Genome of Leptospira borgpetersenii strain 4E, a highly virulent isolate obtained from Mus musculus in southern Brazil.

A previous study by our group reported the isolation and characterisation of Leptospira borgpetersenii serogroup Ballum strain 4E. This strain is of particular interest because it is highly virulent in the hamster model. In this study, we performed whole-genome shotgun genome sequencing of the strain using the SOLiD sequencing platform. By assembling and analysing the new genome, we were able to identify novel features that have been previously overlooked in genome annotations of other strains belonging to the same species.

Reversal of renal tubule transporter downregulation during severe leptospirosis with antimicrobial therapy.

Tubular dysfunction is a hallmark of severe leptospirosis. Antimicrobial therapy is thought to interfere on renal involvement. We evaluated the expression of a proximal tubule type-3 Na+/H+ exchanger (NHE3) and a thick ascending limb Na+-K+-2Cl(-) cotransporter (NKCC2) in controls and treated hamsters. Animals infected by a serovar Copenhageni isolate, were treated or not with ampicillin (AMP) and/or N-acetylcysteine (NAC). Leptospiral antigen(s) and expression of renal transporters were evaluated by immunohistochemistry, and serum thiobarbituric acid (TBARS) was quantified. Infected hamsters had high amounts of detectable leptospiral antigen(s) in target tissues while renal expression of NHE3 and NKCC2 decreased. Ampicillin treatment was associated with minimal or no detection of leptospiral antigens, normal expression of NHE3 and NKCC2 transporters, and reduced levels of TBARS. NAC effect was restricted to lowering TBARS. Early and late AMP treatment rescued tubular defects in severe leptospirosis disease, and there was no evidence of benefit from antioxidant therapy.

Infection with Leptospira kirschneri Serovar Mozdok: First Report from the Southern Hemisphere.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira spp. In this study, we characterized two Leptospira kirschneri serogroup Pomona serovar Mozdok isolates, one obtained from a dog and the other from a patient with severe leptospirosis, 4 years later. Histopathological analysis showed that both isolates caused severe tissue damage when used to infect hamsters. While L. kirschneri serogroup Pomona serovar Mozdok is endemic in animals in Europe, there is only one report of human leptospirosis in the literature. Although strains belonging to L. kirschneri serogroup Pomona have been identified in cases of human leptospirosis in Europe, serovar Mozdok has not yet been implicated. The 4-year interval between isolations and the fact that this is the first report of serovar Mozdok as the causative agent of human leptospirosis in the southern hemisphere, demonstrates its epidemiological importance to public health. Moreover, the presence of serovar Mozdok in Brazil has the potential to affect vaccine and diagnostic test development.

Genome of Leptospira borgpetersenii strain 4E, a highly virulent isolate obtained from Mus musculus in southern Brazil

A previous study by our group reported the isolation and characterisation of Leptospira borgpetersenii serogroup Ballum strain 4E. This strain is of particular interest because it is highly virulent in the hamster model. In this study, we performed whole-genome shotgun genome sequencing of the strain using the SOLiD sequencing platform. By assembling and analysing the new genome, we were able to identify novel features that have been previously overlooked in genome annotations of other strains belonging to the same species.

Campylobacter coli no fluxograma de abate de suínos e pesquisa dos genes cdt / Campylobacter coli in swine slaughtering flowchart and research of cdt genes

Background: Campylobacter spp. are among the microorganisms most commonly associated with foodborne disease. Campylobacter spp. isolation from pigs during the slaughter and final products have been reported in several countries, including Brazil. However, very little is known about the sources of contamination in the slaughtering flowchart and how these microorganisms are spread in processing plants. Considering the possibility of the pigs carry Campylobacter spp. since the farm or its products are contaminated in the slaughterhouse, this study had as aim to track Campylobacter spp. in pig slaughtering flowchart to understand the behavior of these pathogens in the production line.Materials, Methods & Results: Forty animals of 10 lots, four from each lot, were followed during slaughter. Stool samples were collected from the floor of each enclosure where the pigs were housed on the farm and immediately after stunning on slaughterhouse. Samples from carcass surface were collected after removal of the animals from scrap machine, after evisceration and before the refrigeration chamber. It was also collected surface samples from jowls and samples from the scalding tank water before and after the passage of animals. The swabs containing samples were plated onto Columbia agar supplemented with activated charcoal, oxygen reduction solution and antibiotics supplement, and incubated at 42°C for 48 h under microaerobic conditions. The colonies which presented with a shiny and moist appearance were analyzed by Gram staining for identification of Campylobacter by morphology, and then tested for catalase and oxidase. The Campylobacter isolates were identified for species C. jejuni or C. coli by PCR. Bands profiles were determined by rep-PCR and used to compare the strains. Campylobacter was isolated from 19 (9.5%) of the 200 pig samples analyzed, seven (36.8%) of the rectum, seven (36.8%) after evisceration and five (26.3%) before the refrigeration chamber.[...]

Campylobacter coli no fluxograma de abate de suínos e pesquisa dos genes cdt / Campylobacter coli in swine slaughtering flowchart and research of cdt genes

Background: Campylobacter spp. are among the microorganisms most commonly associated with foodborne disease. Campylobacter spp. isolation from pigs during the slaughter and final products have been reported in several countries, including Brazil. However, very little is known about the sources of contamination in the slaughtering flowchart and how these microorganisms are spread in processing plants. Considering the possibility of the pigs carry Campylobacter spp. since the farm or its products are contaminated in the slaughterhouse, this study had as aim to track Campylobacter spp. in pig slaughtering flowchart to understand the behavior of these pathogens in the production line.Materials, Methods & Results: Forty animals of 10 lots, four from each lot, were followed during slaughter. Stool samples were collected from the floor of each enclosure where the pigs were housed on the farm and immediately after stunning on slaughterhouse. Samples from carcass surface were collected after removal of the animals from scrap machine, after evisceration and before the refrigeration chamber. It was also collected surface samples from jowls and samples from the scalding tank water before and after the passage of animals. The swabs containing samples were plated onto Columbia agar supplemented with activated charcoal, oxygen reduction solution and antibiotics supplement, and incubated at 42°C for 48 h under microaerobic conditions. The colonies which presented with a shiny and moist appearance were analyzed by Gram staining for identification of Campylobacter by morphology, and then tested for catalase and oxidase. The Campylobacter isolates were identified for species C. jejuni or C. coli by PCR. Bands profiles were determined by rep-PCR and used to compare the strains. Campylobacter was isolated from 19 (9.5%) of the 200 pig samples analyzed, seven (36.8%) of the rectum, seven (36.8%) after evisceration and five (26.3%) before the refrigeration chamber.[...](AU)

Impacto da salmonelose na suinocultura e suas implicações em saúde pública / Impact of salmonelosis on pork meat industry and its implications on public health

O objetivo deste trabalho foi mostrar a presença da Salmonella na cadeia de produção de suínos e o risco em potencial para a saúde pública. As bactérias do gênero Salmonella são importantes agentes causadores de doenças transmitidas por alimentos (DTAs) em humanos. Estima-se que cerca de 10% do total de casos de salmonelose sejam veiculados por produtos de origem suína. A transmissão ao homem pode ocorrer pelo contato direto com animais, tanto nas granjas quanto nos frigoríficos, mas principalmente devido à ingestão de alimentos contaminados. A infecção de lotes suínos pode ocorrer em qualquer fase zootécnica e o principal ciclo de infecção é fecal-oral, podendo a bactéria se alojar nos linfonodos e ser excretada quando o animal for submetido a um fator estressante, como o transporte e/ou o reagrupamento. O uso inadequado de antimicrobianos também gera cepas multirresistentes. Os equipamentos e utensílios, de uma forma geral, estão relacionados à contaminação cruzada, agindo como veículos de propagação do micro-organismo dentro da indústria. Para garantir a segurança alimentar do consumidor, conclui-se que cuidados como limpeza, desinfecção e biossegurança devem se iniciar na granja de criação, com a utilização correta de antibióticos, passando pelo transporte evitando a superlotação e com medidas higiênico-sanitárias rigorosas durante o abate desses animais.(AU)

The objective of this study was to show the presence of Salmonella in the swine production chain, and its impact on public health. Bacteria of the Salmonella genus are an important cause of food transmitted diseases in Brazil. Around 10% of clinical salmonellosis cases are attributed to swine products. Humans can become infected through direct contact with the animals, both living and in slaughter houses; however, the most common cause of infection is the ingestion of contaminated food. Infection of swine herds can occur in any of the rearing stages, mainly through a fecal-oral cycle, where the bacteria will shelter in the lymph nodes and be excreted when the host is submitted to a stress factor, such as transport and/or regrouping. Equipment and utensils are also associated with cross contamination among animals, acting as vehicles in the propagation of the microorganism inside the industry or rearing facility. Furthermore, inadequate use of antimicrobial agents will contribute to the rise of resistant bacteria. This study shows that, in order to safeguard consumer health, awareness must begin in the rearing facilities, with special care to hygiene, biosafety, and the proper use of antibiotics. Further care must be considered during transportation, and especially rigorous measures should be set during slaughter.(AU)

Impact of salmonelosis on pork meat industry and its implications on public health / Impacto da salmonelose na suinocultura e suas implicações em saúde pública

The objective of this study was to show the presence of Salmonella in the swine production chain, and its impact on public health. Bacteria of the Salmonella genus are an important cause of food transmitted diseases in Brazil. Around 10% of clinical salmonellosis cases are attributed to swine products. Humans can become infected through direct contact with the animals, both living and in slaughter houses; however, the most common cause of infection is the ingestion of contaminated food. Infection of swine herds can occur in any of the rearing stages, mainly through a fecaloral cycle, where the bacteria will shelter in the lymph nodes and be excreted when the host is submitted to a stress factor, such as transport and/ or regrouping. Equipment and utensils are also associated with cross contamination among animals, acting as vehicles in the propagation of the microorganism inside the industry or rearing facility. Furthermore, inadequate use of antimicrobial agents will contribute to the rise of resistant bacteria. This study shows that, in order to safeguard consumer health, awareness must begin in the rearing facilities, with special care to hygiene, biosafety, and the proper use of antibiotics. Further care must be considered during transportation, and especially rigorous measures should be set during slaughter.

O objetivo deste trabalho foi mostrar a presença da Salmonella na cadeia de produção de suínos e o risco em poten cial para a saúde pública. As bactérias do gênero Salmonella são importantes agentes causadores de doenças transmitidas por ali mentos (DTAs) em humanos. Estimase que cerca de 10% do total de casos de salmonelose sejam veiculados por produtos de origem suína. A transmissão ao homem pode ocorrer pelo contato direto com animais, tanto nas granjas quanto nos frigoríficos, mas princi palmente devido à ingestão de alimentos contaminados. A infecção de lotes suínos pode ocorrer em qualquer fase zootécnica e o prin cipal ciclo de infecção é fecaloral, podendo a bactéria se alojar nos linfonodos e ser excretada quando o animal for submetido a um fator estressante, como o transporte e/ou o reagrupamento. O uso inadequado de antimicrobianos também gera cepas multirresistentes. Os equipamentos e utensílios, de uma forma geral, estão relaciona dos à contaminação cruzada, agindo como veículos de propagação do microorganismo dentro da indústria. Para garantir a segurança alimentar do consumidor, concluise que cuidados como limpeza, desinfecção e biossegurança devem se iniciar na granja de criação, com a utilização correta de antibióticos, passando pelo transporte evitando a superlotação e com medidas higiênicosanitárias rigo rosas durante o abate desses animais.

Impact of salmonelosis on pork meat industry and its implications on public health / Impacto da salmonelose na suinocultura e suas implicações em saúde pública

The objective of this study was to show the presence of Salmonella in the swine production chain, and its impact on public health. Bacteria of the Salmonella genus are an important cause of food transmitted diseases in Brazil. Around 10% of clinical salmonellosis cases are attributed to swine products. Humans can become infected through direct contact with the animals, both living and in slaughter houses; however, the most common cause of infection is the ingestion of contaminated food. Infection of swine herds can occur in any of the rearing stages, mainly through a fecaloral cycle, where the bacteria will shelter in the lymph nodes and be excreted when the host is submitted to a stress factor, such as transport and/ or regrouping. Equipment and utensils are also associated with cross contamination among animals, acting as vehicles in the propagation of the microorganism inside the industry or rearing facility. Furthermore, inadequate use of antimicrobial agents will contribute to the rise of resistant bacteria. This study shows that, in order to safeguard consumer health, awareness must begin in the rearing facilities, with special care to hygiene, biosafety, and the proper use of antibiotics. Further care must be considered during transportation, and especially rigorous measures should be set during slaughter.(AU)

O objetivo deste trabalho foi mostrar a presença da Salmonella na cadeia de produção de suínos e o risco em poten cial para a saúde pública. As bactérias do gênero Salmonella são importantes agentes causadores de doenças transmitidas por ali mentos (DTAs) em humanos. Estimase que cerca de 10% do total de casos de salmonelose sejam veiculados por produtos de origem suína. A transmissão ao homem pode ocorrer pelo contato direto com animais, tanto nas granjas quanto nos frigoríficos, mas princi palmente devido à ingestão de alimentos contaminados. A infecção de lotes suínos pode ocorrer em qualquer fase zootécnica e o prin cipal ciclo de infecção é fecaloral, podendo a bactéria se alojar nos linfonodos e ser excretada quando o animal for submetido a um fator estressante, como o transporte e/ou o reagrupamento. O uso inadequado de antimicrobianos também gera cepas multirresistentes. Os equipamentos e utensílios, de uma forma geral, estão relaciona dos à contaminação cruzada, agindo como veículos de propagação do microorganismo dentro da indústria. Para garantir a segurança alimentar do consumidor, concluise que cuidados como limpeza, desinfecção e biossegurança devem se iniciar na granja de criação, com a utilização correta de antibióticos, passando pelo transporte evitando a superlotação e com medidas higiênicosanitárias rigo rosas durante o abate desses animais.(AU)

Highly virulent Leptospira borgpetersenii strain characterized in the hamster model.

Abstract. A recent study by our group reported the isolation and partial serological and molecular characterization of four Leptospira borgpetersenii serogroup Ballum strains. Here, we reproduced experimental leptospirosis in golden Syrian hamsters (Mesocricetus auratus) and carried out standardization of lethal dose 50% (LD50) of one of these strains (4E). Clinical disease features and histopathologic analyses of tissue lesions were also observed. As results, strain 4E induced lethality in the hamster model with inocula lower than 10 leptospires, and histopathological examination of animals showed typical lesions found in severe leptospirosis. Gross pathological findings were peculiar; animals that died early had more chance of presenting severe jaundice and less chance of presenting pulmonary hemorrhages (P < 0.01). L. borgpetersenii serogroup Ballum has had a considerable growth in human leptospirosis cases in recent years. This strain has now been thoroughly characterized and can be used in more studies, especially evaluations of vaccine candidates.

Assessing different chemoprophylactic protocols against bovine tick-borne diseases and their influence on the weight gain of calves / Avaliação de diferentes protocolos de quimioprofilaxia da tristeza parasitária bovina sobre o ganho de peso de novilhos

In the present study, 87 Aberdeen Angus calves were used to assess the effects of low dose, agent-specific drugs on weight gain after a babesiosis and anaplasmosis outbreak. All animals were weighed on weaning (day 34) and again on day zero, with a mean (on day zero) of 223.46 Kg and an average individual daily weight gain (ADG) of 0.258 Kg. The animals were then separated in three groups: G1 was composed of 37 calves with below average ADG; G2 was composed of 35 animals with below average ADG; and G3 was composed of 15 animals with above average ADG. On day zero animals in G1 were treated with 1.17 mg Kg-1 of diminazene diaceturate and 6.7 mg Kg-1 of oxytetracycline; those in G2 were treated with 1.2 mg Kg-1 of imidocarb dipropionate; and those in G3 were not treated. The animals were then monitored daily for the onset of disease, and on days 15 and 34 they were weighed and had their blood harvested. Animals in G1 had the better overall ADG (0.613 Kg day-1) (P 0.05), with no clinical cases during the experiment. The performance in G2 was moderate, not differing from either G1 or G3 (mean ADG = 0.528 Kg day-1), however, this group had two clinical cases of anaplasmosis during the experiment. Animals in G3 had the worst performance, considering ADG (0.343 Kg day-1). When total weight gain per animal is compared for the study period (35 days), those in G1 gained an average...(AU)

No presente estudo foram utilizados novilhos da raça Aberdeen Angus remanescentes de um de surto de babesiose e anaplasmose, expostos a infestações por carrapatos Rhipicephalus (Boophilus) microplus. No dia zero, o lote de 87 novilhos pesando em média 223,46 Kg, apresentava GMD de peso vivo de 0,258 Kg por dia nos últimos 34 dias. O lote foi dividido em 3 grupos: G1, 37 novilhos com GMD abaixo da média; G2, 35 novilhos com GMD abaixo da média; G3, 15 novilhos com GMD acima da média geral do lote. Os novilhos do G1 foram submetidos à quimioprofilaxia com uso de 1,17 mg Kg-1 de diaceturato de diminazeno e 6,7 mg Kg-1 de oxitetraciclina. Os novilhos do G2 foram submetidos à quimioprofilaxia com o uso de 1,2 mg Kg-1 de dipropionato de imidocarb. Os animais do G3 não foram medicados. O G1 obteve o melhor desempenho de ganho de peso no período (0,613 Kg dia-1) (P 0,05) e não apresentou nenhum caso clínico da doença durante o experimento. O protocolo quimioprofilático do G2 teve desempenho mediano, sem diferença dos demais grupos (0,528 Kg dia-1) e o G3 teve o pior desempenho (0,343 Kg dia-1), porém ocorreram 2 quadros clínicos de anaplasmose no final do experimento no G2. Na comparação do desempenho no ganho de peso total por animal no período, o G3 (Controle) obteve 11,667 Kg, O G2 obteve 17,957 Kg e o G1 obteve 20,851 Kg. O protocolo quimioprofilático a base de diaceturato de...(AU)

Ilhas de patogenicidade de Salmonella enterica: uma revisão / Salmonella enterica pathogenicity islands: a review

Salmonella é um bom modelo bacteriano para o estudo das interações entre hospedeiro e agente patogênico. Embora muitos de seus fatores de virulência tenham sido caracterizados, os mecanismos de especificidade aos hospedeiros com o desfecho na doença não estão elucidados. As ilhas de patogenicidade (PAI) são elementos genéticos dos cromossomos de um amplo número de agentes patogênicos. Nas salmonelas, muitos dos fatores de virulência são codificados por genes presentes nas PAI, as quais são referidos como ilhas de patogenicidade da Salmonella (SPI). Nesta revisão, são sumarizados os relatos na literatura específica dos últimos vinte anos sobre o papel das SPI na patogenia da doença e como elas influenciamnos mecanismos envolvidos na invasão e colonização das bactérias patogênicas no hospedeiro.

Ilhas de patogenicidade de Salmonella enterica: uma revisão / Salmonella enterica pathogenicity islands: a review

Salmonella é um bom modelo bacteriano para o estudo das interações entre hospedeiro e agente patogênico. Embora muitos de seus fatores de virulência tenham sido caracterizados, os mecanismos de especificidade aos hospedeiros com o desfecho na doença não estão elucidados. As ilhas de patogenicidade (PAI) são elementos genéticos dos cromossomos de um amplo número de agentes patogênicos. Nas salmonelas, muitos dos fatores de virulência são codificados por genes presentes nas PAI, as quais são referidos como ilhas de patogenicidade da Salmonella (SPI). Nesta revisão, são sumarizados os relatos na literatura específica dos últimos vinte anos sobre o papel das SPI na patogenia da doença e como elas influenciamnos mecanismos envolvidos na invasão e colonização das bactérias patogênicas no hospedeiro. (AU)

Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamsters from challenge.

The bacillus Calmette--Guerin (BCG), a live attenuated Mycobacterium bovis strain is considered a promising candidate as a vector system for delivery of foreign antigens to the immune system. The gene coding for the Leptospira interrogans external membrane protein LipL32, a highly immunogenic antigen found in all pathogenic leptospira, was cloned into several mycobacterial vectors for expression in BCG. Hamsters immunized with recombinant BCG (rBCG) expressing LipL32 were protected against mortality (P

Influence of temperature, incubation time, antigen density and age of the culture in agglutinability of leptospires

In leptospirosis, the Microscopic Agglutination Test (MAT) is the basis of serological diagnosis and the most widely used. In our study, the MAT was applied to evaluate the influence of temperature, incubation time, antigen density and age of the culture in the agglutinability of Leptospires and the interference of these factors in diagnosis. Three serum samples with titers of 100, 800 and 25.600 to the serovar tande (local isolate) of canicola serogroup were used with different combinations among the four factors. There was a significant relation among all factors, specially between the antigen density and age of the culture.

Na leptospirose, o teste de soroaglutinação microscópica (MAT) é a base do diagnóstico sorológico, sendo amplamente usado. Em nosso estudo, o MAT foi aplicado para avaliar a influência da temperatura, tempo de incubação, densidade do antígeno, a idade da cultura na aglutinabilidade das leptospiras e a interferência desses fatores na interpretação do diagnóstico. Três amostras de soro com títulos de 100, 800 e 25600 para o sorovar tande (isolado local), do sorogrupo canicola foram utilizadas com combinações diferentes entre os quatro fatores. Houve uma significante relação entre todos os fatores principalmente a densidade do antígeno e a idade da cultura.

Influence of temperature, incubation time, antigen density and age of the culture in agglutinability of leptospires

Na leptospirose, o teste de soroaglutinação microscópica (MAT) é a base do diagnóstico sorológico, sendo amplamente usado. Em nosso estudo, o MAT foi aplicado para avaliar a influência da temperatura, tempo de incubação, densidade do antígeno, a idade da cultura na aglutinabilidade das leptospiras e a interferência desses fatores na interpretação do diagnóstico. Três amostras de soro com títulos de 100, 800 e 25600 para o sorovar tande (isolado local), do sorogrupo canicola foram utilizadas com combinações diferentes entre os quatro fatores. Houve uma significante relação entre todos os fatores principalmente a densidade do antígeno e a idade da cultura.

Leptospirosis diagnosis using Nested-PCR

Leptospirosis is a worldwide sanitary problem. Its clinical signs resemble that of other diseases like Dengue and Flu, and it is difficult to distinguish between them. Currently available diagnostic methods shown low sensitivity and specificity. Efforts have been made to develop simpler, faster and more efficient diagnostic methods. The aim of this work was to evaluate and optimize a Nested-PCR method for diagnosis of leptospirosis. Primers were designed to amplify a 264 bp region within the LipL32 gene. The sensitivity and specificity of the assay was evaluated using seven saprophytic serovars and 35 pathogenic serovars. This technique showed to be very specific for pathogenic serovars, however it lacked sensitivity. In order to enhance the sensitivity, another primer pair was designed which amplify a 183 bp region within the 264 bp region in lipL32 gene, and used in a Nested-PCR assay. This approach was much more sensitive than traditional PCR.

A leptospirose constitui um problema sanitário de importância mundial. Esta doença caracteriza-se por apresentar sintomas muito parecidos com os de outras doenças como a dengue e a gripe e, clinicamente é difícil de distingui-las. Técnicas de diagnóstico da leptospirose atualmente disponíveis apresentam baixa sensibilidade e ou especificidade. Por isso, tem havido um grande esforço no sentido de desenvolver testes rápidos e eficientes, baseados em técnicas de biologia molecular. Este trabalho objetivou a avaliação e otimização do Nested-PCR, para o diagnóstico de leptospirose. Para isso foi desenhado um par de primers que amplifica uma região de 264 pb do gene LipL32. A sensibilidade e especificidade do teste foram avaliadas utilizando 7 sorovares saprófitas e 35 sorovares patogênicos. Este PCR mostrou-se específico para leptospiras patogênicas, porém a sensibilidade não foi muito alta. Com o objetivo de melhorar a sensibilidade do teste , foi desenhado outro par de primers que amplifica uma região de 183 pb do gene LipL32, interna a de 264 pb para a realização do Nested-PCR. Nesta reação foi utilizado como DNA molde o produto da primeira amplificação. O Nested-PCR mostrou-se mais sensível que o PCR normal, pois foi capaz de detectar um baixo número de células bacterianas.

Leptospirosis diagnosis using Nested-PCR

A leptospirose constitui um problema sanitário de importância mundial. Esta doença caracteriza-se por apresentar sintomas muito parecidos com os de outras doenças como a dengue e a gripe e, clinicamente é difícil de distingui-las. Técnicas de diagnóstico da leptospirose atualmente disponíveis apresentam baixa sensibilidade e ou especificidade. Por isso, tem havido um grande esforço no sentido de desenvolver testes rápidos e eficientes, baseados em técnicas de biologia molecular. Este trabalho objetivou a avaliação e otimização do Nested-PCR, para o diagnóstico de leptospirose. Para isso foi desenhado um par de primers que amplifica uma região de 264 pb do gene LipL32. A sensibilidade e especificidade do teste foram avaliadas utilizando 7 sorovares saprófitas e 35 sorovares patogênicos. Este PCR mostrou-se específico para leptospiras patogênicas, porém a sensibilidade não foi muito alta. Com o objetivo de melhorar a sensibilidade do teste , foi desenhado outro par de primers que amplifica uma região de 183 pb do gene LipL32, interna a de 264 pb para a realização do Nested-PCR. Nesta reação foi utilizado como DNA molde o produto da primeira amplificação. O Nested-PCR mostrou-se mais sensível que o PCR normal, pois foi capaz de detectar um baixo número de células bacterianas.