 0 período de atuação dos desinfetantes foi de 60 minutos, à temperatura de 4 a 8 °C. A interrupção da ação dos desinfetantes foi estabelecida pelo emprego de inativantes. A recuperação dos microrganismos remanescentes foi obtida por cultivo em meio de Lowenstein-Jensen, com quantificação do número de unidades formadoras de colônias (U.F.C.). Os produtos "A", “B" e "C" revelaram possuir atividade micobactericida em presença de matéria orgânica, com percentuais de redução do número de U.F.C. em relação aos controles, superiores a 55,0%. O produto "D" apresentou variabilidade de comportamento frente ao sistema de ensaio adotado. 0 produto "E" mostrou-se destituído de poder micobactericida. A despeito de apresentar percentuais de redução de U.F.C. em relação aos controles, superiores a 90%, o produto "A" teve a sua atividade micobactericida inibida pela matéria orgânica (Teste U de Mann-Whitney: nível de significância de 0,05). 0 produto "B" foi inibido apenas quando a matéria orgânica foi representada por soro bovino. 0 produto "C" não apresentou redução de seu poder micobactericida quando em presença de soro ou fezes.The influence of the organic matter on the mycobactericidal activity of five proprietary disinfectant of veterinary use was evaluated using a 0.0005% (moist weight) suspension of Mycobacterium fortuitum; the organic matter was represented either by bovine faeces or serun. The five disinfectant products tested uere: "A" - sodiun hypochloride; "B" - amixture of phenols and cresylic acid; "C" - a mixture of aldehydes; "D" - iodophor; "E" - phenolyc compounds. The contact period of each disinfectant with the suspension of M. fortuimm in the presence of the organic matter was 60 minutes at temperature between 4 to 8 ºC. The disinfectant action was interrupted after the established time using nutrient broth containing 5% sterilized rabbit serun added with 0.05% Tween 80 for the disinfectants "C", "D" and "E"; disinfectant "B" was neutralized with nutrient broth enriched with rabbit serun added with 3.0% Tween 80; disinfectant "A" was treated by 5% sodium thiosulphate added with 0.05% Tween 80. The isolation of the viable microorganisms was carried out by culturing in the Lowenstein-Jensen medium and counting the Colonies Forming Unit (CFU). Disinfectants "A", "B" and "C" were found to be highly effective even in the presence of the serun or faeces; the percentage of the CFU reduction was above 55.0% when compared to controls. Disinfectant "D" was found to have a varied behavior with the system tested. Product "E" was found to be ineffective, without any mycobactericidal activity. Despite the high percentage of CFU reduction of above 90.0% found with product "A", the mycobactericidal activity was inhibited by both types of organic matter (Mann-Whitney U-test, α = 0.05). The efficacy of product "B" on M. fortuimm was influenced only by the presence of the bovine serum. The mycobactericidal activity of product "C" was not affected by the two type of the organic matter used in this study

Controle da eficácia de bacterinas antileptospirose: relação entre os resultados dos testes de inibição de crescimento de leptospiras in vitro com os do desafio in vivo em hamsters

Foi efetuada a comparação em hamsters da proteção conferida e dos níveis de anticorpos induzidos por duas bacterinas comerciais antileptospirose. Os ensaios empregados foram o teste oficial de potência com desafio (TP), o ensaio proposto, teste de inibição de crescimento de leptospiras in vitro (ICLIV) e a soroaglutinação microscópica (SAM). O protocolo de imunização foi representado por duas aplicações individuais de 0,25 mL das bacterinas, puras ou de suas diluições geométricas de razão dois variando de 200 a 51.200 para a bacterina A e de 200 a 3.200 para a bacterina B, por via subcutânea com o intervalo de 15 dias. Decorridos 15 dias da segunda aplicação de vacina, um grupo de animais foi desafiado com 0,2 mL de cultivos de leptospiras, por indivíduo, respectivamente dos sorovares Canicola (bacterinas A e B) ou Kennewicki (bacterina A). Os números de doses infectantes empregados nos desafios foram de 100 e 631 respectivamente, para os sorovares Canicola e Kennewicki. Decorridos 21 dias do desafio, os grupos de animais utilizados nos testes de ICLIV e SAM foram sangrados e os seus soros foram reunidos em pools (n = 5). No TP, adotando-se os critérios internacionais, as bacterinas foram aprovadas. A comparação do desempenho das bacterinas para os sorovares adotados, segundo sua concentração, por meio das proporções de animais sobreviventes ao TP e a média dos títulos de anticorpos identificados no teste de ICLIV, indicou que um título igual ou superior a 0,77 log corresponde ao nível de aprovação da bacterina no TP.It was performed a comparison between the protection afforded in hamsters and the antibody levels induced by two commercial vaccines against leptospirosis. The assays used were the official challenge test (TP), the in vitro leptospires growth inhibition test (ICLIV) and microscopic agglutination test (MAT). The immunization protocol consisted of two single applications, 15 days from each other, of 0.25 mL of the bacterins, pure or its two-fold serial dilutions: 200 to 51,200 for bacterin A and 200 to 3.200 bacterin B, both of them administered subcutaneously. A group of animals was challenged, after 15 days from the second vaccine application, with 0.2 mL/animal of live leptospire cultures, with Canicola (bacterin A and B) or Kennewicki (bacterin A) serovars. The numbers of infective doses employed in the challenges were 100 and 631 for Canicola and Kennewicki serovars, respectively. After 15 days from the second vaccine dose the groups of animals used in ICLIV and SAM tests were bled and their sera were collected in pools (n = 5). In TP, adopting the criteria established by the Code Federal Regulation, both bacterins were approved. The comparison of the performance of the tested bacterins with the adopted serovars, 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 0.77 log corresponding with the bacterin level of approval in the potency assay

The effects of the leptospiral infection on reproductive performance in sows

A serologic survey was conducted among 76 sows randomly selected from a single farrow-to-finish swine herd, located on the Ibiúna municipality, state of São Paulo, Brazil, to investigate associations between Leptospira spp. seropositivity and reproductive performance. For detection of anti-leptospires antibodies, the microscopic agglutination test (MAT) was carried out using live cultures of 22 pathogenic and two saprophytic Leptospira spp. serovars. Questionnaires with open and closed questions were administered to the manager of the herd for collection of information about the sows included in the study and the reproductive performance of the subsequent offspring. The following variables were evaluated: age of sows, total number of piglets born, number of piglets born alive, number of weak piglets, number of weaned piglets, number of mummified fetuses, number of stillbirths, mass of piglets at birth, mass of piglets at weaning, weaning to service interval (WSI), and occurrence of return to heat, mummified fetuses and stillbirths. The frequency of seropositivity for at least one Leptospira spp. serovar was 18.4%. The reactant serovars and respective frequencies were Bratislava (33.3%), Hardjobovis (33.3%), Shermani (19.1%), Icterohaemorrhagiae (9.5%) and Grippotyphosa (4.8%). Seropositivity for Leptospira spp. was associated with prolonged WSI (P<0.001), decreased number of piglets born (P = 0.001), decreased number of piglets born alive (P<0.001), decreased number of weaned piglets (P = 0.003), low mass of the piglets at birth (P<0.01), and increased number of stillbirths (P = 0.001). Seropositive sows had also significant relative risk (P = 0.003) of stillbirth

Ocorrência e fatores de risco associados à leptospirose em cães atendidos em hospital veterinário no semiárido paraibano

Investigou-se a ocorrência de leptospirose em cães da cidade de Patos, semiárido paraibano, e realizou-se um estudo de fatores de risco associados à infecção. Foram examinadas 152 amostras de soro sanguíneo de cães atendidos em hospital veterinário no período de julho a novembro de 2008. O diagnóstico da leptospirose foi realizado pela técnica de soroaglutinação microscópica, utilizando-se uma coleção de 24 sorovariedades. A frequência de animais soropositivos encontrada foi de 19,73% (IC 95% = 13,73% - 26,96%), e as sorovariedades mais frequentes foram Autumnalis (13,16%) e Grippotyphosa (1,97%). A análise de regressão logística múltipla mostrou que os fatores de risco para a leptospirose foram a não definição da raça (odds ratio = 3,67; P = 0,009) e o contato com caprinos/ovinos (odds ratio = 10; P = 0,034).The occurrence of leptospirosis was investigated in dogs from Patos city, semiarid of Paraíba State, Northeast region of Brazil, and the risk factors associated with the infection were analyzed. One hundred and fifty two blood samples were collected from dogs attended in a veterinary hospital during July to November 2008. The diagnostic method carried out for leptospirosis was the microscopic agglutination test, using a batch of 24 leptospiral serovars. The frequency of seropositive animals was 19.73% (95% CI = 13.73% - 26.96%) and most frequent reactant serovars were Autumnalis (13.16%) and Grippotyphosa (1.97%). The multivariate logistic regression analysis showed that the risk factors for leptospirosis were mixed breed (odds ratio = 3.67; P = 0.009) and the contact with caprine/ovine (odds ratio = 10; P = 0.034)