Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant.

Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen.

Utilização de uma técnica rápida para o diagnóstico de mycobacterium bovis em amostras de leite experimentalmente inoculadas

ABSTRACT The modified Middlebrook 7H11 cultivation technique was comparedwith the tradicional culture techniqueby using the Stonebrink medium. The purpose of this comparison was to to evaluate the sensitivity and the time for the detection of positive cultures of mycobacteria in milk samples experimentally inoculated with Mycobacterium bovis (strain AN5), at a dilution of 10-2 , and submitted to two types of procedures: technique 1 (skin milk) and technique 2 (pellet), decontamined by modified the Petroff´s method, added with Tween 80, and compared with total milk samples submitted to tradicional Petroff method in the same media types. The results of the two tecniques were compared with each other by the non-parametric Wilcoxon test and Mann-Whitney. The results obtained from this experiment showed that: techniques 1 and 2 produced great number of colonies and higher proportion of positive culture than the tradicional one by using total milk; the time needed for the detection of mycobacteria colonies was slightly shorter by the thin layer technique than by the tradicional culture method; in order to improve the epidemiological surveillance, this technique should be used as a complementary method to the tradicional one in the diagnosis of bovine tuberculosis.

RESUMO O meio de Middlebrook 7H11 modificado foi comparado ao meio de Stonebrink, a fim de se avaliar a sensibilidade e o tempo de detecção de micobactérias em amostras de leite, experimentalmente inoculadas com Mycobacterium bovis (estirpe AN5), em uma diluição 10-2, e submetidas a duas diferentes técnicas de processamento: gordura (técnica 1) e sedimento (técnica 2), descontaminadas pelo método de Petroff modificado (adicionado de Tween 80), confrontadas com a técnica do leite total submetida ao método de Petroff tradicional. Os resultados destas técnicas (1 e 2) foram comparados entre si pelos testes não paramétricos de Wilcoxon e de MannWhitney e demonstraram que as técnicas 1 e 2 forneceram maior recuperação de micobactérias e proporção de cultivos positivos nos meios de Stonebrink e Middlebrook 7H11; o meio de Middlebrook 7H11 permitiu a visualização precoce das micobactérias, podendo ser utilizado como uma técnica de diagnóstico rápida da tuberculose bovina, em amostras de leite, para fins de vigilância epidemiológica.

Leptospirosis outbreak in dairy cattle due to leptospira spp. serovar canicola: reproductive rates and serological profile after treatment with streptomycin sulfate

ABSTRACT The objectives of the present trial were to evaluate the reproductive parameters in a Holstein bovine herd affected by leptospirosis during the outbreak of abortion and stillborn fetus and treated with streptomycin sulfate. Animals were divided in 2 groups of 10 cows each: G1, treated with streptomycin sulfate 25 mg/kg/lw for 3 days by IM route; and; G2, untreated control. Serum samples were collected on the onset of abortions and after that every 15 or 30 days until completing 270 days. Samples were analyzed by means of MAT microscopic agglutination test that showed sera conversion to serovar Canicola with titers ranging from 100 to 51,200; 7/20 of the urine samples were positive for Leptospira spp. by PCR After streptomycin sulfate therapy, the outbreak ended and urine samples also got negative to PCR and no fetus samples were positive in either techniques. However, it did not influence the evolution in antibody titers of serological profiles in both groups, which got negative after around 200 days of the onset of symptoms. Although there was no significant difference of the frequency of pregnancy/AI and calving/pregnancy rates between treated and untreated groups (p > 0.05), reproductive rates were lower in the untreated group.

RESUMO O presente trabalho objetivou avaliar o efeito do tratamento com sulfato de estreptomicina sobre o perfil sorológico de um rebanho bovino acometido de um surto de leptospirose pelo sorovar Canicola e avaliar os parâmetros reprodutivos no ciclo reprodutivo subseqüente. Foram formados 2 grupos de 10 fêmeas bovinas: G1-tratado com sulfato de estreptomicina, 25 mg/kg/pv por 3 dias, via IM; G2 sem tratamento. Amostras de soro foram colhidas no início dos abortamentos e posteriormente a cada 15 ou 30 dias num total de 270 dias e examinadas pela reação de soroaglutinação microscópica-SAM. Vinte amostras de urina colhidas no início dos sintomas e 10 -15 dias após tratamento e seis fetos abortados (rins, fígado, pulmão e conteúdo abomasal) foram submetidos ao exame bacteriológico para pesquisa de bactérias causadoras de abortamento e à técnica de PCR para Leptospira spp. Ocorreu soroconversão predominante para Leptospira spp. sorovar Canicola (títulos: 100 a 51.200); 7/20 amostras de urina foram positivas para Leptospira spp. na PCR, sendo todas negativas no exame bacteriológico e nenhuma amostra fetal foi positiva em ambas as técnicas. Os abortamentos cessaram após a antibioticoterapia, e as amostras de urina negativaram-se na PCR. O sulfato de estreptomicina foi eficaz na eliminação da doença, entretanto, não influenciou a evolução dos títulos de anticorpos no perfil sorológico quando comparado ao grupo controle, ambos negativando-se ao redor de 200 dias após o início dos sintomas. Embora não houvesse diferença estatística entre as taxas de prenhez/IA e parto/prenhez do grupo tratado em relação ao não tratado (p > 0,05), talvez devido ao baixo número amostrado, as taxas reprodutivas foram menores no grupo não tratado.

Anticorpos contra agentes bacterianos e virais em suínos de agricultura familiar do município de monte negro, ro

ABSTRACT Sera from swine fromfamily-employed farms in Monte Negro County, state of Rondônia, Brazil, were evaluated againstbacterial and viral agents. Brucellosis was evaluated by Rose-Bengal agglutination (RBA), standard tube agglutination (STA), and mercaptoethanol (ME); and leptospirosis by microscopic agglutination test (MAT) against 24 leptospira serovars. Anti-porcine parvovirus antibodies were tested by haemmaglutination inhibition assay, and ELISA technique was used for detection of antibodies against classical swine fever virus and Aujeszky disease virus. Only 1 (0.9%) from 104 tested sera reacted by RBA, and no samples reacted in STA and ME. Leptospira spp. antibodies were detected in 29 (32.9%) of 88 tested sera and the most frequent serovars detected were Castellonis, Bratislava and Canicola. Antibodies against porcine parvovirus were detected in 7.7% (8/104) of the sera. None of the examined samples reacted to classical swine fever virus or Aujeszky disease virus.

RESUMO Foram avaliados soros de suínos provenientes de propriedades rurais que desenvolvem agricultura familiar no Município de Monte Negro, Rondônia, frente a agentes bacterianos e virais. Para a pesquisa de anticorpos contra Brucella spp., foram utilizados o Antígeno Acidificado Tamponado (AAT), a Soroaglutinação Lenta em Tubos (SAL) e a prova do 2-Mercaptoetanol (2-ME). Para a pesquisa de Leptospira spp. utilizou-se a Soroaglutinação Microscópica (SAM) contra 24 sorovares de leptospira. Anticorpos contra Parvovírus Suíno (PVS) foram pesquisados pela Inibição da Hemaglutinação (IH), enquanto que o vírus da Peste Suína Clássica (PSC) e da Doença de Aukeszky (DA) por Ensaio Imunoenzimático (ELISA). Apenas uma (0,9%) das 104 amostras testadas foi reagente na AAT e nenhuma na SAL e 2-ME. Para Leptospira spp. foram testados 88 soros, sendo detectados anticorpos em 29 amostras (32,9%) e os sorovares mais freqüentes foram Castellonis, Bratislava e Canicola. Foram detectados anticorpos contra PVS em 7,7% (8/104) dos soros testados. Nenhuma amostra foi reagente para PSC e DA.