Occurrence of anti-Leptospira antibodies in dogs in the southwestern region of the state of São Paulo / Ocorrência de anticorpos anti-Leptospira em cães na região sudeste do estado de São Paulo

Introduction: Leptospirosis is an emerging disease with different prevalence in dog populations. Dogs are crucial in the disease epidemiology, acting as accidental or maintenance hosts. Infective serovars present different geographic distribution among these populations, depending on exposure to hosts from infected wild or domestic animal reservoirs. The most common serovars that infect dogs prior to the introduction of the vaccines against leptospirosis were Icterohaemorrhagiae and Canicola. Objective: To analyze the occurrence of anti-leptospira antibodies in dogs from southwestern region of the state of São Paulo, using the microscopic agglutination test (MAT).(AU)

Occurrence of anti-Leptospira antibodies in dogs in the southwestern region of the state of São Paulo / Ocorrência de anticorpos anti-Leptospira em cães na região sudeste do estado de São Paulo

Introduction: Leptospirosis is an emerging disease with different prevalence in dog populations. Dogs are crucial in the disease epidemiology, acting as accidental or maintenance hosts. Infective serovars present different geographic distribution among these populations, depending on exposure to hosts from infected wild or domestic animal reservoirs. The most common serovars that infect dogs – prior to the introduction of the vaccines against leptospirosis – were Icterohaemorrhagiae and Canicola. Objective: To analyze the occurrence of anti-leptospira antibodies in dogs from southwestern region of the state of São Paulo, using the microscopic agglutination test (MAT).

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.

Comparison of agglutinating and growth inhibition antibodies levels in sows immunized with two experimental inactivated monovalent vaccines of Canicola serovar: whole culture with aluminum hydroxide and lipopolysaccharide subunit with lipid A / Comparação dos níveis de anticorpos pós-vacinais, aglutinantes e inibidores do crescimento de leptospiras em fêmeas suínas imunizadas com duas vacinas inativadas experimentais monovalentes do sorovar Canicola: cultivo total inativado com hidróxido de alumínio e subunidade de lipopolissacarídios com monofosforil lipídio A

It was compared the antibody response of sows immunized with two experimental vaccines produced with L.interrogans, serovar Canicola, strain LO-4, isolated in Brazil.One of the vaccines was the usual bacterin (whole culture inactivated with phenol and adjuvanted with aluminum hydroxide -WC-AlOH3) and the other one was a subunit vaccine produced with a lipopolysaccharide (LPS) fraction extracted from the bacteria outer envelop and with the lipid A, also extracted from the leptospira wall as adjuvant (LPS-MPLA). Experiment was as follows: group 1 (n = 11), not immunized control, group 2, (n = 11): two immunization with 30 days interval of LPS-MPLA vaccine and group 3 (n = 11): two immunization with 30 days interval of WC-AlOH3 vaccine All three groups were simultaneously immunized, independently of pregnancy stage. Both agglutinin and neutralizing post vaccination antibodies levels were measured respectively by the microscopic sera agglutination with live antigens test (MAT) and the in vitro leptospira growth inhibition test (GIT). Sera collections were performed each 30 days during four months after the first vaccination. Non vaccinated control group animals presented no agglutinating antibodies against Canicola serovar during the whole experiment. At 32 and 68 post vaccination days the agglutinating antibodies levels of group 2 (LPS-MPLA) were significantly higher than the observed in group 3 (WC AlOH3), respectively, p = 0.013 and p = 0.031. The differences observed in the growth inhibition antibodies titers of the two vaccines tested were not significant (p > 0.05). Despite the peak of post-vaccination agglutinins have been registered at 68 days after first immunization, higher levels growth inhibition antibodies were detected at 30 days of first vaccination. Subunit vaccine presented the same immunogenic capacity for the production of neutralizing antibodies as the whole culture one.

Foi comparada a resposta imune de fêmeas suínas adultas imunizadas contra a leptospirose, com vacinas monovalentes produzidas com L.interrogans, sorovar Canicola estirpe LO4, isolada no Brasil. A vacina foi empregada em duas formas: cultura de bactérias totais inativada e acrescida do adjuvante de hidróxido de alumínio (WC-AlOH3) e a do tipo de subunidade constituída apenas por uma fração de lipopolisacarídios (LPS) extraídos do envelope externo da bactéria tendo como adjuvante o monofosforil lipídio A, também extraído da parede da leptospira (LPS-MPLA). O delineamento experimental incluiu: grupo 1 (n = 11): controle não imunizado; grupo 2 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina LPS MFLA; Grupo 3 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina WC-AlOH3. Todos os grupos foram imunizados simultaneamente sem ser considerado o estágio de gestação dos animais. Os níveis de anticorpos pós-vacinais, aglutinantes e neutralizantes foram avaliados, respectivamente, pelos testes de soroaglutinação microscópica com antígenos vivos (SAM) e o de inibição do crescimento de leptospiras in vitro (ICLIV). O monitoramento sorológico foi efetuado a cada 30 dias durante quatro meses após aplicação da primeira dose da vacina. Os animais do grupo controle, não vacinados, não apresentaram anticorpos aglutinantes para o sorovar Canicola durante todo o período experimental. Aos 32 e 68 dias da primo-vacinação, os níveis de anticorpos aglutinantes do grupo 2 (LPS-MPLA) foram significativamente superiores aos observados no grupo 3 (WC AlOH3), respectivamente p = 0,013 e p = 0,031. As diferenças observadas nos níveis de anticorpos inibidores do crescimento de leptospiras in vitro, induzidos pelas duas vacinas, não foram significativas (p > 0,05). A despeito do pico de anticorpos aglutinantes pós-vacinais ter sido registrado aos 68 dias da primeira imunização, os níveis mais elevados de anticorpos inibidores do crescimento de leptospiras já foram observados aos 30 dias da primo-vacinação. A vacina de subunidade apresentou a mesma capacidade de indução de anticorpos neutralizantes que a vacina de bactérias totais.

COMPARISON OF AGGLUTINATING AND GROWTH INHIBITION ANTIBODIES LEVELS IN SOWS IMMUNIZED WITH TWO EXPERIMENTAL INACTIVATED MONOVALENT VACCINES OF CANICOLA SEROVAR: WHOLE CULTURE WITH ALUMINUM HYDROXIDE AND LIPOPOLYSACCHARIDE SUBUNIT WITH LIPID A

ABSTRACT It was compared the antibody response of sows immunized with two experimental vaccines produced with L.interrogans, serovar Canicola, strain LO-4, isolated in Brazil.One of the vaccines was the usual bacterin (whole culture inactivated with phenol and adjuvanted with aluminum hydroxide -WC-AlOH3) and the other one was a subunit vaccine produced with a lipopolysaccharide (LPS) fraction extracted from the bacteria outer envelop and with the lipid A, also extracted from the leptospira wall as adjuvant (LPS-MPLA). Experiment was as follows: group 1 (n = 11), not immunized control, group 2, (n = 11): two immunization with 30 days interval of LPS-MPLA vaccine and group 3 (n = 11): two immunization with 30 days interval of WC-AlOH3 vaccine All three groups were simultaneously immunized, independently of pregnancy stage. Both agglutinin and neutralizing post vaccination antibodies levels were measured respectively by the microscopic sera agglutination with live antigens test (MAT) and the in vitro leptospira growth inhibition test (GIT). Sera collections were performed each 30 days during four months after the first vaccination. Non vaccinated control group animals presented no agglutinating antibodies against Canicola serovar during the whole experiment. At 32 and 68 post vaccination days the agglutinating antibodies levels of group 2 (LPS-MPLA) were significantly higher than the observed in group 3 (WC AlOH3), respectively, p = 0.013 and p = 0.031. The differences observed in the growth inhibition antibodies titers of the two vaccines tested were not significant (p > 0.05). Despite the peak of post-vaccination agglutinins have been registered at 68 days after first immunization, higher levels growth inhibition antibodies were detected at 30 days of first vaccination. Subunit vaccine presented the same immunogenic capacity for the production of neutralizing antibodies as the whole culture one.

RESUMO Foi comparada a resposta imune de fêmeas suínas adultas imunizadas contra a leptospirose, com vacinas monovalentes produzidas com L.interrogans, sorovar Canicola estirpe LO4, isolada no Brasil. A vacina foi empregada em duas formas: cultura de bactérias totais inativada e acrescida do adjuvante de hidróxido de alumínio (WC-AlOH3) e a do tipo de subunidade constituída apenas por uma fração de lipopolisacarídios (LPS) extraídos do envelope externo da bactéria tendo como adjuvante o monofosforil lipídio A, também extraído da parede da leptospira (LPS-MPLA). O delineamento experimental incluiu: grupo 1 (n = 11): controle não imunizado; grupo 2 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina LPS MFLA; Grupo 3 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina WC-AlOH3. Todos os grupos foram imunizados simultaneamente sem ser considerado o estágio de gestação dos animais. Os níveis de anticorpos pós-vacinais, aglutinantes e neutralizantes foram avaliados, respectivamente, pelos testes de soroaglutinação microscópica com antígenos vivos (SAM) e o de inibição do crescimento de leptospiras in vitro (ICLIV). O monitoramento sorológico foi efetuado a cada 30 dias durante quatro meses após aplicação da primeira dose da vacina. Os animais do grupo controle, não vacinados, não apresentaram anticorpos aglutinantes para o sorovar Canicola durante todo o período experimental. Aos 32 e 68 dias da primo-vacinação, os níveis de anticorpos aglutinantes do grupo 2 (LPS-MPLA) foram significativamente superiores aos observados no grupo 3 (WC AlOH3), respectivamente p = 0,013 e p = 0,031. As diferenças observadas nos níveis de anticorpos inibidores do crescimento de leptospiras in vitro, induzidos pelas duas vacinas, não foram significativas (p > 0,05). A despeito do pico de anticorpos aglutinantes pós-vacinais ter sido registrado aos 68 dias da primeira imunização, os níveis mais elevados de anticorpos inibidores do crescimento de leptospiras já foram observados aos 30 dias da primo-vacinação. A vacina de subunidade apresentou a mesma capacidade de indução de anticorpos neutralizantes que a vacina de bactérias totais.

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.

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.

Utilização de uma técnica rápida em camada delgada para avaliação da atividade micobactericida de desinfetantes químicos

ABSTRACT The mycobactericidal activity of sodium hypochlorite (2.5% of active chlorine) was evaluated against a strain of Mycobacterium bovis using the modified thin-layer Middlebrook 7H11 cultivation technique and it was compared to the test made in tubes with Stonebrink medium. The assays were performed either in the presence or absence of organic matter, at temperatures of 4° C and 21 ± 2° C. The colonycounting results were transformed into reduction percentages (Tabela 1) which were compared between the two techniques, showing the disinfectant had similar reduction percentages between plates and tubes in all conditions tested (Mann-Whitney test p 0.05; = 5%). The 2.5% sodium hypochlorite had a mycobactericidal activity of 100% in all conditions, except when in the presence of organic matter and at 4º C (Kurskall-Wallis test and Dunn test, p > 0.05). The reduction percentage was higher when in the absence of organic matter and at 21 ± 2° C (Mann-Whitney test p > 0.05). The modified thin layer cultivation technique was practicable for the realization of the disinfectant tests and allowed an earlier visualization of the colonies (7 days medium). The 2.5% sodium hypochlorite had its mycobactericidal activity diminished when in the presence of organic matter and at the temperature of 4° C.

RESUMO A atividade micobactericida do hipoclorito de sódio (2,5 % de cloro ativo) foi avaliada frente a uma estirpe de Mycobacterium bovis utilizando-se a técnica de cultivo em camada delgada de ágar Middlebrook 7H11 modificado, a qual foi comparada ao teste padrão realizado em tubos contendo meio de Stonebrink. Os testes foram realizados na presença e ausência de matéria orgânica e em duas temperaturas (21 ± 2° C e à 4° C). Os resultados obtidos nas contagens das unidades formadoras de colônias (U.F.C) nas placas e tubos foram transformados em percentual de redução (Tabela 1) os quais foram comparados entre as duas técnicas, mostrando que o hipoclorito de sódio apresentou percentuais iguais entre as placas e os tubos em todas as condições testadas (teste de Mann-Wihtney p > 0,05; = 5%). O desinfetante atingiu atividade micobactericida de 100 % em todas as condições, exceto quando à temperatura de 4° C e presença de matéria orgânica (teste de Kruskal-Wallis e teste de Dunn; p 0,05 ). O percentual de redução foi maior quando na ausência de matéria orgânica e à temperatura ambiente (teste de Mann-Wihtney; p 0,05). A técnica de cultivo em camada delgada mostrou-se viável para realização de testes de desinfetantes e permitiu uma visualização precoce das micobactérias (média de 7 dias). O hipoclorito de sódio 2,5% teve a atividade micobactericida diminuída quando na presença de matéria orgânica e à temperatura de 4° C.

Soroprevalência de leptospirose em fêmeas bovinas em idade reprodutiva no estado da bahia

ABSTRACT The 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 regions.

RESUMO 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 foram examinadas 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.

Frequency of anti-leptospires agglutinins in sows from a swine herd in the ibiúna municipality, state of são paulo, brazil

ABSTRACT A serologic survey was conducted among 164 sows from a single farrow-to-finish swine herd located in the Ibiúna municipality, state of São Paulo, Brasil, to determine the frequency of antileptospires agglutinins. For detection of anti-leptospires antibodies, the microscopic serumagglutination test (MAT) was carried out using live cultures of 22 pathogenic and two saprophytic Leptospira spp. serovars. The most frequent serovar was found crossing the results of frequency and titer of agglutinins, and sera presenting equal titers for two or more serovars were not considered for this analysis. Of the 164 sows, 27 (16.5%) were seropositive for at least one Leptospira spp. serovar. The most frequent serovar was Hardjo (Hardjobovis), with 13 (54.2%) reactant sera. Other reactant serovars and respective frequency were: Shermani (16.6%), Bratislava (12.5%), Autumnalis (12.5%) and Icterohaemorrhagiae (4.2%).

RESUMO Com o objetivo de determinar a freqüência de aglutininas anti-leptospiras, foi realizado um inquérito sorológico em 164 matrizes de uma granja suína de ciclo completo localizada no Município de Ibiúna, Estado de São Paulo, Brasil. Para a detecção de anticorpos anti-leptospiras, foi utilizada a técnica de soroaglutinação microscópica (SAM), utilizando-se culturas vivas de 22 sorovares patogênicos e dois sorovares saprófitos de Leptospira spp. Para a determinação do sorovar mais provável, foram considerados o título de aglutininas e a freqüência de soros reagentes, e soros que apresentaram títulos iguais para dois ou mais sorovares foram excluídos desta análise. Das 164 matrizes suínas, 27 (16,5%) foram soropositivas para pelo menos um dos sorovares empregados. O sorovar mais provável foi o Hardjo (Hardjobovis), com 13 (54,2%) soros reagentes. Também foram constatadas reações sorológicas para os seguintes sorovares: Shermani (16,6%), Bratislava (12,5%), Autumnalis (12,5%) and Icterohaemorrhagiae (4,2%).