A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells

It has been reported previously that activation of vascular endothelium by outer membrane proteins of the spirochetes Borrelia sp. and Treponema sp. resulted in enhanced expression of endothelial cell adhesion molecules. To investigate the role of leptospiral proteins in this process, a predicted lipoprotein encoded by the gene LIC10365 was selected, which belongs to a paralogous family that presents a domain of unknown function, DUF1565. The LIC10365 gene was cloned and the protein expressed in Escherichia coli C43 (DE3) strain using the vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and was used to assess its ability to activate cultured human umbilical vein endothelial cells. The rLIC10365 activated endothelium in such a manner that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose-dependent fashion. The LIC10365-encoded protein was identified in vivo in the renal tubules of animal during experimental infection with Leptospira interrogans. Collectively, these results implicate the LIC10365-coding protein of L. interrogans as a potential effector molecule in the promotion of a host inflammatory response. This is the first report of a leptospiral protein capable of up-regulating the expression of endothelial cell adhesion molecules ICAM-1 and E-selectin.Instituto de Biotecnologia y Biologia Molecula

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

A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells

It has been reported previously that activation of vascular endothelium by outer membrane proteins of the spirochetes Borrelia sp. and Treponema sp. resulted in enhanced expression of endothelial cell adhesion molecules. To investigate the role of leptospiral proteins in this process, a predicted lipoprotein encoded by the gene LIC10365 was selected, which belongs to a paralogous family that presents a domain of unknown function, DUF1565. The LIC10365 gene was cloned and the protein expressed in Escherichia coli C43 (DE3) strain using the vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and was used to assess its ability to activate cultured human umbilical vein endothelial cells. The rLIC10365 activated endothelium in such a manner that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose-dependent fashion. The LIC10365-encoded protein was identified in vivo in the renal tubules of animal during experimental infection with Leptospira interrogans. Collectively, these results implicate the LIC10365-coding protein of L. interrogans as a potential effector molecule in the promotion of a host inflammatory response. This is the first report of a leptospiral protein capable of up-regulating the expression of endothelial cell adhesion molecules ICAM-1 and E-selectin.Instituto de Biotecnologia y Biologia Molecula

Seroprevalence of leptospirosis in reproductive-age bovine females in the state of Bahia, Northeastern Brazil

O objetivo do presente trabalho foi determinar a soroprevalência da leptospirose em fêmeas bovinas em idade reprodutiva no Estado da Bahia. A amostragem foi delineada para a determinação da prevalência de propriedades positivas (focos) e de animais soropositivos para a leptospirose. O Estado foi dividido em quatro regiões ou estratos amostrais, nos quais foramexaminadas 10.823 fêmeas bovinas com idade ≥24 meses distribuídas em 1.414 propriedades. A reação de Soroaglutinação Microscópica (SAM), empregando 23 sorovares de Leptospira spp. como antígenos, foi utilizada como teste diagnóstico. O rebanho foi considerado foco quando apresentou pelo menos um animal soropositivo. As prevalências de foco e de animais soropositivos no Estado foram de 77,93% [IC 95% = 75,73% – 79.99%] e 45,42% [IC 95% = 42,00% – 48,88%], respectivamente.O sorovar mais frequente foi o Hardjo (Hardjoprajitno), em 34,49% [IC 95% = 31,93% – 37,14%] daspropriedades positivas e 14,95% [IC 95% = 12,59% - 17,67%] de animais soropositivos de diferentes regiõesThe aim of this work was to determine the seroprevalence of leptospirosis in reproductive-age bovine females in Bahia State, Northeastern Brazil. The sampling was delineated for the determination of the prevalence of seropositive animals as well as herds positive for bovine leptospiroses (foci). The state was divided into 4 regions or sampling strata in which 10,823 bovine females aged ≥24 months allocated in 1,414 herds were sampled. The microscopic agglutination test (MAT), using 23 Leptospira spp. serovars as antigens, was employed as a diagnostic test. The herd was considered positive if at least one animal was seropositive. The prevalences of positive herds and seropositive animals in the state were 77.93% [75.73%–79.99%] and 45.42% [42.00%–48.88%], respectively. Serovar Hardjo (Hardjoprajitno) was the most frequent, with 34.49% [31.93%–37.14%] of positive herds and 14.95% [12.59%–17.67%] of seropositive animals in the different region

Toxicity effects of ethanol extract of Simarouba versicolor on reproductive parameters in female Wistar rats

Simarouba versicolor is popularly known as pau-paraiba. It belongs to the Simaroubaceae family and is found in the Northeast of São Paulo and in some parts of the states of Pará and Mato Grosso do Sul. It is known that this and other species from this genus have several activities: insecticides, antiinflammatory, antitumor, anthelmintic, among others. The effects of ethanolic extract of the bark of S. versicolor (Sv-EtOH) on the reproductive system of rats were investigated; also the influence of estrogenic and/or antiestrogenic activity, estrous cycle, pregnancy, lactation and offspring development was observed. In the experimental protocols with ethanolic extract of S. versicolor, the doses were 10, 20 and 40 mg/kg. To evaluate estrogenic and anti-estrogenic activity, uterotrophic assay and histopathology of the uterus were performed. In the estrous cycle tests, animals were treated daily, for 30 days, to count number of cycles and record the biochemical profile. During pre-implantation and post-implantation, the effect of the extract on pregnancy, lactation and development of the progeny was observed. Uterotrophic assay exhibited anti-estrogenic activity at extract doses of 10, 20 and 40 mg/kg. The animals exposed to all doses had no changes in their estrous cycle, pregnancy, lactation and biochemical profile. All results were within normal range; however, creatinine concentration increased significantly at all doses as compared to the control. The ethanol extract of S. versicolor presented antiestrogen activity, low systemic toxicity and had no reproductive toxicity at investigated doses.Key words: Simarouba versicolor, reproduction, rats

Evaluation of the Expression and Protective Potential of Leptospiral Sphingomyelinases

Leptospirosis is a zoonotic disease of global distribution, which affects both animals and humans. Pathogenic leptospires, the bacteria that cause this disease, require iron for their growth, and these spirochetes probably use their hemolysins, such as the sphingomyelinases, as a way to obtain this important nutrient from host red blood cells during infection. We expressed and purified the leptospiral sphingomyelinases Sph1, Sph2, Sph4, and SphH in a heterologous system. However, the recombinant proteins were not able to lyse sheep erythrocytes, despite having regular secondary structures. Transcripts for all sphingomyelinases tested were detected by RT-PCR analyses, but only Sph2 and SphH native proteins could be detected in Western blot assays using Leptospira whole extracts as well as in renal tubules of infected hamsters. Moreover, antibodies present in the serum of a human patient with laboratory-confirmed leptospirosis recognized Sph2, indicating that this sphingomyelinase is expressed and exposed to the immune system during infection in humans. However, in an animal challenge model, none of the sphingomyelinases tested conferred protection against leptospirosis.Instituto de Biotecnología y Biología Molecula

A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells

It has been reported previously that activation of vascular endothelium by outer membrane proteins of the spirochetes Borrelia sp. and Treponema sp. resulted in enhanced expression of endothelial cell adhesion molecules. To investigate the role of leptospiral proteins in this process, a predicted lipoprotein encoded by the gene LIC10365 was selected, which belongs to a paralogous family that presents a domain of unknown function, DUF1565. The LIC10365 gene was cloned and the protein expressed in Escherichia coli C43 (DE3) strain using the vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and was used to assess its ability to activate cultured human umbilical vein endothelial cells. The rLIC10365 activated endothelium in such a manner that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose-dependent fashion. The LIC10365-encoded protein was identified in vivo in the renal tubules of animal during experimental infection with Leptospira interrogans. Collectively, these results implicate the LIC10365-coding protein of L. interrogans as a potential effector molecule in the promotion of a host inflammatory response. This is the first report of a leptospiral protein capable of up-regulating the expression of endothelial cell adhesion molecules ICAM-1 and E-selectin.Instituto de Biotecnologia y Biologia Molecula

Use of Leptospira spp. strains isolated in Brazil in the microscopic agglutination test applied to diagnosis of leptospirosis in cattle herds in eight brazilian states

Sarmento A.M.C., Azevedo S.S., Morais Z.M., Souza G.O., Oliveira F.C.S., Goncales A.P., Miraglia F. & Vasconcellos S.A. 2012. [Use of Leptospira spp. strains isolated in Brazil in the microscopic agglutination test applied to diagnosis of leptospirosis in cattle herds in eight brazilian states.] Emprego de estirpes Leptospira spp. isoladas no Brasil na microtecnica de soroaglutinacao microscopica aplicada ao diagnostico da leptospirose em rebanhos bovinos de oito estados brasileiros. Pesquisa Veterinaria Brasileira 32(7);601-606. Universidade de Sao Paulo, Faculdade de Medicina Veterinaria e Zootecnia, Departamento de Medicina Veterinaria Preventiva e Saude Animal, Av. Prof. Dr. Orlando Marques de Paiva 87, Sao Paulo, SP 05508-270, Brazil. E-mail: [email protected] The aim of this study was to investigate the adequacy of the use of autochthonous strains of leptospires isolated in Brazil, added to antigen collection of the microscopic agglutination test (MAT) applied to the diagnosis of bovine leptospirosis. By means of non-probability sampling, 109 farms and 9,820 cattle, females at reproductive age were chosen from 85 municipalities in the states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Parana, Rio Grande do Sul, Santa Catarina and Sao Paulo. Among the 9,820 examined animals, 5,806 (59.12%) were reactants at MAT for at least one serovar using the 23 reference serovars. Employing the collection of reference serovars and the ten autochthonous strains, 6,400 (65.24%) reactants and significant difference (p=0.001) was found. The most probable serovars identified by the collection of reference antigens were Hardjo (43.03%), Shermani (20%), Wolfi (9.96%), Grippothyphosa (5.42%) and Pomona (4.28%). With the collection amplified with the ten strains isolated in Brazil, the most probable serovars were Hardjo (31%), Guaricura-M4/84 (22.50%), Shermani (15.43%), Wolffi (4.76%), Grippothyphosa (3.71%) and Autumnalis (3.24%). The serovar Guaricura, strain M4/84, isolated from bovines and buffaloes in the State of Sao Paulo, was ranked as one of the three most probable serovars in the states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais and Sao Paulo. The addition of autochthonous strains to the MAT antigen collection provided the confirmation of the diagnosis of leptospirosis in 594 cattle (6%) which have been classified as non-reactants by the reference collection (p=0.001)