DNA nanovaccines prepared using LemA antigen protect Golden Syrian hamsters against Leptospira lethal infection

BACKGROUND Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis. OBJECTIVES We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection. METHODS An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge. FINDINGS rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05). MAIN CONCLUSIONS NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.

Soropositividade para Leptospira sp. e sorogrupos predominantes em cães do Pantanal Brasileiro / Seropositivity for Leptospira sp. and predominant serogroups in dogs from the Brazilian Pantanal

Em face à grande importância que a leptospirose possui no contexto sanitário mundial, tanto no aspecto humano como animal, este estudo teve por objetivo realizar a pesquisa de anticorpos anti-Leptospira sp. pela técnica de Soroaglutinação Microscópica (SAM) em 429 amostras de soros de cães provenientes de quatro municípios (Poconé/MT, Santo Antônio de Leverger/MT, Barão de Melgaço/MT e Corumbá/MS) localizados na região do Pantanal Brasileiro, bem como foram verificadas possíveis associações entre os resultados dos exames sorológicos e respostas aos questionários epidemiológicos aplicados aos proprietários. Do total de cães avaliados pela SAM (título 100), verificou-se que 34 (7,93%; IC 95%: 5,63%-11,00%) cães tinham anticorpos antiLeptospira sp. Os títulos encontrados variaram entre 100 e 1600 e todos os municípios analisados tinham cães sororreagentes ao agente pesquisado. O sorogrupo reator mais frequente foi o Icterohaemorrhagiae, seguido pelo Australis. Por outro lado, foram observadas menores proporções de cães reagentes aos sorogrupos Tarassovi, Hebdomadis, Autumnalis e Grippotyphosa. As variáveis associadas com a ocorrência de leptospirose foram habitat rural (P 0,01) e área alagável (P=0,01).

Given the great importance that leptospirosis has the global health context, both in human and animal aspect, this study aimed tosearch for antibodies anti-Leptospira sp. by the technique of microscopic agglutination test (MAT) in 429 samples of sera from dogsfrom four municipalities (Poconé/MT, Santo Antônio de Leverger/MT, Barão de Melgaço/MT and Corumbá/MS) located in the BrazilianPantanal region, in order for determine associations between the results of the serological tests and answers to epidemiologicalquestionnaires applied to owners. Of the total dogs evaluated by MAT (titer ≥100), it was verified that 34 (7.93%, 95% CI: 5.63%-11.00%) dogs had antibodies against Leptospira sp. The titers found ranged from 100 to 1600 and all municipalities analyzed hadseroreactive dogs for the investigated agent. The most frequent serogroup reactor was Icterohaemorrhagiae, followed by Australis.On the other hand, smaller proportions of reactive dogs were observed for serogroups Tarassovi, Hebdomadis, Autumnalis andGrippotyphosa. The variables associated with the occurrence of leptospirosis were rural habitat (P<0.01) and flooded area (P=0.01).These results demonstrate that dogs from the Pantanal region had contact with agents of the genus Leptospira, which representsinformation relevant to local public health due to the zoonotic importance of the disease.

Occurrence of serological reactions for serogroup Sejroe (CTG and Prajtino) in female buffalo in the state of Pernambuco, Brazil

ABSTRACT The objective of this study was to evaluate the occurrence of anti-Leptospira spp. antibodies in female buffalo in the state of Pernambuco. A total of 123 female buffalo blood samples were collected from five properties distributed in the state of Pernambuco. The microscopic agglutination test was used to study anti-Leptospira spp. antibodies. The occurrence of anti-Leptospira spp. antibodies was 28.5% (35/123; CI 20.7-37.3%) and on different properties, the occurrence ranged from 28.6% to 80.0%, with 100% of the properties showing animals with positive results. The serovars of the serogroup Sejroe with a higher incidence were Hardjoprajtino (CTG strain, 49.1%) and Hardjo (Prajtino genotype, 43.2%), followed by serogroup Grippotyphosa with the Grippotyphosa serovar (3.9%), serogroup Pomona with the Pomona serovar (1.9%), and the Icterohaemorrhagiae serovar Copenhageni (1.9%). This was the first record of the occurrence of anti-Lepstospira spp. antibodies in female buffalo in the state of Pernambuco. Control measures are necessary to prevent health and economic losses, given that the agent involved affects animal reproduction, triggering drops in conception rates or even clinical cases of abortion.

Soropositividade e fatores de risco para leptospirose, toxoplasmose e neosporose na população canina do Estado da Paraíba / Seropositivity and risk factors for leptospirosis, toxoplasmosis and neosporosis in the canine population of Paraiba state, northeastern Brazil

Objetivou-se com este trabalho determinar a frequência de animais soropositivos para Leptospira spp., Toxoplasma gondii e Neospora caninum em cães do Estado da Paraíba, Nordeste do Brasil, bem como identificar fatores de risco. Foram amostrados 1.043 soros de cães procedentes de cinco centros urbanos considerados polos regionais: João Pessoa, Campina Grande, Patos, Sousa e Cajazeiras. Para o diagnóstico sorológico da infecção por Leptospira spp. foi utilizada a soroaglutinação microscópica (SAM) enquanto que para detecção de anticorpos anti-T. gondii e N. caninum empregou-se a reação de imunofluorescência indireta (RIFI). Noventa e sete cães apresentaram aglutininas anti-Leptospiraspp., resultando em frequência de 9,3% (IC 95% = 7,5-11,1%). Os sorovares de maior frequência foram Icterohaemorragiae (47,4%), Copenhageni (16,5%), Bratislava (11,3%), Canicola (10,3%) e Pomona (6,2%). Observou-se soropositividade de 22,1% (231/1.043; IC 95% = 19,6-24,7%) e 7,7% (80/1.043; IC 95% = 6,1-9,3%) para T. gondii e N. caninum, respectivamente. Idade >48 meses (OR=2,92), raça não definida (OR=1,94) e criação com acesso à rua (OR=1,57) foram apontados como fatores de risco para infecção por Leptospira spp. Para toxoplasmose, as categorias idade >48 meses (OR=1,74), alimentação com comida caseira (OR=2,24), alimentação com ração e comida caseira (OR=2,34) e contato com gatos (OR=1,57) foram consideradas fatores de risco, enquanto que a criação com acesso à rua (OR=2,62) foi fator de risco para N. caninum. Conclui-se que cães de cinco centros urbanos do Estado da Paraíba estão expostos às infecções por Leptospiraspp., T. gondii e N. caninum, evidenciadas pela detecção de anticorpos, bem como sugere-se melhor manejo alimentar, controle no acesso a ambientes externos e destino adequado das fezes de gatos.(AU)

The aim of this study was to determine the frequency of seropositive animals for Leptospiraspp., Toxoplasma gondii and Neospora caninum in dogs from Paraiba state, northeastern Brazil, and to identify risk factors. A total of 1,043 sera were sampled from dogs from five urban centers considered as regional poles: João Pessoa, Campina Grande, Patos, Sousa and Cajazeiras. For the serological diagnosis of Leptospira spp. infection the microscopic agglutination test (MAT) was used, and for detecting anti-T. gondii and N. caninum antibodies the indirect fluorescent antiboy test (IFAT) was carried out. Ninety-seven dogs showed anti-Leptospira spp. agglutinins, resulting in a frequency of 9.3% (95% CI=7.5; 11.1%). The most frequente serovars were Icterohaemorragiae (47.4%), Copenhageni (16.5%), Bratislava (11.3%), Canicola (10.3%) and Pomona (6.2%). The seropositivities for T. gondii and N. caninum were 22.1% (231/1043; 95% CI=19.6; 24.7%) and 7.7% (80/1043; 95% CI=6.1; 9.3%) respectively. Age >48 months (OR=2.92), mixed breed (OR=1.94) and access to street (OR=1.57) were identified as risk factors for Leptospira spp. infection. For toxoplasmosis, the categories age >48 months (OR=1.74), homemade food (OR=2.24), comercial and homemade food (OR=2.34) and contact with cats (OR=1.57) were considered risk factors, while access to street (OR=2.62) was risk fator for N. caninum. We conclude that dogs from five urban centers in Paraiba state are exposed to Leptospira spp., T. gondii and N. caninum infections, evidenced by antibody detection, as well as it is suggested a better feed management, control of outside home environment access and proper disposal of cat feces.(AU)

IgM ELISA for leptospirosis diagnosis: a systematic review and meta-analysis / ELISA IgM para diagnóstico de leptospirose: revisão sistemática e meta-análise

Abstract A systematic review with meta-analysis was performed to estimate the accuracy of IgM ELISA for Leptospirosis diagnosis. A search of Medline, Lilacs, Embase, Cochrane Central Register of Controlled Trials and Grey literature (Google Scholar and British Library) was conducted. The medical subject headings (MeSHs) and the words "leptospirosis", "human leptospirosis" and "IgM ELISA" were used. Fifty-two studies were analyzed, which included 10,775 samples. The pooled sensitivity of all the studies was 86% (CI 95%, 85%-87%) and specificity was 90% (CI 95%, 89%-91%). In studies of the acute phase, the sensitivity and specificity were 84% (CI 95%, 82%-85%) and 91% (CI 95%, 90%-91%), respectively. In conclusion, IgM ELISA is sensitive for use as an initial screen for leptospiral infections.

Resumo O objetivo desta revisão sistemática e meta-análise foi avaliar a acurácia do ELISA IgM para o diagnóstico precoce da leptospirose em humanos. A busca foi realizada nas seguintes bases de dados: Medline, PubMed, LILACS, Embase e Cochrane Central Register of Controlled Trials e Grey literature (Google Scholar and British Library). As palavras-chaves usadas foram: "leptospirosis", "human leptospirosis" e "IgM ELI-SA". Foram analisados 52 estudos, que incluíram 10.775 amostras. A sensibilidade e especificidade combinada de todos os estudos foram 86% (CI 95%, 85%-87%) e 90% (CI 95%, 89%-91%), respectivamente. Nos estudos de fase aguda, a sensibilidade e especificidade foram, respectivamente, 84% (CI 95%, 82%-85%) e 91% (CI 95%, 90%-91%). Conclui-se que o ELISA IgM é um teste sensível para rastreamento inicial da leptospirose.

DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.

Leptospira interrogans sorovar Copenhageni e Icterohaemorrhagiae: relação evolutiva, diferenças genéticas e associação com desfecho clínico / Leptospira interrogans serovar Copenhageni and Icterohaemorrhagiae: evolutionary relationship, genetic differences and association with clinical outcome

A leptospirose é a zoonose mais disseminada mundialmente por infectar diversas espécies diferentes de animais mamíferos. Apresenta 22 espécies identificadas, sendo dez patogênicas, cinco intermediarias e sete saprofiticas, além de apresentar mais de 250 sorovares diferentes. Em Salvador, Leptospira interrogans sorovar Copenhageni é a causadora da epidemia urbana na cidade e apresenta ratos como seu hospedeiro reservatório. As formas clínicas da leptospirose podem variar de assintomática a formas graves. As manifestações clínicas mais graves envolve o desenvolvimento da síndrome Hemorrágica pulmonar severa, e óbito do paciente. Estudos para entender as diferenças genéticas entre as diferentes espécies e sorovares é de extrema importância para identificar fatores de virulência da bactéria, genes que possam está associado aos diferentes formas clinicas, e sua capacidade de se adaptar aos diferentes ambientes. Neste trabalho foi estudado o genoma de dois importantes serovares de L. interrogans, o sorovar Copenhageni e o serovar Icterohaemorrhagiae, e suas diferenças genéticas e associação com dados clínicos e epidemiológicos. Um total de 141 isolados...

There are 22 different species of Leptospira spp. in which 10 are pathogenic, 5 intermediate and 7 saprophytic species. In Salvador the Leptospira interrogans sorovar Copenhageni is the main serovar detected, responsible for the urban epidemics, and has rats as their main host. The clinical manifestations of leptospirosis can vary from asymptomatic form to severe disease like pulmonary hemorrhagic syndrome, and death. Studies to understand de genetic differences among the species and serovars are of great importance to identify virulence factors, genes that could be related to the different clinical manifestations and its capacity to adapt in different environments. Here, the genome of two epidemiologically important serovar of the L. interrogans, the serovar Copenhageni and serovar Icterohaemorrhagiae, and their genetic differences and the association of these differences with epidemiological and clinical data were studied. A total of 141 strains...

Caracterização das leptospiras isoladas dos pacientes atendidos no Hospital Couto Maia / Characterization of isolated leptospires of patients seen in the hospital Couto Maia

A leptospirose é uma zoonose de distribuição mundial, com 1,2 milhões de casos registrados a cada ano. De 1996 a 2013, o grupo de pesquisa de leptospirose do CPqGM, realiza uma vigilância ativa no Hospital Couto Maia em Salvador-Ba, onde foi recrutado 4612 casos suspeitos para leptospirose. Destes 4612 foi confirmado o diagnóstico de 1853 (40%) utilizando pelo menos um dos três métodos de diagnóstico (MAT, Hemocultura, qPCR). Dentre os casos confirmados, 1759 (95%) foram confirmados pelo MAT. A sensibilidade do MAT foi diferente entre as amostras aguda e convalescente, sendo 60% na fase aguda e 97% na fase convalescente. O sorogrupo Icterohaemorrhagiae foi o mais prevalente (90%) dos casos confirmados para MAT. Durante o período do estudo foram coletadas 1133 hemoculturas e destas 203 (18%) foram positivas, sendo possível isolar leptospiras de 93/203 (45%) das hemoculturas, as quais foram soro-agrupadas com soros heterológos de coelho. A concordância entre o sorogrupo encontrado no MAT e na soro-agrupagem foi de 80%. Os achados mostram que existe uma concordância significante entre o sorogrupo encontrado pelos dois métodos, o que indica que o painel de cepas utilizado no MAT apresenta uma ótima cobertura para os sorogrupos prevalentes na região. A predominância de um sorogrupo facilitou quanto a tomadas de decisões para prevenção e controle, assim como facilita para o desenvolvimento de novos testes de diagnóstico e vacinas mais direcionados.

Leptospirosis is a re-emerging zoonotic disease of global importance, with 1,2 million cases reported each year. Diagnosis of leptospirosis is often difficult given the nonspecific disease presentation. In order to compare the performance of the two gold standard diagnostic tests for leptospirosis, the group enrolled 4612 patients with suspected leptospirosis during active surveillance at the state infectious disease reference hospital, Hospital Couto Maia, in Salvador, Bahia between 1996 and 2013. Of these, was confirmed Leptospira infection in 1853 (40%) using at least one of three diagnostic methods (microagglutination (MAT), blood culture, and qPCR). Was confirmed 1759 (95%) cases using only the MAT assay, and identified the serogroup Icterohaemorrhagiae as the infective agent in 90% of MAT positive samples. It was determined the sensitivity of the MAT was 60% for acute phase samples and increased to 97% for convalescent samples. Within this study period, it was possible to collect 1133 blood cultures and was isolated leptospires from 93 of 203 (45%) of blood cultures, and determined the serotype using heterologous rabbit sera. The concordance between the infective serogroup identified using hemoculture and MAT techniques was 80%. This result indicates that the panel of 11 strains used in the MAT represents a majority of the infective serogroups causing disease in our study population. The predominance of a single serogroup in symptomatic cases informs the development of new diagnostic tests and novel vaccines to prevent leptospirosis in Brazil.

Identificação de biofilme renal em ratos naturalmente infectados por Leptospira interrogans através de técnicas histoquímicas e de microscopia eletrônica de varredura, Brasil / Identification of renal biofilm in rats naturally infected with Leptospira interrogans using histochemical and scanning electron microscopy techniques, Brazil

INTRODUÇÃO: A leptospirose é um importante agravo de saúde pública e afeta ao menos um milhão de humanos por ano no mundo. Ratos de esgoto (Rattus norvegicus) são os principais reservatórios de leptospiras patogênicas nas áreas urbanas e excretam, através da urina, elevadas quantidades de bactérias diariamente. As leptospiras sobrevivem no ambiente e são transmitidas para novos hospedeiros através do contato com mucosas e pele. Leptospiras patogênicas formam densos biofilmes in vitro. A patogênese da leptospirose em reservatórios crônicos é pouco conhecida. OBJETIVO: Estudar a formação de biofilme por Leptospira interrogans em R. norvegicus capturados em uma área hiperendêmica de Salvador, Bahia, Brasil. MATERIAIS E MÉTODOS: Capturamos 86 ratos, dos quais 76 (88,4%) foram considerados portadores de L. interrogans através da avaliação por imunohistoquímica (IHQ) anti-L. interrogans e/ou Reação em Cadeia da Polimerase quantitativa em Tempo Real (RT-qPCR) utilizando iniciadores específicos para o gene lipL32. RESULTADOS: Ao exame de IHQ anti-L. interrogans, 69 ratos foram positivos. Destes, 24 (35%)...

INTRODUCTION: Leptospirosis is an important public health problem and affects at least one million people each year worldwide. Norway rats (Rattus norvegicus) are the main reservoir hosts of leptospires in urban environments. They excrete large amounts of bacteria daily. Leptospires survive in the environment and are transmitted to new hosts by contact with mucous membranes and skin. Pathogenic leptospires form dense biofilms in vitro. The pathogenesis of chronic leptospirosis in reservoirs is largely unknown. The aim of this work was to study biofilm formation by Leptospira interrogans in R. norvegicus caught in a hyperendemic area in Salvador, Bahia, Brazil. MATERIAL AND METHODS: We caught 86 rats, out of which 76 (88.4%) were carriers of L. interrogans according to immunohistochemistry (IHC) anti-L. interrogans and/or quantitative real time PCR (RT-qPCR) using LipL32 primers. RESULTS: From the 69 rats positive for IHC anti-L. interrogans, 24 (35%)...

Cross-protection between experimental anti-leptospirosis bacterins

We investigated the existence of cross-protection between two anti-leptospirosis monovalent experimental bacterins produced with two strains of Leptospira serogroup Pomona: Fromm strain of serovar Kennewicky, isolated from pigs in the United States, and strain GR6 of serovar Pomona isolated from pigs in Brazil. Both were added of aluminum hydroxide as an adjuvant. Experimental bacterins were tested with the hamster potency test in order to assess protection provided against the disease and against the establishment of kidney infection. Controls were polyvalent commercial vaccine produced with Leptospira strains isolated outside Brazil, which included a representative of Pomona serovar, or Sorensen solution added of aluminum hydroxide adjuvant. The challenge was performed with cross-strains of serogroup Pomona tested in accordance with international standards established for the potency test. After 21 days of the challenge, survivors were killed to evaluate the condition of Leptospira renal carrier. Experimental bacterins protected hamsters against homologous and heterologous strains, demonstrating the existence of cross-protection. The commercial vaccine protected the hamsters challenged with both strains, but there was a high proportion of animals diagnosed as renal carriers when the challenge was performed with strain GR6, isolated from pigs in Brazil.

Immune responses to Leptospira infection: roles as biomarkers for disease severity

Various leptospiral components have been identified and shown to be involved in tissue destruction. In addition, immune responses to leptospires have been implicated in target organ damages in severe leptospirosis cases. Several inflammatory mediators were shown to be higher in susceptible animals than in resistant hosts. Moreover, cytokines/chemokines and serum proteins induced following Leptospira infection were suggested to be biomarkers for disease severity in human leptospirosis. This review focuses on the role of immune responses in the severity of leptospirosis. Studies in both animal models and humans are discussed.

Avaliação da atividade imunogênica de três proteínas de Leptospira interrogans expressas em Escherichia coli / Evaluation of immunogenic activity of three proteins of Leptospira interrogans expressed in Escherichia coli

A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. No mundo, aproximadamente 500.000 casos são reportados a cada ano, com 10% de taxa de mortalidade. Atualmente, vacinas contra leptospirose são compostas por células inativadas e são ineficazes em diferentes aspectos. Após analise do genoma, os genes LIC11121, LIC11087, LIC11228 e LIC11084 foram escolhidos para caracterização da imunogenicidade de suas respectivas proteínas. Esses genes foram clonados no vetor de expressão pAE e as proteínas recombinantes foram purificadas. Os resultados sugerem que essas proteínas podem estar localizadas na membrana externa, são imunogênicas, possivelmente expressas durante a infecção e que podem ter envolvimento em mecanismos de evasão do sistema imune e de patogenicidade da bactéria. Além disso, em um de dois experimentos, a proteína rLIC11084 induziu imunidade protetora parcial em hamsters imunizados frente desafio letal.

Leptospirosis is a zoonotic disease caused by pathogenic bacteria of genus Leptospira. In the world, nearly 500,000 cases are reported each year, with 10% of mortality rate. Currently, vaccines against leptospirosis are composed by inactivated cells that are ineffective in many aspects. After genome analysis, the genes LIC11121, LIC11087, LIC11228 e LIC11084 were chosen for immunogenicity characterization of their respective proteins. These genes were cloned in the pAE expression vector and the proteins encoded by LIC11087, LIC11228 and LIC11084 were purified. The results suggest the localization of these proteins in the bacterial outer membrane, are immunogenic, are possibly expressed during infection and may have involvement in mechanisms of immune system evasion and pathogenicity. Moreover, in one of two experiments, the rLIC11084 protein induced partial protective immunity of immunized hamsters against lethal challenge.

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 / Efficacy of anti-leptospirosis bacterines: relation between the results of the in vitro growth inhibition test and the potency test, in vivo, with challenge in 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.

Identificação de adesinas de Leptospira interrogans por shotgun phage display / Identification of Leptospira interrogans adhesins by shotgun phage display

Em Leptospira interrogans algumas proteínas com capacidade de ligação aos componentes de matriz extracelular foram identificadas e, em sua maioria, são fatores de virulência. Phage display é considerada uma técnica poderosa na identificação de novos ligantes, inclusive de moléculas adesinas, importantes no primeiro estágio de infecção do hospedeiro. A técnica de shotgun phage display foi utilizada visando à obtenção de ligantes à células de mamíferos. Quatro bibliotecas, por inserção de fragmentos aleatórios obtidos por sonicação do DNA de L. interrogans nos fagomídeos pG8SAET (BBT1 e BBT2) e pG3DSS (BBT5 e BBT6), foram construídas. As bibliotecas BBT1 e BBT5 contém insertos maiores e as BBT2 e BBT6 contém insertos menores, com tamanhos médios de 1500 pb e 350 pb, respectivamente. Após ensaio de panning da BBT5 contra células de mamíferos e soro fetal bovino, as sequências de clones selecionados foram analisadas quanto a orientação correta e se a fusão estava em fase com a proteína pIII. As proteínas codificadas pelos genes LIC11719, LIC10769, LIC13143 e LIC12976 foram selecionadas com estas características. Os genes que codificam a LIC12976, LIC10768, LIC10769 e LIC13418, tiveram sua conservação avaliada em diferentes sorovares da espécie patogênica L. interrogans e no sorovar Patoc da espécie de vida livre L. biflexa. As proteínas LIC12976 (selecionada pela técnica de phage display) e LIC13418 (selecionada por ferramentas de bioinformática) tiveram suas sequências amplificadas por PCR, clonadas em pGEM T easy, subclonadas em vetor de expressão pAE e expressas na fração celular correspondente ao corpúsculo de inclusão em E. coli BL21 (DE3) Star pLysS e E. coli BL21 SI, respectivamente. Após renaturação e purificação destas proteínas por cromatografia de afinidade a metal bivalente, um grupo de cinco animais BALB/c fêmeas foi imunizado. Ambas as proteínas se mostraram imunogênicas com títulos dos soros policlonais 1:256000 e 1:512000, respectivamente...

In Leptospira interrogans, proteins capable to bind to extracellular matrix components have been identified and most of them are important virulence factors. Phage display is a powerful technique to identify new ligands, including adhesin molecules that are important in the first stage of host infection. A shotgun phage display technique was used in order to obtain cell ligands. Four libraries were constructed by inserting random fragments obtained by sonication of L. interrogans DNA into phagemids pG8SAET (BBT1 and BBT2) and pG3DSS (BBT5 and BBT6). The libraries BBT1 and BBT5 contain larger inserts and BBT2 and BBT6 contain smaller inserts, with 1500 bp and 350 bp average sizes, respectively. After panning of BBT5 against mammalian cells and bovine fetal serum, the sequences of selected clones were analyzed for correct orientation and fusion with pIII protein. The proteins encoded by genes LIC11719, LIC10769, LIC13143 and LIC12976 were selected. The genes LIC12976, LIC10768, LIC10769 and LIC13418 were evaluated for their conservation in different pathogenic serovars of L. interrogans and free-living L. biflexa serovar Patoc. Proteins LIC12976 (selected by phage display technique) and also LIC13418 that was selected by bioinformatic tools, were amplified by PCR, cloned into pGEM T easy, subcloned into expression vector pAE and expressed in cellular fraction corresponding to the inclusion body in E. coli BL21 (DE3) Star pLysS and E. coli BL21 SI, respectively. After protein renaturation protocol and purification by affinity chromatography, a group of five BALB/c mice was immunized with the purified proteins. Both proteins were shown to be immunogenic with 1:256000 and 1:512000 polyclonal sera titers, respectively. In Western blot the sera were specific to recognize recombinant proteins and native proteins were detected in pathogenic L. interrogans serovars extracts...

Atypical lymphocytosis in leptospirosis: a cohort of hospitalized cases between 1996 and 2009 in State of Rio de Janeiro, Brazil / Linfócitos atípicos na leptospirose: coorte de pacientes hospitalizados entre 1996 e 2009, Estado do Rio de Janeiro

INTRODUCTION: Leptospirosis is a zoonotic disease found in tropical and temperate countries, and its clinical diagnostic confusion with arboviruses (dengue fever, oropouche fever and yellow fever), Brazilian spotted fever, viral hepatitis and hantaviruses has been an ongoing public health concern. The aim of this observational study was to demonstrate an association between findings of atypical lymphocytosis and the progression of endemic leptospirosis. METHODS: A retrospective analysis was performed on the demographic, epidemiological, clinical and laboratory aspects of 27 human leptospirosis cases that occurred over a period of 13 years (1996-2009) with no reported epidemic outbreaks in Rio de Janeiro, Brazil. RESULTS: The overall mortality rate was 11.1 percent in our cohort of hospitalized cases. However, there was no mortality among patients with atypical lymphocytosis (OR = 11.1; 95 percent CI = 1.12-110.9; p = 0.04). Two patients who were in the septicemic phase showed signs of expansion of γδ T cell responses in peripheral blood. CONCLUSIONS: Atypical lymphocytosis may be observed in patients with leptospirosis. Our observations suggest that these atypical leukocyte subsets are associated with partial protection during the disease course of leptospirosis.

INTRODUÇÃO: Leptospirose é uma zoonose que permanece endêmica em regiões tropicais e temperadas. A dificuldade no diagnóstico clínico diferencial entre os quadros de leptospirose humana e as várias arboviroses (dengue, febre amarela, febre de oropouche), febre maculosa brasileira, hepatite viral e hantavirose permanece um problema na Saúde Pública. MÉTODOS: No presente estudo, foi realizada análise retrospectiva de características demográficas, epidemiológicas, clínicas e laboratoriais de 27 casos de leptospirose humana que ocorrerem durante um período de 13 anos sem ocorrência de notificação de surtos epidêmicos no Rio de Janeiro, Brasil (1996-2009). RESULTADOS: A mortalidade da coorte de pacientes com leptospirose correspondeu a 11,1 por cento, sem embargo, o grupo de pacientes com atipia linfocitária não evoluiu para o óbito (OR = 11,1; 95 por cento CI = 1,12-110,9; p = 0.04). Em duas oportunidades, foi observada uma expansão dos linfócitos T gama-delta no sangue periférico de pacientes na fase septicêmica da leptospirose. CONCLUSÕES: Atipia linfocitária pode ocorrer em pacientes com leptospirose. Nossos dados também sugerem que os linfócitos atípicos podem estar envolvidos na patogênese da leptospirose.

Leptospirose em cão errante da região noroeste do Estado do Paraná - relato de caso / Leptospirosis in a stray dog in the northwestern region of Paraná State - case report / Leptospirosis en perro callejero de la région noroeste del estado de Paraná - relato de caso

A leptospirose é uma doença de caráter zoonótico, provocada pela Leptospira spp. Na área urbana, os cães errantes possuem 3,59 vezes mais risco de se infectar com esta enfermidade, pelo fato de formarem grupos quando fêmeas estão no cio, além da exposição com água empoçada e revirar lixos à procura de restos de alimentos que podem estar contaminados com urina de roedores ou animais infectados. O objetivo deste trabalho foi detectar anticorpos contra Leptospira spp. em um cão errante assintomático para avaliar seu potencial zoonótico. Foi coletado sangue de um cão errante assintomático de um canil privado da região Noroeste do estado do Paraná para a pesquisa de anticorpos contra Leptospira spp. pela SAM. A amostra foi considerada reagente, apresentando anticorpos contra o sorovar Canicola com título de 3.200. O resultado encontrado neste trabalho sugere a presença da Leptospira spp., entre os cães assintomáticos do referido abrigo, demonstrando a importância de se conhecer a ocorrência desta enfermidade que é uma zoonose de importância para saúde pública.

Leptospirosis is a zoonotic disease caused by Leptospira spp. In urban areas, stray dogs have 3.59 times higher risk of becoming infected with this disease because they form groups when females are in heat besides their exposure to still water and trash when looking for food scraps that may be contaminated by urine of infected rodents or animals. The objective of this study was to detect antibodies against assymptomatic Leptospira spp. in a stray dog ??to evaluate its zoonotic potential. Blood was collected from an asymptomatic stray dog of a private shelter in the northwest of Paraná state for antibodies against Leptospira spp. by SAM. The sample was considered reactive, with antibodies against serovar Canicola title with 3.200. The findings of this study show the presence of Leptospira spp. among asymptomatic dogs of that shelter, demonstrating that knowing the occurrence of this disease is a zoonosis of public health importance.

La leptospirosis es una enfermedad de carácter zoonótico, provocada por Leptospira spp. En el área urbana, los perros callejeros poseen 3,59 más riesgo de infectarse con esta enfermedad, por el hecho de estar en grupos cuando las hembras están en celo, además de la exposición en agua estancada y revolver basuras en búsqueda de alimentos que pueden estar contaminados con orina de roedores o animales infectados. El objeto de esta investigación fue detectar anticuerpos contra Leptospira spp., en un perro callejero asintomático para evaluar su potencial zoonótico. La sangre fue colectada de un perro callejero asintomático en una perrera privada de la región Noroeste del Estado de Paraná para investigación de anticuerpos contra Leptospira spp., por la SAM. La muestra fue considerada reactiva, presentando anticuerpos contra el serovar Canicola con título de 3.200. El resultado encontrado en esta investigación sugiere la presencia de Leptospira spp., entre los perros asintomáticos del referido abrigo, demostrando la importancia en conocer la ocurrencia de esta enfermedad que es una zoonosis de importancia para la salud pública.

Prevalencia de anticuerpos contra Leptospira en población urbana humana y canina del Departamento del Tolima / The prevalence of antibodies against Leptospira in urban human and canine populations from the Tolima Department

Objetivo Determinar la seroprevalencia de anticuerpos contra Leptospira sp. y los serovares dominantes, en población urbana humana y canina de tres municipios del departamento del Tolima, Colombia. Materiales y Métodos Se realizó un estudio epidemiológico trasversal con selección de sujetos por conveniencia en 62 barrios. Se obtuvieron muestras de 850 personas.y 850 caninos durante los meses de junio, julio y agosto de 2007, las cuales fueron procesadas utilizando la prueba de microaglutinación (MAT). En la prueba se incluyeron 5 serovares: Hardjo, Pomona, Grippotyphosa, Canicola, Icterohaemorrhagiae y Bratislava. La prueba se interpretó como positiva por la presencia de una aglutinación >50 por ciento de las leptospiras con uno o más serovares, en una dilución del suero >100 para las dos especies. Resultados Se encontró evidencia de infección en el 6 por ciento de la población humana y en el 21,4 por ciento de los caninos, con al menos uno de los serovares. La mayor reactividad fue para los serovares Pomona y Grippotyphosa para humanos y caninos. Se presentaron coaglutinaciones en el 13,7 por ciento de los sueros humanos y en el 4,4 por ciento de los caninos. Fue evidente la disminución de la frecuencia de aglutinación de los serovares Canicola e Icterohaemorrhagiae en los perros. Conclusiones Se observa un cambio en el patrón de presentación de la leptospirosis en poblaciones urbanas, siendo necesario fortalecer la vigilancia epidemiológica activa y pasiva, la implementación de pruebas rutinarias de diagnóstico y medidas de prevención específica en los reservorios animales.

Objective Determining Leptospira sp. antibody seroprevalence and prevalent serovars in human and canine populations from three municipalities in the Tolima department of Colombia. Materials and Methods This was a cross-sectional study (subjects being selected by convenience sampling in 62 districts). Samples were collected from 850 dogs and 850 people during June, July and August 2007; these were processed using a microscopic agglutination test (MAT) and five serovars: Hardjo, Pomona, Grippotyphosa, Canicola, Icterohaemorrhagiae and Bratislava. MAT was considered positive when 50 percent or more leptospira were agglutinated with one or more serovars in a 1:100 serum dilution in both species. Results At least one serovar showed evidence of infection in 6 percent of the people and 21.4 percent of canines. The most prevalent serovars were Pomona and Grippotyphosa in two populations. Co-agglutinations were present in 49 percent of human sera and 19.2 percent of canine samples. Decreased Canicola and Icterohaemorrhagiae serovar agglutination frequency was evident in dogs. Conclusions There was a change in leptospirosis pattern in urban populations. Active and passive surveillance must be strengthened, routine diagnostic tests implemented and preventative measures taken in specific animal reservoirs.

Identificación de antígenos inmunorreactivos de Leptospira interrogans / Identification of immunoreactive antigens of Leptospira interrogans

Se estudió un lote de 28 sueros de llama (Lama gama) de la provincia de Jujuy, Argentina, a fin de identificar antígenos inmunorreactivos contra Leptospira interrogans. Se utilizaron distintas preparaciones antigénicas de la bacteria para estudiar la inmunorreactividad mediante microaglutinación (MAT), ELISA y Western inmunoblot. Un pool de sueros bovinos positivos a la MAT fue empleado como control. Todos los sueros de llama fueron negativos mediante MAT e igual resultado se observó mediante ELISA. Dos de los 28 sueros de llama y el pool de sueros bovinos positivos, al ser evaluados por Western inmunoblot, arrojaron resultados positivos y permitieron identificar proteínas inmunorreactivas. Por MALDI-TOF se logró establecer que la proteína asociada a los dos sueros de llama inmunorreactivos era una flagelina periplásmica de Leptospira interrogans serovar Lai STR, mientras que la asociada al pool de sueros bovinos positivos a Leptospira sp. se trataba de una lipoproteína de la membrana externa de Leptospira interrogans serovar Ballum, LipL21. Estas proteínas podrían ser utilizadas en el diseño de un nuevo ELISA aplicado al diagnóstico temprano de leptospirosis, ya sea en distintos tipos de ganado como así también en reservorios silvestres.

A batch of 28 llama (Lama gama) sera from Jujuy province in Argentina was studied in order to identify immune reactive antigens to Leptospira interrogans. Different antigenic preparations from the bacterium were used to study the immune reactivity by the microagglutinattion (MAT), ELISA and Western immunoblot tests. A control pool of positive bovine sera was used. All the llama sera were negative to MAT as well as to ELISA. Two of the llama sera and the positive bovine sera pool rendered positive results when evaluated by Western immunoblot, allowing the identification of immune reactive proteins. These proteins were identified by MALDI-TOF. A periplasmic flagellin of Leptospira interrogans serovar Lai STR called FlaB1 was identified from the reactive llama sera, and an external membrane lipoprotein of Leptospira interrogans serovar Ballum called LipL21 was identified from the pool of bovine positive sera. These proteins could be used in a new ELISA applied to the early diagnosis of leptospirosis in different kind of cattle or wild reservoirs.

Immune strategies using single-component LipL32 and multi-component recombinant LipL32-41-OmpL1 vaccines against leptospira

Genes encoding lipoproteins LipL32, LipL41 and the outer-membrane protein OmpL1 of leptospira were recombined and cloned into a pVAX1 plasmid. BALB/c mice were immunized with LipL32 and recombined LipL32-41-OmpL1 using DNA-DNA, DNA-protein and protein-protein strategies, respectively. Prime immunization was on day 1, boost immunizations were on day 11 and day 21. Sera were collected from each mouse on day 35 for antibody, cytokine detection and microscopic agglutination test while spleen cells were collected for splenocyte proliferation assay. All experimental groups (N = 10 mice per group) showed statistically significant increases in antigen-specific antibodies, in cytokines IL-4 and IL-10, as well as in the microscopic agglutination test and splenocyte proliferation compared with the pVAX1 control group. The groups receiving the recombined LipL32-41-OmpL1 vaccine induced anti-LipL41 and anti-OmpL1 antibodies and yielded better splenocyte proliferation values than the groups receiving LipL32. DNA prime and protein boost immune strategies stimulated more antibodies than a DNA-DNA immune strategy and yielded greater cytokine and splenocyte proliferation than a protein-protein immune strategy. It is clear from these results that recombination of protective antigen genes lipL32, lipL41, and ompL1 and a DNA-protein immune strategy resulted in better immune responses against leptospira than single-component, LipL32, or single DNA or protein immunization.

Títulos de anticorpos aglutinantes induzidos por vacinas comerciais contra leptospirose bovina / Agglutinating antibody titers induced by commercial vaccines against bovine leptospirosis

No presente estudo, 100 fêmeas bovinas foram divididas em cinco grupos de 20 animais cada. Os grupos experimentais receberam quatro diferentes vacinas comerciais (B, C, D e E), e um grupo permaneceu como controle. Amostras foram colhidas no dia da aplicação da primeira dose e nos dias 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 120, 150 e 180 pós-vacinação (PV). A triagem dos animais foi feita pela análise sorológica com 6 antígenos de leptospiras, escolhendo-se os animais não reagentes. Os títulos de anticorpos foram monitorados pela soroaglutinação microscópica (SAM) com os sorovares Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona e Wolffi. Todas as vacinas induziram, aos 3 dias PV, títulos de anticorpos aglutinantes para os sorovares Hardjo e Wolffi, que persistiram até o 150º dia PV. Os sorovares Hardjo e Wolffi induziram os maiores títulos de anticorpos aglutinantes. A vacina D, apesar de não possuir o sorovar Wolffi em sua composição foi capaz de induzir anticorpos aglutinantes contra este sorovar. Somente foram detectados anticorpos contra o sorovar Canicola nos animais vacinados com a bacterina D. A vacina que induziu os maiores títulos médios de anticorpos, considerando todos os sorovares testados foi a D.

In the investigation 100 heifers were used, divided into 5 groups of 20 animals each. The four experimental groups were vaccinated using distinct commercial polyvalent bacterines: B, C, D and E, and A group was the control. Samples were collected at days 0, 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 120, 150 and 180 from the first injection of the vaccine. The selection of the animals for the experimental groups was done based on a serological screening with 6 antigens of Leptospira sp. constituted by non-reagent animals. The vaccine titers were monitored using the microscopic agglutination test (MAT) for Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona and Wolffi serovars. All vaccines used were capable to product agglutinins for the Hardjo and Wolffi serovars observed at 3 days after vaccination, remaining until the 150th day; those serovars induced the highest titres of agglutinins. Vaccine D, in spite of not containing the Wolffi serovar, induced the production of agglutinins to this serovar. Agglutinins to the Canicola serovar were only observed in the animals vaccinated with the D bacterine. Vaccine D induced the highest average titers of antibodies to all tested serovars.