Thesis (MSc (Genetics))--Stellenbosch University, 2008. / South Africa is one of the top ten wine producing countries in the world. The South African wine industry contributes approximately R16.3 billion to South Africa’s annual gross domestic product with 42.8% of wine being exported. To compete with the top wine producing countries and to ensure a viable export market, South Africa needs to ensure that healthy, virus free propagation material is produced and sold. One of the viruses that need to be tested for is Grapevine fanleaf virus (GFLV). Grapevine fanleaf virus causes degeneration and malformation of berries, leaves and canes and is responsible for significant economic losses by reducing crop yields by as much as 80%, reducing the longevity of the vines and affecting fruit quality. It is widespread in the Breede River Valley of the Western Cape where the nematode vector, Xiphinema index, is prevalent. The Breede River Valley contributes approximately 30% of the total production of the local wine industry, and severe losses in this region could threaten the viticulture. The Plant Improvement Act states that all propagation material sold must be tested for GFLV by a reputable scientific technique. The technique commonly used in South Africa is the Double Antibody Sandwich - Enzyme-linked Immunosorbent Assay (DAS-ELISA) and the kits are imported from Europe at a significant cost to the South African viticulture industry. The objective of this study was to produce a reliable and sensitive diagnostic assay specific for the South African strains of GFLV. This project aimed to develop and optimize a DAS-ELISA, by using recombinant DNA technology to produce antibodies against bacterially expressed viral coat protein. Total RNA was extracted from GFLV infected grapevine material and the viral coat protein (CP) amplified. The CP was cloned into the pGex-6P-2 expression vector, fusing a Glutathione STransferase (GST) partner to the viral coat protein enhancing solubility and protein purification. Insufficient amounts of the soluble protein were expressed and purified, preventing the production of antibodies and thus the development of the DAS-ELISA. An alternative diagnostic rapid-direct-one-tube-RT-PCR assay was developed. This rapid-directone- tube-RT-PCR assay was compared to commercially available DAS-ELISA and ImmunoStrip tests (Agdia) to assess the reliability, sensitivity and specificity of the rapid-direct-one-tube-RTPCR assay. Twelve GFLV isolates from South Africa were sequenced to investigate the variability between the isolates as well as the variability between the South African isolates and GFLV sequences available in Genbank. Sequence identities between clones from different GFLV isolates from South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the coat protein gene sequences showed that the South African isolates form two distinct clades or sub-populations. No significant correlation was found between geographical origin and symptoms, nor between geographical origin and sequence variability or between grapevine cultivar and symptom expression. Of the 23 samples tested with all three tests, 21 tested positive with rapid-direct-one-tube-RT-PCR, 19 with the ImmunoStrips and 17 with an imported DAS-ELISA kit (Agdia). Rapid-direct-one-tube-RT-PCR was found to be the most reliable technique for GFLV detection. Although the establishment of a DAS-ELISA directed to the South African strain(s) of GFLV was not successful, an alternative PCR based diagnostic system was developed, and proved to be sensitive and reliable. RT-PCR based diagnostic assays are generally accepted to be more sensitive than DAS-ELISA, but the latter is still used as the diagnostic assay of choice for routine testing due to ease of use. This rapid-direct-one-tube-RT-PCR assay is a rapid, sensitive and reliable diagnostic test, reducing the prevalence of false negatives, contributing to a virus free viticulture industry. The rapid-direct-one-tube-RT-PCR assay is as easy to use as DAS-ELISA, faster and can be performed by semi skilled workers, thus providing all the advantages associated with DAS-ELISA.

>Magister Scientiae - MSc / Glucose oxidase (GOx) and horseradish peroxidase (HRP) are important enzymes for the development of amperometric enzyme linked immunosensors. The selectivity of each enzyme towards its analyte deepens its importance in determining the sensitivity of the resultant immunosensor. In designing immunosensors that have customized transducer surfaces, the incorporation with FAD and iron based enzymes ensures that electron kinetics remains optimal for electrochemical measurement. Various different immobilization strategies are used to produce response signals directly proportional to the concentration of analyte with minimal interferences. The combination of self-assembled monolayers and supramolecular chemistry affords stability and simplicity in immunosensor design. In this work, two electrochemical strategies for the detection of human chorionic gonadotropin(hCG) is presented. This involves the modification of a gold surface with a thiolated β-cyclodextrin epichlorohydrin polymer (βCDPSH) to form a supramolecular inclusion complex with ferrocene (Fc)-functionalised carboxymethyl cellulose polymer (CMC). Cyclic voltammetry indicated that ferrocene is in close proximity to the electrode surface due to the supramolecular complex formed with βCDPSH. Furthermore, strategy (a) for the detection of hCG used α-antihCG labelled (HRP) as reporter conjugate. Strategy (b) maintained the CMC bifunctionalised with Fc and recognition antibody for hCG hormone. However, the system was functionalised with a HRP enzyme and detection is done by using GOx reporter conjugates for in situ production of hydrogen peroxide. The reduction of H2O2 was used for the amperometric detection of hCG by applying a potential of 200 mV. The sensitivity and limit of detection of both strategies were calculated from calibration plots. For strategy (a) the LOD was found to be 3.7283 ng/mL corresponding to 33.56 mIU/mL and a sensitivity of 0.0914 nA ng-1 mL-1. The corresponding values for strategy (b) are 700 pg/mL (6.3 mIU/mL) and 0.94 nA ng-1 mL-1.

Philosophiae Doctor - PhD (Chemistry) / Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (MTB) that usually affects the lungs leading to severe coughing, fever and chest pains. It was estimated that over 9.6 million people worldwide developed TB and 1.5 million died from the infectious disease of which 12 % were co-infected with human immunodeficiency virus (HIV) in the year 2015. In 2016 the statistics increased to a total of 1.7 million people reportedly died from TB with an estimated 10.4 million new cases of TB diagnosed worldwide. The development of the efficient point-of-care systems that are ultra-sensitive, cheap and readily available is essential in order to address and control the spread of the tuberculosis (TB) disease and multidrugresistant tuberculosis.

Magister Scientiae - MSc / The toxicity of microcystin is associated with the inhibition of serine/threonine protein phosphatases 1 and 2A, which can lead to hepatocyte necrosis and haemorrhage. Analysis of microcystin is most commonly carried out using reversed-phase high performance liquid chromatographic methods (HPLC) combined with ultra-violet (UV) detection .The ability of these techniques to identify unknown microcystin in environmental samples is also restricted by the lack of standard reference materials for the toxins. Highly specific recognition molecules such as antibodies and molecularly imprinted polymers (MIPs) have been employed in the pre-concentration of trace levels of microcystin from water and show great potential for the clean-up of complex samples for subsequent analysis. New biosensor technologies are also becoming available, with sufficient sensitivity and specificity to enable rapid ‗on-site‘ screening without the need for sample processing. In this work we constructed a Protein phosphatase biosensor for detection of microcystin-LR in aqueous medium, onto polyamic acid/graphene oxide (PAA: GO) composite electrochemically synthesised in our laboratory. The composites were synthesised at three different ratios i.e. 50:50, 80:20 and 20:80 to evaluate the effect of each component in the search to produce highly conductive mediator platforms. The electrochemistries of the three different composites were evaluated using CV and SWV to study interfacial kinetics of the materials as thin films at the glassy carbon electrode. The phosphatase biosensor parameters were evaluated using CV, SWV, EIS and Uv-vis spectroscopy. The affinity binding of the microcystin-LR to protein phosphatase 2A was investigated using electrochemical impedance spectroscopy which is a highly sensitive method for measuring interfacial kinetics of biosensor systems.

Made available in DSpace on 2015-03-26T13:46:43Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1492276 bytes, checksum: 4d382ffe8ee028de0569c86987765f6b (MD5) Previous issue date: 2004-12-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study was carried out with the purpose to develop an serological diagnosis protocol of bovine cysticercosis for the ELISA test using two metacestodes antigens of Taenia crassiceps and three of Taenia solium. There were used 20 sera of experimentally infected cattle with T. saginata eggs, 60 of cattle with natural infection, diagnosed at slaughterhouses, five of cattle negative for cisticercose, reared in isolation, 55 of negative bovine in slaughterhouses and 10 of bovine with actinomicosis (n=2), actinobacilosis (n=1), fasciolosis (n=1) diagnosed at slaughterhouse and of experimentally infected cattle with Anaplasma marginale (n=3), Babesia sp. (n=2) and concomitant infection for Anaplasma marginale and Babesia bovis (n=1). There were used the total and fluid cistic antigens of T. Crassiceps metacestodes and escólex and membrane antigens of T. solium metacestodes in ELISA test, after previous characterization in polyacrylamide gel electrophoresis (PAGE) in gradient 5 to 20%. The better antigen concentration was 1 µg and the better sera and conjugated dilutions were 1:25 and 1:5.000, respectively. Although the T. solium antigens have provided the most sensibility values, the T. crassiceps antigens also showed good performance to the bovine cysticercosis diagnosis. Different control serum groups employed for the cut-off calculation had changed the ELISA test results. We can concluded that the ELISA test for antibodies detection presents deficiencies in the diagnosis of naturally infected animals that showed low sensibility (5 to 32%) for different antigens. However, for experimentally infected cattle, the sensibility was high, 75 to 90% for different antigens. The test could still be considered useful in the differentiation between the cisticercose and other diseases, due to its high specificity rates (81 to 100%). / A cisticercose bovina é uma zoonose que tem o ser humano como único hospedeiro definitivo. Além de sua importância para a Saúde Pública, esta parasitose acarreta prejuízos econômicos em matadouros, ao levar as carcaças acometidas a julgamento. Este trabalho teve como objetivo o desenvolvimento de um teste de diagnóstico sorológico da cisticercose bovina pelo teste ELISA empregando dois antígenos de larvas de Taenia crassiceps e três de Taenia solium. Foram utilizados 20 soros de bovinos infectados experimentalmente com ovos de T. saginata, 60 de bovinos com infecção natural, diagnosticados em matadouros, cinco de bovinos negativos para cisticercose, criados sob isolamento, 55 de bovinos considerados negativos em matadouros e 10 de bovinos portadores de actinomicose (n=2), actinobacilose (n=1), fasciolose (n=1) diagnosticados em matadouro e de bovinos infectados experimentalmente com Anaplasma marginale (n=3), Babesia sp. (n=2) e infecção mista por Anaplasma marginale e Babesia bovis (n=1). Foram empregados os antígenos total e vesicular de larva de Taenia crassiceps e total, de escólex e de membrana de larva de Taenia solium no teste ELISA, após prévia caracterização em eletroforese em gel de poliacrilamida (SDS-PAGE) sob gradiente 5 a 20%. Após a realização de ensaios de avaliação do desempenho do teste em duas etapas, sempre considerando o critério da amplitude da diferença entre densidades ópticas de soros-controle positivos e negativos, foram obtidos os resultados que se seguem. A concentração de 1 µg de antígeno por orifício foi a que proporcionou, na maioria das vezes, a maior diferenciação entre soros positivos e negativos com todos os cinco antígenos estudados. As diluições 1:25 de soros e 1: 5.000 de conjugado também foram as que se destacaram, à exceção do antígeno de líquido vesicular de larva de T. crassiceps, que teve melhor desempenho quando o conjugado foi diluído a 1:2.500 vezes. O leite desnatado foi a melhor substância bloqueadora dos sítios reativos remanescentes da placa. Embora os antígenos de larva de T. solium tenham proporcionado valores mais elevados de sensibilidade, os antígenos de larva de T. crassiceps também mostraram bom desempenho no diagnóstico de cisticercose bovina. A escolha de diferentes grupos de soroscontrole para o cálculo do ponto de corte interferiu de forma expressiva no desempenho do teste ELISA. Pode-se concluir que o teste ELISA para detecção de anticorpos apresenta deficiências no diagnóstico de animais destinados ao abate, em virtude de sua baixa sensibilidade (5 a 32%) para diferentes antígenos, quando se consideram soros de animais com infecção natural, geralmente discreta. No entanto, no caso de animais infectados experimentalmente, a sensibilidade se mostrou elevada, 75 a 90%, para diferentes antígenos. O teste ainda pode ser considerado útil na diferenciação entre a cisticercose e outras doenças, devido às suas elevadas taxas de especificidade (81 a 100%).

Made available in DSpace on 2015-07-22T20:49:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-27 / Uma cisteína proteinase recombinante, rLdccys1, obtida pela expressão do gene Ldccys1 de Leishmania (Leishmania) chagasi em sistema bacteriano, foi utilizada em ensaios de imunodiagnóstico por ELISA e DTH em cães de uma região endêmica de leishmaniose visceral (LV), Teresina, Piauí. A rLdccys1 também foi utilizada para a imunização de cães e a avaliação da possível proteção dos animais imunizados contra o desafio com a L. (L.) chagasi. Os ensaios por ELISA dos soros de cães com LV mostraram que a sensibilidade para a detecção de anticorpos anti-L. (L.) chagasi utilizando a rLdccys1 e os extratos de promastigotas e amastigotas de L. (L.) chagasi foi de 98%, 86% e 89%, respectivamente. A rLdccys1 não apresentou reatividade cruzada com os soros de cães com doenças comuns nas regiões endêmicas de LV, tais como erliquiose, babesiose e doença de Chagas e a especificidade dos ensaios de ELISA utilizando-se a rLdccys1 e os extratos de amastigotas e promastigotas de L. (L.) chagasi foi de 96%, 69% e 68%, respectivamente. As respostas de DTH foram avaliadas nos cães após a injeção subcutânea da rLdccys1 ou do extrato dos amastigotas de L. (L.) chagasi. Todos os cães assintomáticos apresentaram resposta de DTH positiva à rLdccys1 com a formação de nódulos em torno de 10 mm 48 horas após a injeção do antígeno, enquanto que os cães sintomáticos não apresentaram reatividade significante à rLdccys1 nos ensaios de DTH. As respostas de DTH foram mais intensas quando se utilizou a rLdccys1 comparadas às observadas com o extrato dos parasitas. A análise histológica mostrou áreas de necrose e hemorragia nos nódulos induzidos pelo extrato dos parasitas e reação granulomatosa típica, com predomínio de células mononucleares, nos cortes dos nódulos induzidos pela rLdccys1. A análise dos dados obtidos nos ensaios de ELISA e DTH realizados com a rLdccys1 e os extratos de amastigotas de L. (L.) chagasi mostrou a correlação inversa entre as respostas humoral e celular no desencadeamento da LV canina. As respostas celulares induzidas nos cães pela rLdccys1 foram avaliadas após a imunização dos animais com o antígeno recombinante juntamente com a Propionibacterium acnes. Os linfócitos de sangue periférico dos cães imunizados com a rLdccys1 + P. acnes apresentaram significantes índices de estimulação quando reestimulados in vitro com a rLdccys1 ou o extrato dos amastigotas de L. (L.) chagasi. A dosagem de citocinas nos sobrenadantes dessas culturas mostrou a secreção de níveis significantes de IFN-γ, enquanto IL-10 não foi detectada. Nos experimentos em que cães foram imunizados com a rLdccys1 + P. acnes e desafiados com a injeção intraperitoneal de 1x104 amastigotas isolados de cão infectado com a L. (L.) chagasi 3 de 4 animais sobrevieram dez semanas após o desafio, enquanto que os animais controles que receberam PBS e a P. acnes morreram em no máximo 4 e 6 semanas, respectivamente, após o desafio. Nos cães imunizados com a rLdccys1 o número de amastigotas de L. (L.) chagasi foi significantemente reduzido no baço, fígado e medula óssea comparado ao observado nos controles. A dosagem sérica de IFN-γ nos animais imunizados com a rLdccys1 mostrou a secreção significante dessa linfocina, enquanto que níveis basais de IL-10 foram detectados. Todos os cães imunizados com a rLdccys1 e desafiados pela picada das fêmeas de Lutzomyia longipalpis sobreviveram até dezesseis semanas após o desafio e os animais controles injetados com PBS e P. acnes morreram sete e nove semanas, respectivamente, após o desafio. Os cães controles apresentaram número significante de amastigotas de L. (L.) chagasi no fígado e baço, porém nos animais imunizados com a rLdccys1 nenhum parasita foi observado. No grupo imunizado com a rLdccys1 houve aumento crescente dos níveis séricos de IFN-γ durante a imunização que atingiu um pico uma semana após o desafio, enquanto que e a concentração de IL-10 foi mantida em níveis basais. Os dados do presente trabalho mostraram o potencial da rLdccys1 de L. (L.) chagasi para o diagnóstico e a imunoprofilaxia da LV canina, abrindo perspectivas para a imunização dos cães em larga escala nas regiões endêmicas de LV e a avaliação do impacto da proteção conferida nos animais imunizados na incidência da doença. / A recombinant protein, rLdccys1, produced by expression of the gene encoding a 30 kDa cysteine proteinase from Leishmania (Leishmania) chagasi, was used to detect specific antibodies in serum by enzyme-linked immunosorbent assays and to test for reactivity in delayed-type hipersensitivity (DTH) responses of dogs from an endemic region of visceral leishmaniasis (VL), Teresina, Piauí State, Brazil. The recombinant protein was also used for immunization of dogs and evaluation of its possible protective role against L. (L.) chagasi infection. The sensitivity for detection of specific antibodies to L. (L.) chagasi using rLdccys1 and lysates from L. (L.) chagasi promastigotes and amastigotes was 96%, 68%, and 69%, respectively. No cross reactivity between rLdccys1 and Chagas disease was observed, and little reactivity was found with sera from dogs with babesiosis and ehrlichiosis. The specificity of ELISA assays using rLdccys1, lysates from L. (L.) chagasi promastigotes and amastigotes was 96%, 69%, and 68%, respectively. DTH responses were determined after subcutaneous injection of rLdccys1 or L. (L.) chagasi amastigote extract and the induration area was measured at 24, 48 and 72 h after injection. All asymptomatic dogs showed a positive intradermal response to rLdccys1 (10 mm) which peaked at 48 h, whereas no significant reactivity to the recombinant antigen was found in the symptomatic group. DTH responses to rLdccys1 were higher than those induced by amastigote extract. Histological analysis of the intradermal induration showed a predominance of necrotic and hemorrhagic areas in sections from asymptomatic dogs injected with L. (L.) chagasi amastigote extract, whereas a typical granulomatous reaction mediated by mononuclear cells was observed in sections from asymptomatic animals injected with rLdccys1. Data analysis from ELISA and DTH assays with rLdccys1 and L. (L.) chagasi amastigote extracts showed that humoral and cellular responses were inversely correlated during the development of canine VL. Cellular immune responses induced by the recombinant antigen were evaluated after immunization of dogs with rLdccys1 plus Propionibacterium acnes. Peripheral blood mononuclear cells isolated from rLdccys1-immunized dogs showed significant stimulation indexes after in vitro incubation with either rLdccy1 or L. (L.) chagasi amastigote extracts. Cytokine dosages in the supernatants from lymphocyte cultures showed significant levels of IFN-γ, whereas IL-10 was not detected. Whereas 3 from 4 dogs immunized with rLdccys1 plus P. acnes and challenged by intraperitoneal injection of 1x104 L. (L.) chagasi amastigotes survived ten weeks after challenge, control dogs which received either PBS or P. acnes died after four and six weeks, respectively. The load of L. (L.) chagasi amastigotes in spleen, liver, and bone marrow from rLdccys1-immunized dogs was significantly reduced in comparison to that of non immunized controls. A significant concentration of IFN-γ and basal levels of IL-10 were detected in sera from dogs immunized with rLdccys1. All dogs immunized with rLdccys1 plus P. acnes and challenged by the bite of L. (L.) chagas infected Lutzomyia longipalpis survived until sixteen weeks after challenge, whereas control dogs injected with PBS or P. acnes died after seven and nine weeks, respectively. Control dogs showed a significant number of L. (L.) chagasi amastigotes in liver and spleen, but no parasites were found in rLdccys1- immunized dogs. During immunization with rLdccys1 there was an increase of serum levels of IFN-γ in the immunized dogs that peaked one week after challenge. In contrast, a very low concentration of IL-10 was detected in these animals. Overall, these findings indicate that L. (L.) chagasi recombinant cysteine proteinase is potentially useful for diagnosis and immunoprophylaxis of canine VL. We believe that results obtained open perspectives for immunization of dogs in the field and evaluation of the impact on the disease incidence. / TEDE / BV UNIFESP: Teses e dissertações

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-16T11:43:38Z No. of bitstreams: 1 mauro-franca-da-silva.pdf: 1113117 bytes, checksum: 4721a993f32ab60140b4d0cf8c0f704f (MD5) / Made available in DSpace on 2012-11-16T11:43:39Z (GMT). No. of bitstreams: 1 mauro-franca-da-silva.pdf: 1113117 bytes, checksum: 4721a993f32ab60140b4d0cf8c0f704f (MD5) Previous issue date: 2007 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O vírus atenuado da febre amarela, subcepa 17DD, é utilizado por Bio-Manguinhos para a produção da vacina contra a febre amarela. Esta vacina tem sido utilizada para a imunização humana com um excelente histórico de eficácia e segurança. Entretanto, nos últimos anos, devido à ocorrência de alguns casos de eventos adversos associados ao vírus vacinal cepa 17D e subcepa 17DD, apontou-se a necessidade de desenvolvimento de uma vacina inativada. Para a implementação desta nova vacina torna-se necessário o desenvolvimento de métodos de quantificação de antígenos virais. Diferentes metodologias de quantificação podem ser utilizadas na produção de vacinas inativadas, sendo as mais comuns o teste imunoenzimático (ELISA) e o teste de dose-resposta. O presente estudo teve como objetivo o estabelecimento de um ELISA visando à detecção do antígeno do vírus da febre amarela inativado. Para este propósito, foram obtidos estoques de partículas virais da subcepa 17DD, a partir de culturas de células Vero, os quais foram purificados e quantificados por métodos bioquímicos e virológicos clássicos, respectivamente. Para o desenvolvimento do teste utilizamos diferentes anticorpos como capturana fase sólida. Os resultados obtidos para os testes utilizando o anticorpo 2D12 como captura mostraram um limite de detecção do antígeno no ELISA foi de 2,21 log 10 PFU/0,1mL e (1,55 µg/0,1mL). A partir deste valor, foi estabelecido um controle positivo contendo o vírus 17DD atenuado com título de 3,06 log10 PFU/mL e (29µg/0,1mL). Os resultados mostram, também, que o ELISA foi capaz de detectar o vírus 17DD inativado por formaldeído até a diluição 1:16 (52,9 µg/0,1mL). Baseado nos resultados obtidos acredita-se que o desenvolvimento de um teste de ELISA para detecção e quantificação do antígeno 17DD possa representar umimportante avanço tecnológico no controle da produção de uma vacina inativada contraa febre amarela. / The attenuated 17DD substrain of yellow fever virus is used in Bio-Manguinhos for yellow fever vaccine production. This vaccine has been used for human immunization with an excellent history of efficacy and safety. However, in the latest years, the occurrence of adverse events associated with 17D and 17DD substrain pointed to the necessity of developing technologies for the production of an inactivated vaccine. The implementation of this new vaccine will require methods for antigen quantification. Different methodologies of quantification can be used, being the most commonlyused the Enzyme-Linked Immunosorbent Assay (ELISA) and dose response test.The aim of this study was the establishment of an ELISA for the detection of inactivated yellow fever virus antigen. For this purpose, 17DD virus was obtained from Vero cell cultures, purified and quantified by biochemical and virological classical methods, respectively. The results showed that ELISA test using the 2D12 capture antibody presented a limit of 2,21 log10PFU/0.1mL of viral titer and (1,55 µg viral protein/0.1mL). Based on this value, a positive control was established which contained the attenuated 17DD substrain of yellow fever virus with a titer of 2,95log 10 PFU/mL and (29 µg/0.1mL). The results also showed that the ELISA was able to detect 17DD virus inactivated by formaldehyde up the dilution 1:16 (52,9 µg protein/0,1mL). The development of an ELISA test for the detection and quantification of 17DD antigen can represent an important step in the production control of the inactivated vaccine against of yellow fever.

Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-05-22Bitstream added on 2014-06-13T19:12:39Z : No. of bitstreams: 1 ribeiro_d_me_araca.pdf: 985135 bytes, checksum: 3aa44a75a9180f7b89be1f30675f165b (MD5) / A Linfadenite Caseosa (LC) é uma doença crônica contagiosa, causada pela Corynebacterium pseudotuberculosis que acomete ovinos e caprinos acarretando perdas econômicas importantes. O diagnóstico é baseado no cultivo e identificação bioquímica, no entanto a fase subclínica e/ou visceral requerem métodos alternativos para sua detecção. Apesar dos métodos de diagnóstico já existentes, raramente pesquisas investigaram a utilização de material proveniente de punção aspirativa por agulha fina (PAAF) de linfonodo de ovinos aplicadas a outras técnicas de diagnóstico. O presente trabalho objetivou avaliar a sensibilidade e especificidade de métodos de diagnóstico para LC em ovinos sintomáticos (n=26) e assintomáticos (n=129), utilizando material colhido através de PAAF de linfonodo. As técnicas basearam-se na amplificação por PCR do gene alvo pld (fosfolipase D), no cultivo bacteriano associado a identificação bioquímica, no citodiagnóstico, bem como na pesquisa do agente etiológico. O teste sorológico por ELISA indireto foi adaptado para a espécie ovina. As amostras clínicas recuperadas da PAAF forneceram material adequado e suficiente para realização dos testes propostos, implementando a rotina do diagnóstico para LC. Dentre os métodos testados, o ELISA e a PCR foram os que apresentaram maior sensibilidade (92%). A maior especificidade foi verificada no cultivo bacteriano (98%), seguido do exame citológico (94%). / Caseous Lymphadenitis (CL) is considered a cronic contagious disease caused by Corynebacterium pseudotuberculosis affecting sheep and goats causing economical losses. The diagnostic is based on microorganism culture and respective identification. However no clinical and/ or visceral cases need alternative methods for detection. In spite of a range of diagnostic methods, few studies demonstrate the usefulness of fine-needle aspiration biopsy (FNAB) method. The aim of this study was to evaluate the sensitivity and specificity of diagnostic methods for CL in symptomatic (n=26) and asymptomatic (n=129) sheeps, using specimes collected from lymph nodes by FNAB assay. These techniques were performed using PCR targeting the pld gene (phospholipase D), on biochemical identification and culture, cytodiagnostic searching the aetiological agent. The serologic test of ELISA method was also applied in all ovine sera. Clinical samples recovered in FNAB given suitable and enough samples to perform the identification of C. pseudotuberculosis, implementing CL diagnostics. Among all techniques used here, ELISA and PCR demonstrated higher sensitivity (92%), whereas microorganism culture (98%) and cytodiagnostic (94%) presented higher specificity.

A tuberculose é uma doença crônica granulomatosa caracterizada por um déficit de imunidade antígeno específica do hospedeiro, cuja resposta imune é ativamente regulada por citocinas. No Brasil há mais de 50 milhões de habitantes infectados pelo Mycobacterium tuberculosis. O objetivo foi avaliar a linfoproliferação e a produção de citocinas por células mononucleares do sangue periférico (PBMC) estimuladas por quatro diferentes antígenos do M. tuberculosis, um complexo, o antígeno sonicado, e três purificados, ESAT-6, antígeno 85B e antígeno HBHA, eventuais candidatos à vacina anti-tuberculose. Para avaliação da produção de IFN-g e IL-10 foram utilizados dois métodos: Elispot e Elisa à partir de sobrenadante de cultura de PBMC. Para essas avaliações, os pacientes com tuberculose ativa (TB-A) foram comparados a dois subgrupos de indivíduos controles. O primeiro subgrupo foi constituído por indivíduos saudáveis PPD+ e o segundo por indivíduos curados de um episódio de tuberculose (TB-C). Nossos resultados de linfoproliferação e de Elisa revelaram diminuição da resposta linfoproliferativa e da produção de IFN-g dos pacientes em comparação com os indivíduos PPD+, enquanto os indivíduos TB-C apresentaram em geral resultados intermediários. Observou-se também que as respostas à PHA não diferiam significativamente entre os grupos, ressaltando a natureza antígeno específica da hiporreatividade na tuberculose. Adicionalmente, verificamos maior reatividade ao antígeno complexo, sonicado, que aos antígenos purificados, e entre estes, a reatividade foi maior para ESAT-6 e 85B que para HBHA, A resposta ao HBHA pode ter sido eventualmente subestimada por razões técnicas, como utilização de dose sub-ótima ou perda da atividade biológica. Em relação ao Elispot para IFN-g, não pudemos observar diferenças entre os grupos, tanto quando se considerou o número total de spots, como quando se contou apenas spots com diâmetro > 65 mm, apresentando portanto uma sensibilidade aparentemente menor comparado aos outros 2 métodos. A comparação entre os métodos revelou pouca correlação entre seus resultados, que pode ser eventualmente explicado pela diferente contribuição das populações celulares (T CD4+ e T CD8+) para cada uma das provas munológicas. Finalmente, a análise da produção de IL-10 medida por Elisa no sobrenadante de cultura e por spots de IL10, também não revelou diferenças entre os grupos. Convém notar que o Elisa detectou baixas concentrações de IL-10 nos sobrenadantes, porém o Elispot demonstrou número elevado de spots e boa correlação entre as resposta aos antígenos. Em conclusão, nossos resultados sugerem que métodos \'clássicos\', e já estabelecidos, como linfoproliferação e Elisa, persistem válidos para se avaliar a imunidade celular, e que em nossas condições laboratoriais, a técnica de Elispot não representou, até o momento, uma melhora na qualidade da avaliação imunológica. / Tuberculosis is a chronic granulomatous disease characterized by a deficit of the antigen-specific immunity of the host, whose immune response is actively regulated by cytokines. In Brazil there are 50 million people infected with Mycobacterium tuberculosis. The objective of the present work was to evaluate the lymphoproliferative response e the IFN-g response by peripheral blood mononuclear cells (PBMC) indiced with 4 different antigens isolated from Mycobacterium tuberculosis: a complex, crude, the sonicate antigen, and 3 other, purified ones, Esat-6, 85B, and HBHA, the last 3 eventual candidates to the design of a vaccine against tuberculosis. We used 2 methods to evaluate the IFN-g and IL-10 productions, namely Elispot and Elisa of supernatant of PBMC cultures. We studied a group of active tuberculosis patients (TB-A), and compared them with controls individuals comprising 2 groups, one made of healthy PPD+ individuals and the second one of individuals who have been cured from an episode of tuberculosis in the past (TB-C). Our results of lymphoproliferation and Elisa revealed decrease in the lymphoproliferative and IFN-g responses by patients\' PBMC as compared to the PPD+ group, with the TB-C group in general presenting intermediate results. We also observed that the responses to the mitogen PHA were not statisically different among the groups, denoting the antigen-specific nature of the immune deficit in tuberculosis. In addition, we verified that stronger reactivity to the complex antigen than with the purified antigens, and, among the latter, the reactivity was stronger with Esat-6 and 85B as compared to HBHA, Reactivity to HBHA may have been understimated due to technical reasons, such as loss of .the biological activity of the molecule or use of a sub-optimal dose. By using the Elispot for IFN-g we were not able to detect differences among the groups, even when we counted all spots formed or spots with more than > 65 mm in diameter. Thus our Elispot for IFN-g apparently showed lower sensitivity than the other 2 methods. Furthermore, comparisons between the methods revealed low correlation between their results, a finding that may be explained by the differning contribution of different subpopulations (T CD4+ and T CD8+) to each of the results. Finally, analysis of the production of IL-10 as measured by Elisa in the culture supernatants as well as by Elispot revealed no differences among the groups. It is noteworthy that the levels of IL-10 detected by Elisa were low, but the Elispot revealed high number of spots and a good correlation between the antigen responses. In conclusion, we may say that our well standardized \'classical\' methods Elisa and lymphoproliferation persist useful to evaluate cellular immunity responses, and that the Elispot technique, up to now and in our laboratorial conditions, did not represent an improvement in the quality of the immunological evaluation.

Resumo: A Linfadenite Caseosa (LC) é uma doença crônica contagiosa, causada pela Corynebacterium pseudotuberculosis que acomete ovinos e caprinos acarretando perdas econômicas importantes. O diagnóstico é baseado no cultivo e identificação bioquímica, no entanto a fase subclínica e/ou visceral requerem métodos alternativos para sua detecção. Apesar dos métodos de diagnóstico já existentes, raramente pesquisas investigaram a utilização de material proveniente de punção aspirativa por agulha fina (PAAF) de linfonodo de ovinos aplicadas a outras técnicas de diagnóstico. O presente trabalho objetivou avaliar a sensibilidade e especificidade de métodos de diagnóstico para LC em ovinos sintomáticos (n=26) e assintomáticos (n=129), utilizando material colhido através de PAAF de linfonodo. As técnicas basearam-se na amplificação por PCR do gene alvo pld (fosfolipase D), no cultivo bacteriano associado a identificação bioquímica, no citodiagnóstico, bem como na pesquisa do agente etiológico. O teste sorológico por ELISA indireto foi adaptado para a espécie ovina. As amostras clínicas recuperadas da PAAF forneceram material adequado e suficiente para realização dos testes propostos, implementando a rotina do diagnóstico para LC. Dentre os métodos testados, o ELISA e a PCR foram os que apresentaram maior sensibilidade (92%). A maior especificidade foi verificada no cultivo bacteriano (98%), seguido do exame citológico (94%). / Abstract: Caseous Lymphadenitis (CL) is considered a cronic contagious disease caused by Corynebacterium pseudotuberculosis affecting sheep and goats causing economical losses. The diagnostic is based on microorganism culture and respective identification. However no clinical and/ or visceral cases need alternative methods for detection. In spite of a range of diagnostic methods, few studies demonstrate the usefulness of fine-needle aspiration biopsy (FNAB) method. The aim of this study was to evaluate the sensitivity and specificity of diagnostic methods for CL in symptomatic (n=26) and asymptomatic (n=129) sheeps, using specimes collected from lymph nodes by FNAB assay. These techniques were performed using PCR targeting the pld gene (phospholipase D), on biochemical identification and culture, cytodiagnostic searching the aetiological agent. The serologic test of ELISA method was also applied in all ovine sera. Clinical samples recovered in FNAB given suitable and enough samples to perform the identification of C. pseudotuberculosis, implementing CL diagnostics. Among all techniques used here, ELISA and PCR demonstrated higher sensitivity (92%), whereas microorganism culture (98%) and cytodiagnostic (94%) presented higher specificity. / Orientador: Maria Cecília Rui Luvizotto / Coorientador: Vasco Ariston de Carvalho Azevedo / Banca: Márcio Garcia Ribeiro / Banca: Tereza Cristina Cardoso da Silva / Mestre