Biopolymer Xanthan: A New Adjuvant for DNA Vaccines

Abstract DNA vaccines have been evaluated as an option to prevent several diseases. In this study, the capacity of the xanthan biopolymer to improve the DNA vaccines immune response, administered intramuscularly, was evaluated. The experimental vaccines consisted of genes encoding fragments of the proteins LigA and LigB of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130. The humoral immune response was evaluated by indirect ELISA. Cytokine expression levels were determined by RT-qPCR. Compared to the control group, the IgG antibody levels of animals immunized with pTARGET/ligAni and pTARGET/ligBrep plasmids associated with xanthan biopolymer were significantly higher than the control group. Additionally, there was a significant increase in IL-17 expression in animals vaccinated with pTARGET/ligBrep and xanthan.

Alterações histopatológicas e macroscópicas em um cão sorologicamente positivo para Leptospira spp / Case report: Macroscopic and histopathological changes in a dog serologically positive for Leptospira spp

A leptospirose é uma zoonose de distribuição mundial com elevada incidência em regiões tropicais.Nos cães ela se apresenta com grande polimorfismo clínico e estes animais são consideradosfonte de infecção da leptospirose humana, mesmo quando não apresentam qualquer sinal clínicocaracterístico. O processo de patogênese é caracterizado pelo desenvolvimento de vasculite, lesãoendotelial e infiltrado inflamatório composto por células monocíticas, plasmócitos, histiócitos eneutrófilos. O objetivo deste estudo foi relatar o caso de um cão sorologicamente positivo paraleptospirose. Logo após o óbito, o animal foi encaminhado ao Departamento de Patologia Animaldo Hospital Veterinário da Universidade Federal de Uberlândia. Na necropsia foram diagnosticadasicterícia hemolítica e nefrite intersticial crônica moderada. As alterações macroscópicas, histológicase sorológicas do animal foram sugestivas de doença hemolítica grave compatível com leptospirose.

Leptospirosis is a zoonosis of worldwide distribution with high incidence in tropical regions.Leptospirosis in dogs presents with large clinical polymorphism and these animals are consideredsources of infection for human leptospirosis, even without showing any clinical signs. Pathogenesisis characterized by the development of vasculitis, endothelial injury and infiltration of monocyticcells, plasma cells, histiocytes and neutrophils. The objective of this study was to report the caseof a dog serologically positive for leptospirosis. The animal died and then was submitted to theAnimal Pathology Department of the Veterinary Hospital of the Federal University of Uberlândia At necropsy hemolytic jaundice and moderate chronic interstitial nephritis were diagnosed. Themacroscopic, histological and serological changes suggest severe hemolytic disease consistent withleptospirosis.

Tissue apoptosis in mice infected with Leptospira interrogans serovar Icterohaemorrhagiae

Background:This investigation aimed to evaluate the occurrence of some apoptotic features induced by Leptospira interrogans serovar Icterohaemorrhagiae infection in young BALB/c mice during 2, 4, 7, 10, 14 and 21 days post-infection (dpi).Methods:The animals were euthanized and lung, liver and kidneys were harvested to histopathology analysis and immunohistochemistry to caspase-3 antigen detection was performed.Results:Chromatin condensation in kidney and liver tissues, but not in lung tissue, was observed. Caspase-3 reactive cells, mainly characterized as renal epithelial cells, were detected in the days 14 and 21 at high levels when compared to days 2,4 and 7 (p = 0.025; p <0.05). Lung sections revealed caspase-3 labeled alveolar cells in 10 and 14 days post-infection was higher than observed at 7 days (p = 0.0497; p < 0.05). Liver sections demonstrated reactive cells at a highest level at 14 and 21 days post-infection when comparison to 2,4, 7 and 10 days (p = 0.0069; p<0.05).Conclusions:Our results suggest that infection of L interrogans induce in kidney, liver and lung an activation of apoptosis mediated by caspase-3 dependent pathway in later phases of infectious process.(AU)

Tissue apoptosis in mice infected with Leptospira interrogans serovar Icterohaemorrhagiae

Background: This investigation aimed to evaluate the occurrence of some apoptotic features induced by Leptospira interrogans serovar Icterohaemorrhagiae infection in young BALB/c mice during 2, 4, 7, 10, 14 and 21 days post-infection (dpi). Methods: The animals were euthanized and lung, liver and kidneys were harvested to histopathology analysis and immunohistochemistry to caspase-3 antigen detection was performed. Results: Chromatin condensation in kidney and liver tissues, but not in lung tissue, was observed. Caspase-3 reactive cells, mainly characterized as renal epithelial cells, were detected in the days 14 and 21 at high levels when compared to days 2,4 and 7 (p = 0.025; p <0.05). Lung sections revealed caspase-3 labeled alveolar cells in 10 and 14 days post-infection was higher than observed at 7 days (p = 0.0497; p < 0.05). Liver sections demonstrated reactive cells at a highest level at 14 and 21 days post-infection when comparison to 2,4, 7 and 10 days (p = 0.0069; p<0.05). Conclusions: Our results suggest that infection of L interrogans induce in kidney, liver and lung an activation of apoptosis mediated by caspase-3 dependent pathway in later phases of infectious process.

Genotipos de cepas de Leptospira spp. aisladas de perros en Buenos Aires, Argentina / Genotypes of Leptospira spp. strains isolated from dogs in Buenos Aires, Argentina

La leptospirosis es una enfermedad infecciosa de amplia distribución global; endémica en Argentina. El objetivo de este estudio fue obtener los perfiles genéticos de las cepas de Leptospira spp. aisladas de casos clínicos de perros provenientes de la provincia de Buenos Aires, empleando el análisis de repeticiones en tándem de número variable en múltiples locus [multiple-locus variable-number tandem repeats analysis (MLVA)]. Fueron genotipificadas por MLVA ocho cepas aisladas de perros. Se obtuvo un perfil idéntico al de Leptospira interrogans serovar Canicola Hond Utrecht IV en las cepas denominadas Dogy y Mayo. Las cepas denominadas Bel, Sarmiento, La Plata 4581 y La Plata 5478 mostraron un perfil idéntico al genotipo de L. interrogans serovar Portlandvere MY 1039. La cepa denominada Avellaneda presentó un perfil idéntico al genotipo L. interrogans serovar Icterohaemorrhagiae RGA, y la cepa denominada SB mostró un perfil idéntico al genotipo de L. interrogans serovar Pomona Baires y similar al serovar Pomona Pomona. Sería de gran utilidad incluir un mayor número de cepas provenientes de distintas poblaciones caninas de diversas provincias de Argentina a fin de conocer los perfiles de las cepas circulantes en el país. La información así obtenida contribuirá al control de la leptospirosis en la población canina

Leptospirosis is an infectious disease of wide global distribution, which is endemic in Argentina. The objective of this study was to obtain the genetic profiles of Leptospira spp. strains isolated from clinical cases of dogs in the province of Buenos Aires by the multiple-locus variable-number tandem repeat analysis (MLVA). Eight isolated canine strains were genotyped by MLVA, obtaining the identical profile of Leptospira interrogans serovar Canicola Hond Utrecht IV in the strains named Dogy and Mayo. The strains named Bel, Sarmiento, La Plata 4581 and La Plata 5478 were identical to the profile of the genotype of L. interrogans serovar Portlandvere MY 1039.The strain named Avellaneda was identical to the genotype profile of L. interrogans serovar Icterohaemorrhagiae RGA and the strain named SB had the same profile as the L. interrogans serovar Pomona Baires genotype and was similar to the profile of serovar Pomona Pomona genotype. It would be useful to include a larger number of isolates from different dog populations in various provinces of Argentina and to characterize the genetic profiles of the strains circulating in the country. The information obtained will be useful for the control of leptospirosis in the dog population

Genotypes of pathogenic Leptospira spp isolated from rodents in Argentina

Leptospirosis is the most widespread zoonosis in the world and significant efforts have been made to determine and classify pathogenic Leptospira strains. This zoonosis is maintained in nature through chronic renal infections of carrier animals, with rodents and other small mammals serving as the most important reservoirs. Additionally, domestic animals, such as livestock and dogs, are significant sources of human infection. In this study, a multiple-locus variable-number tandem repeat analysis (MLVA) was applied to genotype 22 pathogenic Leptospira strains isolated from urban and periurban rodent populations from different regions of Argentina. Three MLVA profiles were identified in strains belonging to the species Leptospira interrogans (serovars Icterohaemorrhagiae and Canicola); one profile was observed in serovar Icterohaemorrhagiae and two MLVA profiles were observed in isolates of serovars Canicola and Portlandvere. All strains belonging to Leptospira borgpetersenii serovar Castellonis exhibited the same MLVA profile. Four different genotypes were isolated from urban populations of rodents, including both mice and rats and two different genotypes were isolated from periurban populations.

A importância de Leptospira interrogans sorovariedades Icterohaemorrhagiae e Canicola na zona litorânea e nos campos sulinos do Rio Grande do Sul / The importance of Leptospira interrogans serovars Icterohaemorrhagiae and Canicola in coastal zone and in southern fields of Rio Grande do Sul, Brazil

This study aimed to describe the occurrence of Leptospira interrogans serovars Icterohaemorrhagiae and Canicola, in coastal zone and in southern grasslands of Rio Grande do Sul, Brazil. In each one of the four analyzed farms blood samples were collected from free-living wild animals, domestic animals and humans to perform serological testing for leptospirosis. The presence of antibodies was verified by microscopic agglutination test (MAT). The criterion adopted to consider a serum as agglutination reactant was at least 50% of leptospira for a microscopic field of 100x. From 17 blood samples collected at Chuí, five (29.41%) were positive, three (60.00%) for serovar Icterohaemorrhagiae and two (40.00%) for Canicola. From 21 samples collected in the County of Santana da Boa Vista, six (28.57%) were positive, four (66.67%) for serovar Canicola and two (33.33%) for serovar Icterohaemorrhagiae. From 32 samples collected at Alegrete, 10 (31.25%) were positive, seven (70.00%) for serovar Icterohaemorrhagiae and three (30.00%) foro serovar Canicola. From 17 blood samples collected in Cruz Alta, three (17.64%) were positive, two (66.67%) for serovar Icterohaemorrhagiae and one (33.33%) for Canicola. It is necessary to improve sanitary practices on farms in the state of Rio Grande do Sul, in order to achieve success in leptospirosis control programs.

O presente estudo teve como objetivo descrever a ocorrência de Leptospira interrogans sorovariedades Icterohaemorrhagiae e Canicola, na zona litorânea e nos campos sulinos do Estado do Rio Grande do Sul. Em cada uma das quatro propriedades foram realizadas colheitas de sangue de animais selvagens de vida livre, de animais domésticos e de seres humanos para realização de sorologia para leptospirose. A presença de anticorpos foi verificada pela técnica de Soroaglutinação Microscópica (SAM). O critério adotado para considerar um soro como reagente foi aglutinação de pelo menos 50% das leptospiras no campo microscópico no aumento de 100x. Das 17 amostras de sangue colhidas na propriedade pertencente ao Município de Chuí, cinco (29,41%) foram positivas, três (60,00%) à sorovariedade Icterohaemorrhagiae e duas (40,00%) à Canicola. Das 21 amostras colhidas em Santana da Boa Vista, seis (28,57%) foram positivas, quatro (66,67%) à Canicola e duas (33,33%) à Icterohaemorrhagiae. Das 32 amostras colhidas em Alegrete, 10 (31,25%) foram positivas, sete (70,00%) à Icterohaemorrhagiae e três (30,00%) à Canicola. Das 17 amostras colhidas em Cruz Alta, três (17,64%) foram positivas, duas (66,67%) à Icterohaemorrhagiae e uma (33,33%) à Canicola. É necessário melhorias nas práticas sanitárias em propriedades rurais do Estado do Rio Grande do Sul, a fim de se obter sucesso em programas locais de controle da leptospirose.

Anticorpos revelados pelo teste de inibição do crescimento de leptospiras in vitro (TICL) contra os sorovares Canicola, Icterohaemorrhagiae e Copenhageni em cães adultos revacinados anualmente com vacina comercial contendo bacterinas dos sorovares Canicola, Icterohaemorrhagiae, Grippotyphosa e Pomona / Antibody revealed by growth inhibition test the of leptospires in vitro (GIT) against serovars Canicola, Icterohaemorrhagiae and Copenhageni in adult dogs revaccinated annually with commercial vaccine containing serovars Canicola, Icterohaemorrhagiae, Grippotyphosa and Pomona bacterins

Na atualidade, o sorovar Copenhageni é o representante do sorogrupo Icterohaemorrhagiae, mantido por roedores sinantrópicos, que tem prevalecido nos cães e seres humanos das grandes metrópoles brasileiras. A despeito de alguns autores sugerirem a existência de proteção cruzada entre sorovares incluídos em um mesmo sorogrupo esta condição ainda não foi suficientemente esclarecida para os sorovares Icterohaemorrhagiae e Copenhageni. No presente trabalho cães adultos com dois a seis anos de idade primo-vacinados com três doses intervaladas de 30 dias a partir dos 60 dias de idade e revacinados anualmente com vacina anti-leptospirose polivalente contendo os sorovares Canicola, Icterohaemorrhagiae, Grippotyphosa e Pomona foram revacinados com a mesma vacina e aos 30 dias da revacinação foram submetidos aos testes de soroaglutinação microscópica (SAM) e de inibição do crescimento de leptospiras in vitro (TICL), para avaliação comparativa dos níveis de anticorpos produzidos para os sorovares Canicola, Icterohaemorrhagiae e Copenhageni. Os resultados obtidos indicaram que a imunidade conferida pela vacina para o sorovar Icterohaemorrhagiae é mais duradoura que a observada para o sorovar Canicola, já que títulos de anticorpos neutralizantes >1,0 log10 foram observados antes do reforço vacinal não havendo substancial aumento após a revacinação. Quanto ao sorovar Canicola, a revacinação resultou em considerável aumento do título de anticorpos neutralizantes quando comparado ao momento anterior a revacinação (p=0,001). A análise dos valores encontrados após a revacinação demonstrou claramente que cães revacinados com bacterina produzida com o sorovar Icterohaermorrhagiae não apresentam aumento do título de anticorpos inibidores do crescimento contra o sorovar Copenhageni, em nível suficiente para inibir o crescimento de leptospiras. Apesar disso, os títulos de anticorpos inibidores de crescimento anti-Copenhageni encontrados antes e após a revacinação demonstraram que, pelo menos certo grau de proteção contra a infecção por esse sorovar pode ser esperado para os cães vacinados com bacterinas do sorovar Icterohaemorrhagiae, não sendo, no entanto, uma proteção cruzada completa.

Currently, the serovar Copenhageni is the representative of serogroup Icterohaemorrhagiae maintained in synanthropic rodents found most frequently in dogs and humans in metropolitan areas of Brazil. Despite some authors have suggested the existence of cross-protection between serovars included in the same serogroup, this condition has not yet been sufficiently clarified for serovars Icterohaemorrhagiae and Copenhageni. In the present work, 2 to 6-year-old dogs, vaccinated at 60, 90 and 120 days of age and thereafter, revaccinated annually with commercial vaccine containing Canicola, Icterohaemorrhagiae, Grippotyphosa and Pomona bacterins were evaluated as to the immune status against leptospirosis before and 30 days after revaccination. Mycroscopic agglutination test (MAT) and in vitro growth inhibition test (GIT) were performed to search for agglutinating anti-Leptospira antibodies and neutralizing anti-Leptospira antibodies, respectively for serovars Canicola and Icterohaemorrhagiae, and additionally, for serovar Copenhageni, not included in the vaccine. The results showed that the immunity conferred by the vaccine to serovar Icterohaemorrhagiae is more lasting than that observed for serovar Canicola, since neutralizing antibody titers >1.0 log10 were observed before the booster vaccination with no substantial increase after revaccination. As for the serovar Canicola, revaccination resulted in a considerable increase in neutralizing antibody titer when compared to the one observed previously to the revaccination (p=0.001). The analysis of the data obtained by GIT allowed us to conclude that dogs given vaccine containing Icterohaemorrhagiae bacterin did not produce neutralizing antibodies against serovar Copenhageni enough to inhibit leptopiral growth at the same level as occurred for the homologous serovar. Despite this, the GIT titer found for serovar Copenhageni before and after revaccination showed that at least, some level of protection could be expected for dogs vaccinated with serovar Icterohaemorrhagiae bacterin, not a complete cross protection.

Detection of reactive canines to Leptospira in Campeche City, Mexico / Detection of reactive canines to Leptospira in Campeche City, Mexico.

Leptospira reactivity in stray and household dogs in Campeche as well as associated risk factors to the seropositivity in household dogs have been herein determined. The survey included 323 dogs, 142 of which were stray dogs and 181 household dogs. Nine Leptospira interrogans serovars were tested by the microagglutination test. Reactivity was 21.3

corresponded to household dogs and 26.7

), and Leptospira Icterohaemorrhagiae (16.12

) were the most common serovars reacting against the serum of household animals, while Leptospira Canicola (15.78

), and Leptospira Pomona (7.89

) were those reacting in stray dogs. Results showed that all dogs have been in contact with different Leptospira serovars and outdoor exposure is the main infection risk factor.

Characterization of a virulent Leptospira interrogans strain isolated from an abandoned swimming pool

Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.

Soroprevalência de leptospirose suína na região noroeste do Paraná / Seroprevalencia de leptospirosis porcina en la región noroeste de Paraná / Seroprevalence of swine leptospirosis in northwest region Paraná, Brazil

A leptospirose é uma doença zoonótica causada por bactéria da família Leptospiraceae, gênero Leptospira representando risco para a saúde pública, principalmente de médicos veterinários, magarefes, e funcionários de granjas suínas que estão sujeitos ao contato direto com o agente, e trazendo grandes prejuízos reprodutivos para os produtores tais como: aborto no terço final de gestação, repetição de cio, fetal mumificações ou natimortos, nascimento de leitões fracos, baixo número de leitões, descarga vulvar e morte embrionária. Atualmente existem 23 sorogrupos de Leptospira interrogans com aproximadamente 200 sorovares diferentes, neste trabalho usaram-se culturas vivas de 16 sorogrupos de Leptospira spp em amostras de 297 animais da região Noroeste do Paraná submetidas a prova de soroaglutina microscópica com o objetivo de identificar os sorovares predominantes na região. Obteve-se 19,865% de amostras soropositivas para um ou mais sorovares com titulações de 1:100, 1:200 e 1:400, na qual tiveram maior prevalência para o sorovar hardjo com 57,627%, seguido pelos sorovares pyrogenes 30,508%, hardjo C.T.G. 13,559%, icterohaemorrhagiae 5,084%, djasiman 1,694% e copenhageni 1,694%, concluindo que o sorovar Hardjo como o mais prevalente na região noroeste do Paraná.

Leptospirosis is a zoonotic disease caused by a bacteria of the family Leptospiraceae, genus Leptospira that represent risks to public health, especially veterinarians, butcher, and employees from pig farms that are subject to direct contact with the agent, and causing high damages to reproductive producers such as abortion in the final of gestation, re-heat, fetal mummification or stillbirths, birth of weak piglets, low number of piglets, vulvar discharge and embryonic death. Currently there are 23 serogroups of Leptospira interrogans with approximately 200 different serovars, in this study we used live cultures of 16 serogroups from Leptospira spp in samples of 297 animals of the northwest of Paraná, Brazil subjected to microscopic evidence of serum agglutination to identify the predominant serovars in the region. There were 19.865% of samples seropositive for one or more serovars with titers of 1:100, 1:200 and 1:400 which were predominant for serovar hardjo 57.627% , followed by pyrogenes serovars with 30.508%, hardjo CTG 13.559%, icterohaemorrhagiae 5.084%, djasiman 1.694% and copenhageni 1.694%, concluding that the serovar hardjo is the most prevalent in the northwest of Paraná.

La leptospirosis es una enfermedad zoonótica causada por bacteria de la familia Leptospiraceae, género Leptospira representando riesgo para la salud pública, principalmente de médicos veterinarios, carniceros, y funcionarios de granjas porcinas que están sujetos al contacto directo con el agente, trayendo grandes daños reproductivos para los productores tales como: aborto en el último tercio de la preñez, repetición de celo, momificaciones de fetos o mortinatos, nacimientos de lechones débiles, bajo número de lechones, secreción vulvaria y la muerte embrionaria. Actualmente hay 23 serogrupos de Leptospira interrogans con aproximadamente 200 serovares diferentes, en esta investigación se usó culturas vivas de 16 serogrupos de Leptospira spp en muestras de 297 animales de la región Noroeste de Paraná, sometidas a la prueba de seroaglutina microscópica con el objetivo de identificar los serovares predominantes en la región. Se alcanzó 19,865% de muestras seropositivas para un o más serovares con titulaciones de 1:100, 1:200 y 1:400, en la cual tuvieron mayor prevalencia para el serovar hardjo con 57,627%, seguido por los serovares pyrogenes 30,508%, hardjo C.T.G. 13,559%, icterohaemorrhagiae 5,084%, djasiman 1,694% y copenhageni 1,694%, concluyendo que el serovar Hardjo es los más prevalente en la región noroeste de Paraná.

Ophthalmic alterations in horses with leptospirosis by serovar Icterohaemorrhagiae in Rio de Janeiro, Brazil / Alterações oftálmicas em cavalos com leptospirose por serovar Icterohaemorrhagiae no Rio de Janeiro

The objective of the study was to determine the association between clinical ophthalmic alterations and seroreactivity to leptospirosis by serovar Icterohaemorrhagiae in horses in Rio de Janeiro, Brazil. A total of 199 horses were studied. A microscopic agglutination test (MAT) was used to detect specific anti-Leptospira antibodies in blood serum. A total of 107 (53.8 percent) horses were seroreactive (titres > 200); 54 had high (> 800) titres, of which 44 were against serovar Icterohaemorrhagiae. Forty-two out of these 44, plus 40 seronegative horses (titers < 100) were given detailed ophthalmic examinations. Epiphora, ocular congestion, blepharospasm, photophobia, and peripapillary focal depigmentation were the most frequent alterations in seroreactive horses. Most ocular alterations were significantly more frequent in seroreactive horses. Horses seroreactive for leptospirosis (serovar Icterohaemorrhagiae) had a significantly higher prevalence of ophthalmic alterations than seronegative horses, providing additional evidence for an association between leptospirosis and equine uveitis.

O objetivo do estudo foi determinar a associação entre as alterações clínicas oftalmológicas e sororeatividade a leptospirose por serovar Icterohaemorrhagiae em cavalos no Rio de Janeiro. Um total de 199 animais foi estudado. O Teste da Aglutinação Microscópica foi utilizado para detectar anticorpos específicos anti-Leptospira no soro dos animais. Um total de 107 (53,8 por cento) dos cavalos foram sororeativos (títulos >200); 54 tinham títulos elevados (>800), dos quais 44 foram reativos contra o serovar Icterohaemorrhagiae. Quarenta e dois dentre estes 44, mais 40 cavalos soronegativos (títulos <100) foram submetidos ao exame oftalmológico. Epífora, congestão ocular, blefarospasmo, fotofobia, e despigmentação focal peripapilar foram as alterações mais frequentes nos animais sororeativos. Muitas alterações oculares foram significativamente mais frequentes em cavalos sororeativos. Animais sororeativos para leptospirose (serovar Icterohaemorrhagiae) tinham uma prevalência significativamente elevada de alterações oftálmicas em relação aos animais soronegativos, fornecendo evidências adicionais para associação entre leptospirose e uveíte equina.

Characterization of Leptospira borgpetersenii isolates from field rats (Rattus norvegicus) by 16S rRNA and lipL32 gene sequencing

The main goal of this study was to evaluate the prevalence of leptospirosis among field rodents of Tiruchirappalli district, Tamil Nadu, India. In total 35 field rats were trapped and tested for seroprevalence by the microscopic agglutination test (MAT). Isolation of leptospires was performed from blood and kidney tissues and characterized to serovar level. Genomospecies identification was carried out using 16S rRNA and lipL32 gene sequencing. The molecular phylogeny was constructed to find out species segregation. Seroprevalence was about 51.4 percent, and the predominant serovars were Autumnalis, Javanica, Icterohaemorrhagiae and Pomona. Two isolates from the kidneys were identified as serovar Javanica of Serogroup Javanica, and sequence based molecular phylogeny indicated these two isolates were Leptospira borgpetersenii.

Rapid serodiagnosis of leptospirosis by latex agglutination test and flow-through assay.

PURPOSE: Diagnosis of leptospirosis facilitates patient management and initiation of therapy. The microscopic agglutination test (MAT) is the serological test used in reference laboratories because of its high degree of sensitivity and specificity. But the results are not available quickly for patient management. In the present study, in order to develop a simple, rapid immunodiagnostic assay, one of the outer membrane proteins (OMPs), recombinant LipL41 (rLipL41) has been utilised in latex agglutination test (LAT) and flow-through assay. METHODS: Part of LipL41 gene was expressed in Escherichia coli system and purified. The rLipL41 antigen of pathogenic Leptospira interrogans serovar Icterohaemorrhagiae, which is conserved in all pathogenic Leptospira spp. was used as capture antigen in the LAT and flow-through test. Both tests are very rapid and could be completed within 5 minutes. The sensitivity and specificity of rLipL41 was assessed and evaluated in LAT and flow-through assay in comparison with standard MAT. RESULTS: The sensitivity and specificity of the LAT were 89.70 and 90.45% and flow-through assay were 89.09 and 77.70%, respectively. CONCLUSIONS: The developed LAT and flow-through assays were simple, rapid and economical for the detection of leptospira infection and suitable for large-scale screening of samples in endemic areas without any sophisticated equipment.

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.

Proteome and immunome of pathogenic Leptospira spp. revealed by 2DE and 2DE-immunoblotting with immune serum.

In this study, proteomes of two pathogenic Leptospira spp., namely L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni and L. borgpetersenii, serogroup Tarassovi, serovar Tarassovi, were revealed by using two dimensional gel electrophoresis (2DE)-based-proteomics. Bacterial cells were disrupted in a lysis buffer containing 30 mM Tris, 2 M thiourea, 7 M urea, 4% CHAPS, 2% IPG buffer pH 3-10 and protease inhibitors and then subjected to sonication in order to solubilize as much as possible the bacterial proteins. The 2DE-separated components of both Leptospira homogenates were blotted individually onto membranes and antigenic components (immunomes) were revealed by probing the blots with immune serum of a mouse readily immunized with the homogenate of L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni. The immunogenic proteins of the two pathogenic Leptospira spp. could be grouped into 10 groups. These are: 1) proteins involved in the bacterial transcription and translation including beta subunit transcription anti-termination protein of DNA polymerase III, elongation factors Tu and Ts, and tRNA (guanine-N1)-methyltransferase; 2) proteins functioning as enzymes for metabolisms and nutrient acquisition including acetyl-Co-A acetyltransferase, putative glutamine synthetase, glyceraldehyde-3-phospahte dehydrogenase, NifU-like protein, 3-oxoacyl-(acyl-carrier-protein) reductase, oxidoreductase, sphingomyelinase C precursor, spermidine synthase, beta subunit of succinyl-CoA synthetase, and succinate dehydrogenase iron-sulfur subunit; 3) proteins/enzymes necessary for energy and electron transfer, i.e. electron transfer flavoprotein, and proton-translocating transhydrogenase; 4) enzymes for degradation of misfolded proteins, i.e. ATP-dependent Clp protease; 5) molecular chaperone, i.e. 60 kDa chaperonin; 6) signal transduction system, i.e. response regulator; 7) protein involved in immune evasion in host, i.e. peroxiredoxin; 8) cell structure proteins including MreB (cytoskeletal) and flagellin/ periplasmic flagellin; 9) lipoproteins/outer membrane proteins: LipL32, LipL41, LipL45 and OmpL1; and 10) various hypothetical proteins. Many immunogenic proteins are common to both Leptospira spp. These proteins not only are the diagnostic targets but also have potential as candidates of a broad spectrum leptospirosis vaccine especially the surface exposed components which should be vulnerable to the host immune effector factors.

OmpL1 DNA vaccine cross-protects against heterologous Leptospira spp. challenge.

Available leptospirosis vaccines made up of inactivated bacteria or their membrane components elicit immunity which is serovar specific and unsatisfactory immunological memory. A vaccine that protects across Leptospira serogroups/serovars, i.e. broad spectrum, and induces long-lasting memory is needed for both human and veterinary uses. In this study, a plasmid DNA vaccine was constructed from cloning gene encoding a transmembrane porin protein, OmpL1, of pathogenic Leptospira interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni into a mammalian expression vector pcDNA3.1(+). The protective efficacy of the ompL1-pcDNA3.1(+) plasmid DNA vaccine was studied by immunizing hamsters intramuscularly with three doses of the vaccine (100 microg per dose) at two week intervals. The empty pcDNA3.1(+) and PBS were used as mock as negative vaccine controls, respectively. All animals were challenged with the heterologous Leptospira interrogans, serogroup Pomona, serovar Pomona (10 LD50), at one week after the last vaccine booster. The ompL1-pcDNA3.1(+) plasmid DNA vaccine rescued some vaccinated animals from the lethal challenge and delayed death time, reduced morbidity, e.g. fever, and/or the numbers of Leptospira in the tissues of the vaccinated animals. While the results are encouraging, further studies are needed to optimize the immunization schedule, vaccine dosage and formulation in order to maximize the efficacy of the vaccine.

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.