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.

A cross sectional study of leptospirosis and fetal death in Yucatan, Mexico / Estudio transversal de leptospirosis y muerte fetal en Yucatán, México

Introduction: Leptospirosis is a zoonotic disease affecting mainly to low income human population. Acute leptospiral infection during pregnancy has been associated with spontaneous abortion and fetal death during the first trimester and the abortion may occur as consequence of systemic failure. Objective: To estimate the frequency of Leptospira interrogans infection in women with spontaneous abortion in the state of Yucatan, Mexico. Methods: A cross sectional study on women with spontaneous abortion was conducted. Serum samples were tested for Leptospirosis by the microaglutination test, to estimate the frequency of the infecting serovar. The indirect ELISA IgM was used to detect recent infection by L. interrogans. DNA was extracted from paraffin-embedded tissue of placenta for PCR detection of L. interrogans. Results: Overall frequency of infection with L. interrogans in the 81 women with abortion was 13.6%. Five of the 12 serovars evaluated were found and included. Two of the 11 women with abortion and positive to microaglutination test were also positive to the ELISA IgM test. None samples were positive for PCR Leptospira diagnosis. Conclusion: two women could be associated with spontaneous abortion due to leptospirosis, because they showed antibodies against L. interrogans in the microaglutination test and ELISA IgM assays. Differences between regions were found with respect to the prevalences of lesptospirosis.

Introducción: Leptospirosis es una enfermedad zoonótica que afecta principalmente la población humana de bajos recursos. Infección aguda por leptospirosis durante el embarazo se ha asociado con aborto espontáneo y muerte fetal durante el primer trimestre del embarazo. Objetivo: Estimar la frecuencia de infección por Leptospira interrogans en mujeres con aborto espontáneo en el estado de Yucatán, México. Métodos: Se efectuó un estudio trasversal en 81 mujeres con aborto espontáneo. La prueba de referencia para Leptospirosis, prueba de microaglutinación, se utilizó para estimar la frecuencia de la serovar infectante. El ELISA IgM indirecto se utilizó para detectar infección reciente por L. interrogans. Se extrajo ADN a partir de tejido embebido en parafina de placenta para la detección de L. interrogans por PCR. Resultados: La frecuencia global de la infección con L. interrogans en 81 mujeres con aborto, fue del 13.6%. Se encontró cinco de 12 serovares de Leptospira. Dos de las 11 mujeres con aborto espontáneo y positivo a microaglutinación también fueron positivas a la prueba ELISA IgM. Ninguna muestra fue positiva hacia el diagnóstico PCR de Leptospira. Conclusion: Dos mujeres podrían estar asociados con el aborto espontáneo debido a la leptospirosis, porque mostraron anticuerpos contra L. interrogans en la microaglutinación y ensayos de ELISA IgM. Diferencias entre regiones fueron encontradas con respecto a las prevalencias de leptospirosis.

Occurrence of antibodies anti -Toxoplasma gondii, Neospora caninum and Leptospira interrogans in a captive deer herd in Southern Brazil / Ocorrência de anticorpos anti -Toxoplasma gondii, Neospora caninum e Leptospira interrogans em um rebanho de cervídeos de cativeiro do sul do Brasil

Abstract A large number of Brazilian zoos keep many endangered species of deer, however, very few disease surveillance studies have been conducted among captive cervids. Blood samples from 32 Brazilian deer (Blastocerus dichotomus, Mazama nana and Mazama americana) kept in captivity at Bela Vista Biological Sanctuary (Foz do Iguaçu, Brazil) were investigated for 10 ruminant pathogens, with the aims of monitoring deer health status and evaluating any potential zoonotic risk. Deer serum samples were tested for Brucella abortus, Leptospira (23 serovars), Toxoplasma gondii, Neospora caninum, bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, foot-and-mouth disease virus, western equine encephalitis virus, eastern equine encephalitis virus and Venezuelan equine encephalitis virus. Antibodies against T. gondii (15.6%), N. caninum (6.2%) and L. interrogans serogroup Serjoe (3.1%) were detected. The serological results for all other infectious agents were negative. The deer were considered to be clinically healthy and asymptomatic regarding any disease. Compared with studies on free-ranging deer, the prevalences of the same agents tested among the captive deer kept at the Sanctuary were lower, thus indicating good sanitary conditions and high-quality management practices at the zoo.

Resumo Um grande número de zoológicos brasileiros abriga espécies de cervídeos ameaçados de extinção, entretanto, estudos de vigilância de doenças em cervídeos de cativeiro são escassos. Amostras de sangue de 32 cervídeos brasileiros (Blastocerus dichotomus, Mazama nana e Mazama americana), mantidos em cativeiro no Refúgio Biológico Bela Vista (Foz do Iguaçu, Brasil), foram investigados para 10 patógenos de ruminantes, visando monitorar o estado de saúde dos cervídeos e avaliar a presença de agentes zoonóticos. As amostras de soro foram testadas para Brucella abortus, Leptospira (23 sorovares), Toxoplasma gondii, Neospora caninum, diarreia viral bovina, rinotraqueíte infecciosa bovina, febre aftosa, encefalomielite equina do oeste, encefalomielite equina do leste e encefalomielite equina venezuelana. Foram detectados anticorpos para T. gondii (15,6%), N. caninum (6,2%) e para L. interrogans sorogrupo Serjoe (3,1%). As sorologias apresentaram resultado negativo para as demais doenças. Os cervídeos foram considerados clinicamente sadios e assintomáticos para doenças. Comparados aos estudos de populações de vida livre, as soroprevalências para os mesmos agentes testados foram menores para os cervídeos mantidos no Refúgio, indicando as boas condições sanitárias e a qualidade das práticas de manejo no zoológico.

Serosurvey of Leptospira interrogans, Brucella abortus and Chlamydophila abortus infection in free-ranging giant anteaters (Myrmecophaga tridactyla) from Brazil / Inquérito sorológico de Leptospira interrogans, Brucella abortus e Chlamydophila abortus em tamanduás-bandeira (Myrmecophaga tridactyla) de vida livre no Brasil

A serological survey for antibodies against Leptospira interrogans, Brucella abortus, and Chlamydophila abortus was conducted in 21 clinically healthy, free-ranging giant ant- eaters (Myrmecophaga tridactyla) from Parque Nacional das Emas (Goiás State, Brazil; n=6), Parque Nacional da Serra da Canastra (Minas Gerais State, Brazil; n=9), and RPPN SESC Pantanal (Mato Grosso State, Brazil; n=6) between July 2001 and September 2006. Sera were screened for antibodies against 22 serovars of Leptospira interrogans with a microscopic agglutination test. Twelve tested positive for L. interrogansserovars sentot (n=5 in PN Emas, n=2 in PN Serra da Canastra), butembo (n=2 in PN Serra da Canastra), autumnalis, bataviae, and shermani/icterohaemorrhagiae(n=1 each in SESC Pantanal)One adult female tested positive for B. abortus with the buffered plate antigen test. All sera were negative for C. abortususing the complement fixation text. This is the first report of pathogens that may interfere with the reproduction and population dynamics of free-ranging giant anteaters.

Inquéritos sorológicos para detecção de anticorpos contra Leptospira interrogans, Brucella abortus, e Chlamydophila abortus foram realizados em 21 tamanduás-bandeira (Myrmecophaga tridactyla) de vida livre do Parque Nacional das Emas (Goiás, Brasil, n=6), o Parque Nacional da Serra da Canastra (Minas Gerais, Brasil, n=9) e RPPN SESC Pantanal (Mato Grosso, Brasil, n=6) entre julho de 2001 e setembro de 2006. Os sor os foram testados para anticorpos contra 22 sorotipos de Leptospira interrogans com um teste de aglutinação microscópica. Doze animais foram considerados positivos para L. interrogans sorovares sentot (n=5 em PN Emas, n=2 em PN Serra da Canastra), butembo (n=2 em PN Serra da Canastra), autumnalis, bataviae e shermani/icterohaemorrhagiae(n=1 para cada sorovar em SESC Pantanal). Uma fêmea adulta testou positivo para B. abortuscom o teste do antígeno tamponado. Todos os soros se mostraram negativos para C. abortusatravés do teste de fixação do complemento. Este é o primeiro relato de patógenos que podem interferir na dinâmica reprodutiva de populações de tamanduás em estado selvagem.

Using a top predator as a sentinel for environmental contamination with pathogenic bacteria: the Iberian wolf and leptospires

The Iberian wolf (Canis lupus) is the top predator in the Iberian environments in which it lives, feeding on a wide range of species, thus encountering a wide range of disease agents. Therefore, the wolf can serve as sentinel of environmental contamination with pathogens. We investigated the exposure of free-living wolves to 14 serovars of Leptospira interrogans sensu lato. Kidney samples from 49 wolves collected from 2010-2013 in northwestern Spain were analysed by culture, direct immunofluorescence and polymerase chain reaction. Tissue fluids were analysed for antibodies by a microscopic agglutination test. Ten wolves (observed prevalence: 20%, 95% confidence interval = 11-33%) showed evidence of contact with leptospires, eight through direct detection and nine through serology (7 wolves were positive according to both techniques). Titres below the cut-off level were also detected in seven cases. Serovars confirmed were Canicola (n = 4), Icterohaemorrhagiae (n = 3) and Sejroë, Ballum and Grippotyphosa (n = 1 each), indicating that wolves were infected with serovars for which dogs, rodents and ungulates, are the natural hosts and supporting the utility of the wolf and other large predators as environmental sentinels for pathogens.

Survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction

This work reports a survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction (PCR). Seventy pampas deer were captured in the dry season and surveyed using PCR, microscopic agglutination test (MAT) (n = 51) and by both techniques (n = 47). PCR detected infections in two pampas deer and MAT detected infections in three. Through sequencing and phylogenetic analyses, the PCR-amplified fragment detected in deer was identified as Leptospira interrogans. Serovars Pomona and Butembo were detected using MAT and the highest titre was 200 for serovar Pomona. Epidemiological aspects of the findings are discussed.

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.

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.

Hemorrhagic pulmonary leptospirosis: three cases from the Yucatan peninsula, Mexico / Leptospirose hemorrágica pulmonar: três casos na península de Yucatán, México

Three leptospirosis cases with lung involvement are reported from the Yucatan Peninsula, Mexico. All three patients were admitted to the intensive care unit due to acute respiratory failure. Treatment with antibiotics resulted in favorable evolution despite the negative prognosis. Leptospirosis should be included in the differential diagnosis of patients with fever and lung involvement.

Analisamos três casos de leptospirose com envolvimento pulmonar na Península Yucatán, México. Os três pacientes com seqüelas pulmonares entraram na unidade de cuidados intensivos devido à insuficiência respiratória grave. Todos os casos evoluíram favoravelmente ao tratamento com antibióticos, apesar do prognóstico negativo. Leptospirose deve ser incluída no diagnóstico diferencial de pacientes com febre e comprometimento pulmonar.

Diagnosis of leptospirosis by recombinant antigen based single serum dilution ELISA.

BACKGROUND & OBJECTIVES: Leptospirosis, a zoonosis with a worldwide distribution is an acute febrile illness caused by spirochaetes of the pathogenic Leptospira interrogans. Microscopic agglutination test (MAT), the reference method for diagnosis was successively done to evaluate the modified ELISA which was developed with the recombinant LipL32 antigen for the detection of anti-leptospiral antibodies in human serum samples. METHODS: The recombinant LipL32 antigen was developed from the serovar Pomona strain Pomona of the pathogenic L. interrogans species. The predicted titre at a single working dilution was plotted against the observed antiserum titre. Subsequently, predicted antibody activity titres were determined directly from the standard curve by solving the regression line equation. The relative sensitivity, specificity and accuracy of the single dilution ELISA for the detection of anti-leptospiral antibodies were determined in comparison to the MAT. RESULTS: A linear relationship was found between the predicted antibody titres at a single working dilution of 1:250 and the corresponding observed serum titres by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. A high level of sensitivity of 96 per cent and specificity of 91 per cent between ELISA and MAT titres was found. The kappa value was almost 1.0 indicating perfect agreement. INTERPRETATION & CONCLUSIONS: The r LipL32 ELISA was proved to be sensitive, specific and accurate as compared to the standard MAT and the test could be efficiently utilized as a screening test for a large number of human serum samples for the detection of leptospiral antibodies.

Perfil dos cães sororreagentes para aglutininas anti-Leptospira interrogans em Belo Horizonte, Minas Gerais, 2001/2002 / Serological profile of seropositive dogs to anti-Leptospira interrogans agglutinins in Belo Horizonte, Minas Gerais, Brazil, 2001-2002

The serological profile of seropositive dogs according to anti-Leptospira agglutinins was checked in Belo Horizonte including variables such as race, sex, age and whether the dog had an owner or not. The dogs were captured by the Zoonosis Control Center in nine neighborhoods around the city and were separated in two categories - with owners or captured on the streets. The prevalence of anti-Leptospira agglutinins was evaluated in 3,417 blood samples using the microscopic agglutination test (MAT) from September 2001 to September 2002. It was found that 13.1 percent of the dogs had seropositive results with the most reactive serovars being Canicola (7.0 percent), Ballum (6.1 percent), Pyrogenes (3.2 percent) and Icterohaemorrhagiae (2.9 percent). The prevalence of other serovars was less than 1.0 percent. Greater prevalence was found in male, crossbred dogs, without owners. There were no significant results due to age in 95 percent (P=0.808) of the cases. According to the results, more research should be done in order to isolate and classify the serovars in positive dogs, especially Ballum and Pyrogenes, which will suggest their inclusion in the commercial vaccines against leptospira used in dogs in this city

Situación de la leptospirosis en el Urabá antioqueño colombiano: estudio seroepidemiológico y factores de riesgo en población general urbana / Leptospirosis in Uraba, Antioquia, Colombia: a seroepidemiological and risk factor survey in the urban population

Leptospirosis es una zoonosis de gran incidencia en regiones tropicales. Su prevalencia es desconocida en la región del Urabá colombiano. Entre marzo y octubre del año 2000 se realizó un estudio descriptivo de corte para determinar la seroprevalencia de anticuerpos contra Leptospira spp. y describir algunos factores de riesgo en nueve municipios del Urabá. La población incluida fue de 582 personas a las cuales se les tomó una muestra de sangre y se le aplicó una encuesta sobre factores de riesgo. La detección de anticuerpos contra Leptospira spp. fue realizada por inmunofluorescencia indirecta y por microaglutinación. La seroprevalencia general en la zona fue 12,5 por ciento (IC95 por ciento: 10,01-15,5). No hubo diferencias en cuanto al sexo, raza, oficio, edad, años de residencia en la zona y características de la vivienda. L. interrogans serovar Grippotyphosa fue la especie más prevalente, identificándose en 53 de los seropositivos. En 38 seropositivos los títulos detectados fueron iguales o mayores a 1:400. En conclusión, existe alta prevalencia de anticuerpos contra Leptospira spp. Es necesario orientar las medidas de control para disminuir el riesgo de exposición ambiental a leptospirosis por parte de los habitantes de la zona.

Leptospirosis is a widespread zoonosis in tropical regions. The prevalence is unknown in the Colombian region of Uraba. A cross sectional study was conducted from March to October 2000 in order to determine the prevalence of Leptospira spp. antibodies and describe risk factors in nine counties in the region. The sample consisted of 582 individuals, who answered a questionnaire and had blood samples drawn to determine risk factors. Detection of Leptospira spp. antibodies was based on indirect inmunofluorescence and microagglutination. Seroprevalence was 12.5 percent (95 percentCI: 10.01-15.5). No differences were observed according to race, gender, occupation, age, living conditions, or time of residence in the area. L .interrogans serovar Grippotyphosa was the most prevalent species, identified in 53 individuals. Titers were > 1:400 in 38 seropositive individuals. In conclusion, there is a high prevalence of Leptospira spp. antibodies in the area, where it is thus necessary to establish control measures to decrease the risk of environmental exposure to leptospirosis.

Immunoreactive outer membrane proteins of Leptospira interrogans serovar Canicola strain Hond Utrecht IV.

BACKGROUND & OBJECTIVES: Leptospirosis is a severe and complex zoonotic disease prevalent in many countries including India. Current leptospiral research is focussed on the identification of the outer membrane proteins (OMPs) of the organism that could be used in developing diagnostic assays for leptospirosis. METHODS: The Leptospira interrogans serovar Canicola was grown in EMJH medium and the cells were subjected to sarcosyl detergent treatment. The sarcosyl soluble (SS) and sarcosyl insoluble (SI) fractions were analyzed by SDS-PAGE and immunoblotting to deduce their protein profile and identifying various immunodominant antigens. RESULTS: The protein profile of SS fractions indicated the presence of three major bands of 41, 32 and 25 kDa and minor bands of 85 and 46 kDa. The SI fraction in serovar Canicola revealed the presence of 112, 93, 77, 43, 36, 29 and 22.5 kDa as major bands and minor bands of 102 and 53 kDa. In immunoblotting, the SS proteins of 41, 32 and 25 kDa and SI proteins of 112, 77, 36 and 22.5 kDa were detected to be major immunogenic proteins. INTERPRETATION & CONCLUSION: In our study immunogenic proteins were extracted from SS and SI fractions and OMPs were similar to those reported in other pathogenic Leptospira strains. These OMPs being unique to all the pathogenic leptospires, can be targeted for diagnostic purpose. Further analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira cells.

Presença de anticorpos da classe IgM de Leptospira interrogans em animais silvestres do Estado do Tocantins, 2002 / Presence of IgM antibodies for Leptospira interrogans in wild animals from Tocantins State, 2002

Quatrocentos e vinte e sete amostras de soro provenientes de animais silvestres foram testadas frente a 18 sorovariedades de Leptospira interrogans. De 286 amostras de Cebus apella, 46 (16,1 por cento) foram positivas para as sorovariedades pomona, brasiliensis, mini, swajizak, grippothyphosa, sarmin, fluminense, autumnalis, hebdomadis, guaratuba, javanica e icterohaemorhagiae. Das 82 de Alouatta caraya, 2 (2,4 por cento) foram positivas para as sorovariedades mangus e fluminense. Das 31 de Nasua nasua, 4 (12,9 por cento) foram positivas para as sorovariedades fluminense e javanica. Das 10 amostras de Cerdocyon thous, 2 (20 por cento) foram positivas para as sorovariedades fluminense e brasiliensis. Sete de Dasyprocta sp, 6 de Tamandua tetradactila e 5 de Euphractus sexcintus não apresentaram reatividade.

Inmunogenicidad y capacidad protectora en hamsters de vacunas antileptospirósicas monovalentes de células enteras del serogrupo Ballum / Immunogenicity and protective capacity of leptospiral whole-cell monovalent serogroup Ballum vaccines in hamsters

El serogrupo Ballum de Leptospira constituye en la actualidad la primera causa de leptospirosis humana en Cuba. Vacunas de células enteras químicamente inactivadas fueron formuladas a partir de dos cepas clínicas de Leptospira interrogans serogrupo Ballum empleando como adyuvante hidróxido de aluminio. Los niveles de aglutininas inducidos en hamsters por una u otra preparación vacunal fueron estimados mediante aglutinación microscópica y la actividad IgG específica fue cuantificada mediante ELISA. La capacidad de protección homóloga y heteróloga contra la infección letal y subletal se determinó mediante el desafío con 100 y 10 000 DL50 de cinco cepas virulentas pertenecientes a los serogrupos Ballum, Canicola, Icterohaemorrhagiae y Pomona. Las evaluaciones realizadas demostraron que ambas vacunas fueron inmunogénicas e indujeron una completa protección homóloga en el modelo animal empleado. La protección cruzada frente a serogrupos heterólogos solo fue significativa en una de las preparaciones monovalentes frente al desafío con 100 DL50 de Canicola. Como resultado de este estudio se pudo comprobar la alta inmunogenicidad y capacidad protectora en hamsters de vacunas monovalentes de células enteras formuladas a partir de dos cepas candidatas vacunales del serogrupo de Leptospira de mayor circulación en humanos en Cuba no incluido en la vacuna actualmente disponible.

Leptospira serogroup Ballum is at present the first cause of human leptospirosis in Cuba. Killed whole-cell vaccines were formulated with two clinical isolates of Leptospira interrogans serogroup Ballum using aluminum hydroxide as adjuvant. Agglutinins levels induced by each vaccine in hamsters were estimated by microscopic agglutination test and specific IgG activities were quantified by a whole cell-based enzyme-linked immunosorbent assay. Homologous and cross protective capacity against lethal and sublethal infection were determined in vaccinated animals by challenge with 100 and 10 000 LD50 of five virulent strains belonging to serogroups Ballum, Canicola, Icterohaemorrhagiae and Pomona. Both monovalent serogroup Ballum vaccines were immunogenic and induced complete homologous protection in the animal model. Cross-protection was only significant in one of the two vaccines against challenge with 100 LD50 of serogroup Canicola. The results of this study demonstrate the high immunogenicity and protective capacity in hamsters of whole-cell monovalent vaccines formulated with two vaccine candidate strains belonging to the most prevalent serogroup of Leptospira in Cuba.

Leptospira interrogans activation of peripheral blood monocyte glycolipoprotein demonstrated in whole blood by the release of IL-6

Glycolipoprotein (GLP) from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC) and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml) of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control) or medium (negative control), and supernatants were collected after 6, 20/24, and 48 h, and kept at -80°C until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml)-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.

Two simple immunoassays using endemic leptospiral antigens for serodiagnosis of human leptospirosis.

Two simple enzyme immunoassays, a conventional microplate and dot-ELISA, were developed to detect specific IgM antibodies using pool sonicated antigen prepared from three of the most reactive serovars of Leptospira associated with disease in Thailand. Both assays were evaluated and compared with the standard microscopic agglutination test (MAT) performed with 343 serum samples. A battery of 16 pathogenic serovars of L. interrogans were used as antigens in the MAT assay. The result of MAT at serum titers > or = 1:400 showed three pathogenic serovars of leptospira, Bratislava (71.88%), Sejroe (63.54%) and Pyrogenes (36.46%), were among the most commonly reacted serovars and they were selected for preparation of pool sonicated antigen for both IgM ELISA tests. The microplate IgM-ELISA, performed with sera at 1:80 dilution using the cutoff OD of 0.60, demonstrated sensitivity, specificity and efficiency of 87.50, 97.57, and 94.75%, respectively. The same values for IgM dot-ELISA performed with sera at 1:160 dilution were 98.96, 93.93, and 95.33%, respectively. Both ELISA methods showed results with statistically significant differences from MAT (p < 0.05). The agreement rate of IgM dot-ELISA compared with microplate IgM-ELISA was 0.85 by Kappa analysis. Both assays offered relatively high negative predictive values (95.26-99.57%), thus making the assays ideally suited for rapid screening. Future applications of the IgM dot-ELISA as a test kit would be suitable for use at the peripheral level as a rapid screening test for human leptospirosis.

Evaluation of enzyme-linked immunosorbent assay and indirect hemagglutination assay for detection of leptospiral antibody by using three different antigens.

Evaluation of enzyme linked immunosorbent assay (ELISA) and indirect hemagglutination assay (IHA) for detection of leptospiral antibody were performed using three different soluble antigens extracted from Leptospira interrogans serovar Bataviae including heat extracted antigen (HEA), sonicated antigen (SA) and deoxycholate extracted antigen (DEA). One hundred seventy-eight sera from 85 confirmed leptospirosis cases and 202 non-leptospirosis cases were examined SA-IHA showed the highest sensitivity of 98.8% followed by SA-ELISA-IgM and DEA-ELISA-IgM which showed an equal sensitivity of 97.6%. SA-ELISA-IgM was the most specific (96.5%) test followed by DEA-ELISA-IgM and SA-IHA which gave an equal specificity of 94.1%. The IgG-ELISA tests were less sensitive and specific than the others. These data indicated that the IgG-ELISA tests were not suitable as diagnostic tests for acute leptospirosis in humans. Using acute phase sera the sensitivity of DEA-ELISA-IgM, SA-ELISA-IgM and HEA-ELISA-IgM were 22.4%, 20.0% and 20.0% respectively, while those of the IgG ELISA tests were 32.9%, 17.6% and 10.6% respectively, the sensitivity of SA-IHA and DEA-IHA were 22.4% and 10.6% respectively. In conclusion, both the IgM-ELISA tests using SA and DEA and the IHA using SA are not different in diagnosis of human leptospirosis. SA is the most suitable antigen for detection of leptospiral antibody by IgM ELISA and IHA.

Caracterización microbiológica de cepas candidatas vacunales de Leptospira interrogans serogrupo Ballum

Se caracterizaron microbiológicamente 2 cepas candidatas vacunales de Leptospira interrogans serogrupo Ballum, como parte de los trabajos encaminados a la obtención de nuevas formulaciones vacunales antileptospirósicas para uso humano. Se evaluó la cinética de crecimiento de ambas cepas en medio proteico EMJH y en 3 medios libres de proteínas. Se estimó la virulencia en hámsters mediante el cálculo de la dosis letal media. Fueron analizados los perfiles antigénicos celulares y extracelulares mediante SDS-PAGE unidimensional y comparados con aquellos de cepas pertenecientes a los serogrupos Canicola, Icterohaemorrhagiae y Pomona. La homología antigénica entre serogrupos heterólogos se analizó mediante western blotting con suero de hámsters vacunados con vax-SPIRAL®. Los resultados obtenidos demostraron un crecimiento fastidioso de ambas cepas de Ballum en los medios de cultivo estudiados, una alta virulencia en el modelo animal y una gran homología antigénica, con cepas pertenecientes a otros serogrupos de Leptospira de mayor circulación en Cuba.

Two candidate vaccines of Ballum serogroup Leptospira interrogans were microbiologically characterized as part of the work directed to the obtention of new antileptospirosis vaccine formulations for human use. The growth kynetics of both strains was evaluated in EMJH protein medium and in 3 protein free media. The virulence was estimated in hamsters by the calculation of the mean lethal dose. The cellular and extracellular antigenic profiles were analyzed by unidimensional SDS-PAGE and compared with those from strains of Canicola, Icterohaemorrhaagiaae and Pomona serogroups.The antigenic homology among heterologous groups was analyzed by western blotting with serun from hasterms vaccinated with vax-SPIRAL®. The results obtained showed a fastidious growth of both strains of Ballum in the studied media, a high virulence in the animal model and a large antigenic homology with strains from other serogroups of Lepstospira prevailing in Cuba.

Leptospirose-infecção e forma subclínica em crianças de Salvador, Bahia / Leptospiral infection and subclinical presentation among children in Salvador, Bahia

Os objetivos deste trabalho foram estimar a freqüência da leptospirose e os fatores de risco em crianças de 2 a 15 anos de idade, de ambos sexos, contactantes-domiciliares dos pacientes internados (casos-índices) no Hospital Couto Maia, com diagnóstico de leptospirose (ELISA-IgM positivo). De 148 pessoas, dos domicílios dos 25 casos-índices, selecionados aleatoriamente, 73 (49,3 por cento) contatos-domiciliares foram submetidos à avaliaçäo clínico-epidemiológica e ao mesmo teste diagnóstico em soros pareados. Os resultados das IgM e IgG classificaram os 73 contatos-domiciliares em três grupos: A (infecçäo aguda ou recente), 30 (41,1 por cento) casos; B (näo-infectados), 34(46,6 por cento) crianças e C (infecçäo antiga), nove (12,3 por cento). Entre os do Grupo A, cinco (16,7 por cento) apresentaram manifestaçöes de resfriado comum. O sexo, o grupo racial, a idade e a presença de cäo no domicílio näo apresentaram diferenças estatísticas significantes, entre os grupos A e B. Todavia, nas crianças do grupo A predominaram (p<0,05): contato com água na natureza; irmäo caso-índice e menor de 15 anos; sexo feminino e maior de 9 anos de idade e, principalmente, a falta de coleta do lixo nos locais de residência. Em conclusäo, a leptospirose em crianças de Salvador é subestimada, porque é oligossintomática ou similar ao resfriado comum