Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

Anticorpos contra Leptospira spp em bovinos leiteiros vacinados com bacterina polivalente comercial: perfil sorológico frente a dois esquemas de vacinação

Mediante a utilização da prova de soroaglutinação microscópica (SAM), foi pesquisada a indução de anticorpos contra leptospira em bovinos vacinados com uma bacterina polivalente comercial. Procurou-se avaliar a resposta sorológica homóloga frente a dois esquemas de vacinação. Os animais utilizados foram fêmeas adultas em produção leiteira oriundas de seis propriedades da região noroeste do Estado de São Paulo. Vinte animais de cada propriedade foram escolhidos após três exames sorológicos com 24 sorovares de leptospiras com intervalo de 20 dias, através de triagem sorológica com 24 antígenos de leptospiras. Os grupos foram constituídos de animais não reagentes (I, II e III) e animais reagentes (IV, V e VI). Posteriormente os animais foram subdivididos em grupos controle (I e IV), os que receberam somente uma dose de vacina (II e V) e que receberam duas uma doses de vacina com e dose de reforço após 30 dias (III e VI). Os animais foram monitorados por meio da SAM nos dias 0, 15, 30, 45 e 60 após a primeira aplicação da vacina. Os resultados obtidos revelaram que não houve diferença significativa (p>0,05) entre os animais vacinados e não vacinados. Não houve diferença significativa (p>0,05) nas respostas de títulos vacinais com relação ao perfil sorológico apresentados pelos animais. A vacinação com reforço apresentou melhor desempenho e a indução produção de aglutininas somente ocorreu contra os sorovares hardjo, wolffi, icterohaemorrhagiae e pomona. Há a necessidade de maiores estudos sobre o poder imunogênico da vacina utilizada no experimento.

Detection of Leptospira spp. in the semen and urine of bulls serologically reactive to Leptospira interrogans serovar hardjo

O presente trabalho avaliou a PCR na detecção de leptospiras em sêmen e urina de dez touros sorologicamente reagentes, comparando seus resultados com aqueles obtidos por outras técnicas de diagnóstico. Foram realizadas duas colheitas de materiais em dias alternados. As amostras de sêmen e de urina foram separadas em alíquotas para visualização direta em microscopia de campo escuro, inoculação em hamsters (apenas para o sêmen), isolamento em meio de cultura e PCR. Nenhum hamster apresentou positividade na prova de soroaglutinação microscópica (SAM); fragmentos de rins e fígado desses animais foram utilizados para a tentativa de isolamento em meio de cultura, sendo positivo o cultivo a partir do rim de hamster inoculado com semen de um touro, e do fígado de hamsters inoculados com o semen de três touros. O isolamento em meio de cultura foi negativo para todas as amostras de sêmen, mas foi positivo para cinco amostras de urina. Na PCR não houve resultado positivo para as amostras de sêmen, e apenas uma amostra de urina apresentou resultado positivo, sendo coincidente com uma das culturas positivas. Não foi possível visualizar leptospiras em nenhuma das amostras por exame direto em microscopia de campo escuro.

Leptospira spp. and Toxoplasma gondii antibodies in vampire bats (Desmodus rotundus) in Botucatu region, SP, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Frequency of Leptospira spp. in sheep from Brazilian slaughterhouses and its association with epidemiological variables

A leptospirose é uma antropozoonose mundialmente distribuída que infecta animais de produção, incluindo as ovelhas como carreadores para outros animais e o homem. O presente estudo objetivou determinar a prevalência de Leptospira spp. em ovinos de dois abatedouros do estado de São Paulo e sua associação com algumas variáveis epidemiológicas estudadas. Amostras de soro de 182 ovinos foram pesquisadas para a presença de anticorpos para Leptospira spp. pela soroaglutinação microscópica (SAM). Os resultados indicaram 34/182 (18,68%; IC95% 13,70-24,98%) amostras positivas, principalmente para o sorovar Copenhageni (17/34; 50%; IC95% 33,99-66,01%). Crescimento bacteriano no meio de Fletcher foi observado em amostras de 13/34 (38,24%; CI95% 23.87-55.08%) animais, e confirmados pela Reação em Cadeia pela Polimerase (PCR) e seqüenciamento para somente duas amostras renais de dois animais. Assim, o tratamento e vacinação dos ovinos, além do controle de roedores, pode ser útil na prevenção da infecção na região estudada, visto que os ovinos são importantes carreadores de Leptospira spp. para o homem, e sua transmissão aos trabalhadores de abatedouros ocorre principalmente pela manipulação das vísceras.

Leptospira spp. antibodies in captive coatis (Nasua nasua Storr, 1780) (Carnivora: Procyonidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An in vitro growth inhibition test for measuring the potency of Leptospira spp. Sejroe group vaccine in buffaloes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

SEROLOGIC SURVEY FOR LEPTOSPIRA SPP. IN CAPTIVE NEOTROPICAL FELIDS IN FOZ do IGUACU, PARANA, BRAZIL

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Seroprevalence of anti-Leptospira spp. antibodies in dogs in Bahia, Brazil

The aim of this study was to determine the prevalence of anti-Leptospira spp. antibodies in dogs living in the urban area of the city of Ilheus, Bahia, Brazil using the microscopic agglutination test (MAT) to investigate 24 serovars. A semi-structured questionnaire was administered to dog owners to collect data about demography, husbandry and environmental factors. The prevalence of anti-Leptospira spp. antibodies in the population of 282 dogs was 7.1% (95% confidence interval: 4.4-10.7%). Serovar Copenhageni was the most prevalent, followed by serovars Bratislava, Canicola and Gryppotyphosa. No risk factor was detected with regard to demography (age, gender and breed), husbandry (Leptospira vaccinations, food and water exposure through their environment, hunting habits, contact with other animals and contact with rats) and environmental factors (sewage network, garbage collection, history of flooding, river proximity and wastelands). Despite the low prevalence found in this study, the seroprevalence of Leptospira spp. in healthy dogs in Ilheus indicates the presence of this agent in the environment, which may be a source of human infection. Knowledge of the serovars present in this environment is important for understanding the epidemiology of leptospirosis and establishing public health policies aimed at its control. (C) 2012 Elsevier B.V. All rights reserved.

Epidemiological Aspects in the Leptospira spp. and Toxoplasma gondii Infection in Horses from Botucatu, São Paulo, Brazil

Leptospirosis and toxoplasmosis are zoonoses with high importance because of the economic and public health impact. This study was aimed to determine the seroprevafence of leptospirosis and toxoplamosis in 714 serum samples of horses from different farms from Botucatu, São Paulo, Brazil. The samples were researched for Toxoplasma gondii antibodies by indirect immunofluorescence antibody test (IFAT) and for Leptospira spp. antibodies by microscopic agglutination test. of 714 serum samples, 128 (17.9%; 95% Cl: 15.3%-20.9%) were positive for one or more serovars of Leptospira spp., with icterohaemorrhagiae, canicola, and castellonis as the most prevalent serovars, whereas 42 (5.9%; 95% CI: 4.4%-7.9%) were positive for T gondii, of which 33 samples (78.57%; 95% CI: 64.0%-88.2%) presented a titer of 16, 7 (16.7%; 95% CI: 8.4%-30.7%) a titer of 64, and 1 (2.38%; 95% CI: 0.6%-12.3%) a titer of 256. No significant difference was found among the results obtained and the associated variables such as age and sex. (C) 2012 Elsevier B.V. All rights reserved.

Spatial Distribution of Seropositive Dogs to Leptospira spp. and Evaluation of Leptospirosis Risk Factors Using a Decision Tree

Background: Leptospirosis is an important zoonotic disease associated with poor areas of urban settings of developing countries and early diagnosis and prompt treatment may prevent disease. Although rodents are reportedly considered the main reservoirs of leptospirosis, dogs may develop the disease, may become asymptomatic carriers and may be used as sentinels for disease epidemiology. The use of Geographical Information Systems (GIS) combined with spatial analysis techniques allows the mapping of the disease and the identification and assessment of health risk factors. Besides the use of GIS and spatial analysis, the technique of data mining, decision tree, can provide a great potential to find a pattern in the behavior of the variables that determine the occurrence of leptospirosis. The objective of the present study was to apply Geographical Information Systems and data prospection (decision tree) to evaluate the risk factors for canine leptospirosis in an area of Curitiba, PR.Materials, Methods & Results: The present study was performed on the Vila Pantanal, a urban poor community in the city of Curitiba. A total of 287 dog blood samples were randomly obtained house-by-house in a two-day sampling on January 2010. In addition, a questionnaire was applied to owners at the time of sampling. Geographical coordinates related to each household of tested dog were obtained using a Global Positioning System (GPS) for mapping the spatial distribution of reagent and non-reagent dogs to leptospirosis. For the decision tree, risk factors included results of microagglutination test (MAT) from the serum of dogs, previous disease on the household, contact with rats or other dogs, dog breed, outdoors access, feeding, trash around house or backyard, open sewer proximity and flooding. A total of 189 samples (about 2/3 of overall samples) were randomly selected for the training file and consequent decision rules. The remained 98 samples were used for the testing file. The seroprevalence showed a pattern of spatial distribution that involved all the Pantanal area, without agglomeration of reagent animals. In relation to data mining, from 189 samples used in decision tree, a total of 165 (87.3%) animal samples were correctly classified, generating a Kappa index of 0.413. A total of 154 out of 159 (96.8%) samples were considered non-reagent and were correctly classified and only 5/159 (3.2%) were wrongly identified. on the other hand, only 11 (36.7%) reagent samples were correctly classified, with 19 (63.3%) samples failing diagnosis.Discussion: The spatial distribution that involved all the Pantanal area showed that all the animals in the area are at risk of contamination by Leptospira spp. Although most samples had been classified correctly by the decision tree, a degree of difficulty of separability related to seropositive animals was observed, with only 36.7% of the samples classified correctly. This can occur due to the fact of seronegative animals number is superior to the number of seropositive ones, taking the differences in the pattern of variable behavior. The data mining helped to evaluate the most important risk factors for leptospirosis in an urban poor community of Curitiba. The variables selected by decision tree reflected the important factors about the existence of the disease (default of sewer, presence of rats and rubbish and dogs with free access to street). The analyses showed the multifactorial character of the epidemiology of canine leptospirosis.

Epidemiological aspects in leptospirosis. Research of anti-Leptospira spp antibodies, isolation and biomolecular research in bovines, rodents and workers in rural properties from Botucatu, SP, Brazil.

Leptospirosis is a worldwide infection, transmitted between man and animals that causes a decrease in the production of bovine flocks, and offer risks for public health, as an important zoonosis. The rodents are the main reservoirs of leptospires. It was studied 27 dairy farm properties located in or near from Botucatu-SP, Brazil. In these farms were collected blood and kidney samples from rodents, blood and urine samples from bovines and blood samples from the workers. The serology was performed with microscopic agglutination test (MAT). Samples of bovine urine and rodent kidneys were cultivated searching for leptospires isolation. The polymerase chain reaction (PCR) of the kidneys of the rodents was performed. In MAT, 46/ 140 (32.85%) bovine and 8/34 (23.53%) human sera samples were positive, respectively. In human samples, the serovar Brastilava (37.51%) presented the highest occurrence, while in bovines, the serovars Hardjo and Castellonis were most frequent, with 26.08% each one. All of the rodents were negatives in serology. No leptospire was isolated, and kidney samples were negative in PCR. In bovines, the dam water and the bad hygiene quality of milking process were considered important risks of infection in the affected properties (p<0.05), where other reproductive problems, except abortion, can be related. In other side, to human beings the drainage system was the most important risk factor in the studied properties. Thus, it was verified the necessity of an improvement in zoosanitary handling of the properties, mainly of water supply.

Detection of Leptospira spp. from pure cultures and from experimentally contaminated bovine semen by polymerase chain reaction

Considerando a importância do sêmen na transmissão da leptospira bovina, foi realizado o presente estudo que teve como objetivo aplicar a reação em cadeia pela polimerase (PCR) para a detecção de leptospiras em sêmen bovino experimentalmente contaminado. A reação de PCR foi capaz de amplificar um fragmento de DNA específico de 330 pares de bases a partir de cultivos puros de 26 sorovares de Leptospira spp. A contaminação experimental de sêmen com Leptospira interrogans serovar hardjo revelou que a técnica de PCR conseguiu detectar 10 bactérias/ml, concentração sensivelmente mais baixa que as 1.000 bactérias/ml detectadas através do cultivo microbiológico. Os resultados observados revelam o grande potencial da reação de PCR para a detecção de Leptospira spp. em sêmen bovino, notadamente em centrais de inseminação artificial.

Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxoplasma gondii and Leptospira spp. infection in free-ranging armadillos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)