Evaluation of the elastinolytic activity and protective effect of Leptallo I, a protein composed by metalloprotease and FA5/8C domains, from Leptospira interrogans Copenhageni.

Leptospirosis is a re-emergent zoonosis, caused by pathogenic spirochetes from the genus Lepstospira. To date, there is no protein described to be involved in leptospiral hemorrhagic manifestations, although several proteases have been reported for other bacterial infections. In this study we identified 12 putative metalloproteases from the genome of Leptospira interrogans, and characterized for the first time a putative metalloprotease, here named Leptallo I, as a potential Zn(2+) dependent glycylglycine protease belonging to the M23 metalloendopeptidase family. The native protein was detected in extracts from several pathogenic Leptospira species and further shown to be secreted to the culture medium. We expressed the recombinant protein and its C-terminal fragment containing the metalloprotease domain, and both presented regular secondary structures. The sera of humans with leptospirosis were able to recognize rLeptallo I, indicating that the native protein is expressed and presented to the immune system during infection. The recombinant proteins displayed a significant, though relatively low, elastinolytic activity, and the challenge of hamsters immunized with rLeptallo I conferred 33% protection, suggesting a significant importance of this protein in the pathogenesis. The elastinolytic activity may be important for leptospires-host interaction, because elastin constitutes a significant proportion of total lung and blood vessel proteins.

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.

Fatores de risco para a leptospirose em fêmeas bovinas em idade reprodutiva no Estado de São Paulo / Risk factors to leptospirosis in cows in São Paulo state, Brazil

Risk factors associated to leptospirosis were identified in cows from São Paulo State. The State was divided into seven productive circuits from which 8,216 cows older than 24 months of age from 1,021 herds were sampled. For serological diagnosis of Leptospira spp. infection, the microscopic agglutination test (MAT) was carried out. Of the investigated herds, 718 (71.3%; 95% CI = 68.5% - 74.0%) presented at least one reactant animal at MAT to any serovar. Serovar Hardjo was the most prevalent, with 55.2% (95% CI = 51.4% - 58.9%) of the positive herds. Herd size, animal purchase, share pasture, presence of ovine and swine, and utilization of artificial insemination were identified as risk factors. Utilization of maternity pens was a protective factor against leptospirosis.

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection.

In LipL32, the major leptospiral lipoprotein, the C terminus is the primary immunogenic domain and mediates interaction with collagen IV and plasma fibronectin

LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the LipL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.

Fatores de risco associados à ocorrência de anticorpos anti-Leptospira spp. em cães do município de Monte Negro, Rondônia, Amazônia Ocidental Brasileira / Risk factors associated with anti-Leptospira spp antibodies occurrence in dogs from Monte Negro County, Rondônia, Brazilian Western Amazon

Foram testados, pela técnica de soroaglutinação microscópica aplicada à leptospirose, 329 cães provenientes do município de Monte Negro, RO, dos quais 156 eram da área urbana e 173 da área rural. Simultaneamente foi aplicado questionário para verificar a existência de possíveis fatores de risco pela regressão logística. Abordaram-se questões referentes a idade, sexo, dieta, tipo de criação, ambiente, contato com outras espécies e hábito de caça. Foram detectadas reações com títulos >100 em 27,3 por cento, com 90 cães positivos ao agente, das quais 23,7 por cento eram da área (37/156) urbana e 30,6 por cento (53/173), da área rural (P>0,05). Os sorovares predominantes, reatores com títulos mais elevados, foram Autumnalis (22 por cento), Pyrogenes (12 por cento), Canicola (10 por cento) e Shermani (7,5 por cento). Cães com idade acima de 12 meses apresentaram maior ocorrência de anticorpos quando comparados aos cães mais jovens (P<0,05). Dentre os fatores de risco analisados, foram significativos a alimentação (dieta à base de ração comercial - odds ratio: 3,3; intervalo de confiança: 95 por cento: 1,2 - 9,2; P=0,02) e o sexo (macho - odds ratio: 2,3; intervalo de confiança: 95 por cento: 1,3 - 3,9; P=0,003). Ressalta-se a ocorrência de reações para sorovares mantidos na natureza por animais silvestres.

The microscopic agglutination test was applied to 329 samples taken from 156 dogs from urban area and 173 from rural areas of Monte Negro County, Rondônia State, Brazil, to determine anti-Leptospira spp. antibodies. A survey was concomitantly done to detect eventual risk factors thru the use of logistic regression. Studied variables were age, sex, diet, management, environment, contact with other species and hunting habit. Titers >100 were detected in 27.3 percent (90/329) of the dogs, being 23.7 percent (37/156) urban and 30.6 percent (53/173) rural dogs. The most frequent serovars were Autumnalis (22.0 percent), Pyrogenes (12.0 percent), Canicola (10.0 percent) and Shermani (7.5 percent). Dogs older than 12 months showed higher positive frequencies than younger animals (P<0.05). Risk factor were associated with commercial food as compared to homemade food (odds ratio: 3.3; 95 percent confidence interval : 1.3 - 3.9; P=0.02) and associated with sex, with higher occurrence in males than in females (odds ratio: 2.3; 95 percent confidence interval: 1.3 - 3.9; P=0.003). It was emphasized the serological occurrence of serovars kept and spread in nature by wild animals.

Perfil de aglutininas anti-Leptospira em bezerras búfalas vacinadas com bacterina pentavalente comercial contra leptospirose / Serological response anti-Leptospira in female buffalo calves vaccinated with commercial pentavalent bacterin against leptospirosis

Avaliou-se o perfil sorológico de 17 bezerras búfalas vacinadas nos dias zero, 30 e 210 com bacterina pentavalente comercial contra leptospirose, empregando-se a prova de soroaglutinação microscópica com antígenos vivos, durante o período de 360 dias. Utilizaram-se quatro bezerras-controle, e todos os animais foram negativos no dia zero. Nos animais vacinados, foram observadas reações aos 60 dias para todos os sorovares, associadas a títulos máximos para Canicola, Grippothyphosa e Pomona. No 90° dia, houve declínio acentuado dos títulos para todos os sorovares e ausência de títulos para Hardjo. Nos dias 120°, 150°, 180° e 210°, ocorreram oscilações de títulos para todos os sorovares. No 240° dia, foram encontrados títulos máximos para Hardjo, elevação para os outros sorovares e ausência de título para Canicola. Nos dias 270°, 300° e 330°, foi observado declínio gradativo dos títulos. No 360° dia, foram encontrados títulos residuais para Icterohaemorrhagiae, Grippothyphosa e Pomona e ausência de reação para os outros sorovares. Inferiram-se a reduzida persistência dos títulos vacinais mesmo após o reforço e revacinação e a baixa interferência da vacinação no diagnóstico sorológico.

The serological profile of 17 buffalo calves vaccinated at zero, 30 and 210 days of age using a commercial pentavalent bacterin against leptospirosis was evaluated using microscopic seroagglutination, during 360 days post-vaccination (PV). Four non-vaccinated buffalo calves were used as control. All animals studied showed a negative reaction at day zero. In the vaccinated animals, the serological responses for all serovars were detected at the 60th day PV, with maximum agglutinin levels for Canicola, Grippothyphosa and Pomona. The antibody levels decreased for all serovars at day 90 with zero titters for Hardjo serovar. At days 120, 150, 180 and 210 PV were observed oscillations of titters for all serovars. At day 240, maximum antibody levels for Hardjo, an increase of titters for others serovars and no reaction for Canicola were found. At days 270, 300 and 330 a gradual decrease of antibody levels were observed. At day 360 residual titters for Icterohaemorrhagiae, Grippothyphosa and Pomona, and no reaction for the other serovars were observed. The results indicate a short persistency of seric agglutinin levels induced by vaccination, even after improvement and preparation of the vaccine reinforcement, and low interference of vaccination with serodiagnosis.

Soroprevalência e fatores de risco para a leptospirose em cães de Campina Grande, Paraíba / Soroprevalence and risk factors for leptospirosis in dogs from Campina Grande, State of Paraíba, Brazil

Investigou-se a prevalência de leptospirose em cães da cidade de Campina Grande, PB, e realizou-se um estudo de fatores de risco para a infecção. Foram examinadas 285 amostras de soro sangüíneo de cães colhidas durante a campanha de vacinação anti-rábica animal, conduzida em setembro de 2003. O diagnóstico da leptospirose foi realizado pela técnica de soroaglutinação microscópica, utilizando-se uma coleção de 22 sorovares. Para a caracterização do sorovar mais provável, levou-se em conta a titulação e a freqüência. A prevalência encontrada foi de 21,4% (IC 95% = 16,8%-26,6%), com maior freqüência dos sorovares autumnalis (7,4%), copenhageni (6%) e canicola (2,1%). A análise de regressão logística multivariada mostrou que os fatores de risco para a leptospirose foram: idade superior a um ano (odds ratio = 3,00; P = 0,006), raça não definida (odds ratio = 4,02; P = 0,011) e ocorrência de enchentes (odds ratio = 2,32; P = 0,039).

The prevalence of leptospirosis was investigated in dogs from Campina Grande city, State of Paraíba, Brazil, and the risk factors for infection were analyzed. Two hundred and eighty five blood samples were collected from dogs during the rabies vaccination campaign, in September 2003. The diagnostic method run for leptospirosis was the microscopic agglutination test, using a batch of 22 leptospiral serovars. The most prevalent serovar was found crossing the results of frequency and titer of agglutinins. The prevalence was 21.4% (95% CI = 16.8%-26.6%) and most frequent reactant serovars were autumnalis (7.4%), copenhageni (6%) and canicola (2.1%). The multivariate logistic regression analysis showed that the risk factors for leptospirosis were: age older than one year (odds ratio = 3.00; P = 0.006), mixed breed (odds ratio = 4.02; P = 0.011) and occurrence of floods in the area (odds ratio = 2.32; P = 0.039).

Soroprevalência e fatores de risco para a leptospirose em cães de Campina Grande, Paraíba / Soroprevalence and risk factors for leptospirosis in dogs from Campina Grande, State of Paraíba, Brazil

Investigou-se a prevalência de leptospirose em cães da cidade de Campina Grande, PB, e realizou-se um estudo de fatores de risco para a infecção. Foram examinadas 285 amostras de soro sangüíneo de cães colhidas durante a campanha de vacinação anti-rábica animal, conduzida em setembro de 2003. O diagnóstico da leptospirose foi realizado pela técnica de soroaglutinação microscópica, utilizando-se uma coleção de 22 sorovares. Para a caracterização do sorovar mais provável, levou-se em conta a titulação e a freqüência. A prevalência encontrada foi de 21,4 (IC 95 = 16,8-26,6), com maior freqüência dos sorovares autumnalis (7,4), copenhageni (6) e canicola (2,1). A análise de regressão logística multivariada mostrou que os fatores de risco para a leptospirose foram: idade superior a um ano (odds ratio = 3,00; P = 0,006), raça não definida (odds ratio = 4,02; P = 0,011) e ocorrência de enchentes (odds ratio = 2,32; P = 0,039).

Detection of leptospires in bovine semen by polymerase chain reaction.

OBJECTIVE: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. DESIGN: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. RESULT: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. CONCLUSION: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.

[Tuberculosis lymphadenitis in slaughtered swine from the State of São Paulo, Brazil: microscopic histopathology and demonstration of mycobacteria]. / Linfadenites tuberculóides em suínos abatidos no estado de São Paulo, Brasil: aspectos macroscópicos histopatológicos e pesquisa de micobactérias.

INTRODUCTION: As the occurrence of tuberculosis lymphadenitis in swine constitutes a public health risk, especially in immunosuppressed individuals, the distribution of tuberculoid lesions and the presence of microbacteria in lymph nodes and hepatic and muscular tissue in swine for slaughter, in the State of S. Paulo, SP (Brazil), in the period from 1993 to 1994, were studied. MATERIAL E METHOD: Tuberculous lesions in 60 carcasses of swine, slaughtered at abattoirs during the period 1993-1994, were studied. When macroscopic lesions were observed, a representative sampling of lymph nodes, hepatic and muscular tissues (masseter and diaphragm) were examined using histopathological and microbacterial isolation techniques. RESULTS: The macroscopic lesions were found predominantly in mesenteric lymph nodes. The MAI Complex (M.avium-intracellulare) was isolated mainly in the carcass group that showed lesions and was not found in the control group. Microorganisms of the MAI Complex were not isolated from hepatic and muscular tissues. CONCLUSION: No clear relationship between the type of mycobacteria isolated and the macroscopic lesions observed during the carcass inspection was found.

Avaliaçäo quantitativa da concentraçäo de micobactérias em órgäos e humores de hamster experimentalmente infectados com Mycobacterium bovis, estirpe AN 5 / Quantitative evaluation of mycobacterial concentration in organs and fluids of experimentally infected hamster with Mycobacterium bovis, strain AN 5

Descreve-se a tuberculose desencadeada pela inoculaçäo intraperitoneal de 1,0 mg de Mycobacterium bovis (cepa AN 5) em hamsters através de exames bacteriológicos e histopatológicos, realizados 1, 15, 29 e 45 dias após a infecçäo. A bactéria se multiplicou no baço, rins, fígado e pulmöes dos animais infectados, induzindo a formaçäo de lesöes granulomatosas. Um dia após a infecçäo o baço albergou um maior número de bactérias e 45 dias após a infecçäo houve diferença nas concentraçöes bacterianas apenas entre baço e rim, com um maior número de bactérias para o baço. Foram observadas duas fases de bacteremia e a micobactéria foi cultivada a partir do líquido peritoneal em todas as etapas de experimento. Os animais perderam peso em decorrência da doença e morreram em média 50,6 dias após a infecçäo

Influência da matéria orgânica na atividade micobactericida do hipoclorito de sódio comercial com 2, 5 por cento de cloro ativo / Influence of organic soil on the mycobactericidal activity of the commercial sodium hypochlorite with a 2, 5 per cent of active chlorine

Foi investigada a maior diluiçäo do hipoclorito de sódio comercial com 2,5 por cento de cloro ativo (água de lavadeira) capaz de inativar o Mycobacterium fortuitum em presença de níveis variáveis de fezes de bovino como fonte de matéria orgânica. Os ensaios foram conduzidos de modo que o desinfetante atuasse durante 60 minutos à temperatura de 4 a 8§C. A neutralizaçäo da açäo do desinfetante foi obtida com a soluçäo de tiossulfato de sódio a 0,5 por cento e tween-80 a 0,05 por cento (v/v). Os microrganismos sobreviventes foram recuperados em meio de Lowenstein-Jensen. A quantificaçäo das unidades formadoras de colônias (U.F.C.) foi analisada pelo Teste "U" de Mann-Whitney. Os resultados obtidos demonstraram que a presença de fezes bovinas prejudicou a atividade micobactericida do desinfetante ensaiado. Foi possível constatar-se que as diluiçöes de trabalho capazes de determinar a reduçäo de 70 por cento do número de U.F.C. foram de 1:64 e 1:16, respectivamente, em ausência e presença de matéria orgânica. Em diluiçöes de trabalho superiores ao valor de 1:64 a atividade micobactericida do hipoclorito de sódio foi muito irregular

Investigaçäo sobre a presença de leptospiras nos ovários de hamsters experimentalmente infectados com Leptospiras interrogans sorotipo pomona / Investigation on the presence of leptospires in ovaries of hamsters experimentally infected with Leptospiras interrogans serovar pomona

De 90 hamsters primíparas com 80 a 120 gramas de peso vivo, 75 foram inoculadas com a dose individual de 0,5 ml de estirpe virulenta do sorotipo pomona (30 a 40 leptospiras ativas por campo microscópico, no aumento de 200 vezes) e as 15 remanescentes constituíram o grupo testemunho näo infectado. Todos os animais tratados com leptospiras apresentaram os sinais da infecçäo (prostraçäo, taquipnéia, eriçamento do pelame, icterícia e hemorragias nasal, bucal e perineal) e foram sacrificados por ocasiäo da fase agônica da doença, situada entre o quarto e o sétimo dia da inoculaçäo. Nesta oportunidade, os ovários foram colhidos em condiçöes assépticas e submetidos à técnica de visualizaçäo de leptospiras (exame direto em microscopia de campo escuro, coloraçäo argêntica de Levaditi e reaçäo de imunofluorescência direta), cultivo em meio de Fletcher e exame histopatológico (coloraçäo de hematoxilina e eosina). A ocorrência de uma possível contaminaçäo estabelecida durante a retirada dos ovários foi investigada através da lavagem em soluçäo salina tamponada estéril. As leptospiras foram demonstradas em todos os ovários do grupo de animais experimentalmente inoculados (lavados e näo lavados), através da coloraçäo de Levaditi, reaçäo imunofluorescência direta e também no cultivo em meio de Fletcher. O exame direto em microscopia de campo escuro mostrou ser uma técnica muito pouco sensível. O exame das preparaçöes submetidas à coloraçäo argêntica possibilitou a visualizaçäo de leptospiras em diferentes estruturas dos ovários, incluindo: o interstício, a zona pelúcida e o interior dos óvulos. Os exames histopatológicos permitiram observar as alteraçöes morfológicas típicas de um processo inflamatório agudo em 57 por cento dos ovários examinados