Toxoplasma gondii é o agente etiológico da toxoplasmose, podendo causar diferentes formas da doença, entre elas, a toxoplasmose congênita cuja frequência varia significativamente de acordo com o país estudado. Na França, foi estimada em 1-3 casos/1.000 nativivos, enquanto nos EUA cerca de 1/10.000, porém não existem estimativas da prevalência de infecção congênita no Brasil. Toxoplasma gondii possui três linhagens clonais conhecidas como genótipos I, II e III que se relacionam aos três tipos descritos de acordo com a classificação da patogenicidade do parasita em camundongos, estando os três tipos associados a infecções humanas de maior patogenicidade, menor patogenicidade e patogenicidade intermediária, respectivamente. Os três genótipos já foram identificados na América do Norte e Europa, sendo o genótipo II predominante em infecções congênitas. O presente estudo teve como objetivo determinar a freqüência dos genótipos I, II e III de T. gondii em 85 amostras de líquido amniótico de gestantes brasileiras com toxoplasmose confirmada e adquirida durante a gestação, todas oriundas de São Paulo. Para tal, foi utilizada a técnica de multiplexnested- PCR-RFLP com amplificação simultânea de quatro fragmentos pertencentes a três genes do parasita: 3-SAG2, 5-SAG2, SAG3 e GRA6 Das 85 amostras iniciais, 76 foram positivas pelo sistema de multiplexnested- PCR (92,7%), tendo sido possível analisar o maior número de amostras no sistema que amplifica a região do gene SAG3 (69,7%), seguido do sistema 3-SAG2 (44,7%), do sistema 5-SAG2 (40,8%), e finalmente do sistema GRA6 (25%). Nas 76 amostras submetidas à genotipagem foram encontrados: 1,3% (1 amostra) do genótipo I; 71,1% (54 amostras) do genótipo II; 6,6% (5 amostras) do genótipo III; e 21% (16 amostras) para as quais não foi possível definir o genótipo parasitário após clivagem enzimática, tendo sido as mesmas submetidas ao sequenciamento. Das 16 amostras, duas não puderam seqüenciadas por falta de DNA remanescente e das 14 analisadas: duas foram definidas como tipo III, apresentando 100% de homologia com o protótipo VEG, e duas foram definidas como tipo II apresentando 100% de homologia com o protótipo ME49. Em 10 amostras foram observadas misturas de duas ou mais bases, em uma ou mais posições nucleotídicas da sequência de DNA do fragmento amplificado, sendo identificadas como recombinantes. Além disso, seis amostras identificadas pela técnica de multiplex-nested-PCR-RFLP como tipo II foram selecionadas aleatoriamente para a confirmação dos resultados e, em quatro das seis amostras, foi observada mistura de bases em outras posições nucleotídicas do fragmento amplificado, regiões estas não analisadas pela RFLP. A técnica de multiplex-nested-PCR seguida de RFLP mostrou-se útil, porém apresenta limitações no que se refere à genotipagem de amostras de líquido amniótico, uma vez que, após sequenciamento, foram reveladas várias recombinações genéticas nos fragmentos amplificados que haviam sido previamente identificados como tipo II. Sendo assim, sugerimos que, a genotipagem de T. gondii, diretamente de amostras biológicas ou após isolamento do parasita deva ser realizada pela amplificação de múltiplos genes de T. gondii e sequenciamento de todos os fragmentos amplificados / Toxoplasma gondii is the etiologic agent of toxoplasmosis and may cause different forms of the disease, including congenital toxoplasmosis whose frequency varies significantly according to the country. In France, it was estimated in 1-3 cases/1,000 live births, while in the U.S. it is 1/10.000. The prevalence of congenital infection in Brazil is unknown. Toxoplasma gondii has three clonal lineages known as genotypes I, II and III which are related to the three types described according to the pathogenicity in mice, associated with human infections of high pathogenicity, low pathogenicity and intermediate pathogenicity, respectively. The three genotypes have been identified in North America and Europe, and the genotype II is the predominant one in congenital infections. This study aimed at determining the frequency of genotypes I, II and III of T. gondii in 85 amniotic fluid samples from pregnant women living in Sao Paulo, Brazil who presented with seroconversion to Toxoplasma gondii during gestation. The multiplex-nested- PCR-RFLP was performed using four different markers: 3\'-SAG2, 5\'-SAG2, SAG3 and GRA6. Eighty-five samples were initially included, but only 76 were positive in the multiplex-nested-PCR (92.7%). It was possible to examine the larger number of samples by the SAG3 gene system (69.7%), followed by 3\'-SAG2 gene (44.7%), 5\'-SAG2 gene (40.8%), and finally GRA6 gene (25%). From the 76 samples that were genotyped by the multiplexnested- PCR-RFLP, 1.3% (1 sample) was genotype I; 71.1% (54 samples) were genotype II; 6,6% (5 samples) were genotype III, and 21% (16 samples) could not be identified by RFLP and were therefore sequenced. Sequencing analysis revealed: two genotype II samples, presenting with 100% of homology with the ME49 prototype, and two were genotype III showing 100% of homology with the VEG prototype. Ten samples presented with mixture of two or more nucleotide bases in one or more nucleotide positions of the total amplified DNA sequence, so they were classified as recombinant. In addition, six samples identified by the multiplex-nested-PCR-RFLP as type II were randomly selected to confirm the results, and in four of the six samples, we observed a mixture of nucleotide bases in positions that were not analyzed by the RFLP. The technique of multiplex-nested-PCR-RFLP has proved to be useful, however, it is limited when amniotic fluid samples genotyping is concerned because after sequencing, several genetic recombinations were evidenced in samples that had been previously identified as genotype II. Therefore, we suggest that the genotyping of T. gondii, directly from biological samples or after isolation of the parasite should be performed using different targets to amplify multiple T. gondii genes followed by sequencing of all amplified fragments

Les molécules du Complexe Majeur d'Histocompatibilité de classe I (CMH I) contrôlent la plasticité synaptique dans le système nerveux central (SNC) et plusieurs travaux expérimentaux suggèrent des interactions antigène-dépendantes entre des neurones infectés par des virus et les lymphocytes T CD8. Cependant, le rôle de la présentation des antigènes par le CMH I des neurones sur la physiopathologie de l'infection par Toxoplasma gondii (T. gondii) n'a pas encore été clarifié. Après la dissémination aigue sous forme de tachyzoites, T. gondii se convertit en bradyzoites, forme persistante dans les neurones du SNC. Chez les individus immunocompétents, la toxoplasmose latente est associée à des variations des fonctions cognitives ainsi qu'à des pathologies neuropsychiatriques. Les sujets dont le système immunitaire est dysfonctionnel peuvent développer une encéphalite létale causée par T. gondii, qui est caractérisée par une réplication du parasite, une infiltration massive et des agrégats leucocytaires et l'activation des cellules gliales. Les lymphocytes T (LT) CD8 et le CMH I sont des facteurs-clés contrôlant la résistance à l'encéphalite. Utiliser les LT CD8 pour éliminer les kystes chez des sujets à risque est une piste thérapeutique intéressante en raison de l'absence d'approches pharmacologiques ciblant les bradyzoites. A ce jour, les mécanismes et la pertinence fonctionnelle de la présentation des antigènes dérivés de T. gondii par les neurones restent à déterminer, ainsi que la contribution des différents stades parasitaires au contrôle de l'infection. L'utilisation de nouveaux parasites exprimant un antigène immunodominant uniquement au stade tachyzoite a permis de montrer que la reconnaissance par les LT CD8 d'antigènes issus des tachyzoites est suffisante pour une protection efficace contre l'encéphalite.[...] / In the Central Nervous System (CNS), Major Histocompatibility Complex class I (MHC I) molecules regulate synaptic plasticity and evidence suggests antigen-specific interactions between virus-infected neurons and CD8 T cells. Yet, little is known about the impact of neuronal MHC I presentation on the pathophysiology of infection by the neurotropic Toxoplasma gondii (T. gondii) parasite. Following acute dissemination as tachyzoites, T. gondii converts into bradyzoites that persist inside cysts within neurons of the CNS. In immunocompetent hosts, latent toxoplasmosis is associated with cognitive changes and neuropsychiatric disorders. Hosts with sub-optimal immune responses may develop a lethal T. gondii Encephalitis (TE), characterized by parasite replication, granuloma-like structures with massive immune cell influx and glial cell activation. CD8 T cells and MHC I are key determinants of TE resistance. Harnessing CD8 T cells in at-risk individuals may turn helpful in the future as we are currently lacking an effective pharmacological approach to eradicate bradyzoites. Yet the mechanisms and functional relevance of neuronal MHC I presentation of T. gondii remain unexplored, as well as which stage of the parasite contributes to efficient control of the infection. Using new T. gondii parasites with restricted expression of the immunodominant antigen at the tachyzoite stage, this work showed that CD8 T cell recognition of tachyzoite antigens at early stages of brain invasion is enough to protect from TE. Interestingly, by comparing situations of toxoplasmosis with varying TE severity and by pioneering antigen presentation assays with T. gondii-infected primary neurons, we revealed that TE susceptibility may be underlied by sub-optimal MHC I presentation of tachyzoites antigens by neurons. At last, we describe a mouse model that allows conditional deletion of a MHC I allele that is essential for TE resistance (H-2Ld). [...]

Publicación a texto completo no autorizada por el autor / La presente investigación es una revisión de literatura actualizada sobre los cuadros de Toxoplasma gondii en animales de compañía; principalmente en los aspectos epidemiológicos, signos clínicos y técnicas diagnósticas que se vienen utilizando. / Trabajo de suficiencia profesional

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

La toxoplasmose est un problème majeur de Santé Publique et notamment en médecine humaine. Le développement de vaccins est donc d’une grande priorité. L’efficacité de la stratégie vaccinale contre Toxoplasma gondii dépend de l’induction des réponses immunitaires muqueuse et systémique Th1. Les cellules dendritiques (CDs) ont un rôle essentiel dans l'orchestration de l’immunité innée et l’induction de l’immunité adaptative spécifique à Toxoplasma gondii. Dans cette étude, nous explorons une stratégie de vaccination originale qui consiste en l’administration par voie systémique et muqueuse de protéines de fusion capables de cibler l’antigène de surface SAG1 aux CDs, en utilisant un fragment d'anticorps de type scFv dirigé contre le récepteur d'endocytose DEC205. Nos résultats montrent que le ciblage de SAG1 aux DCs par le fragment scFv via la voie intranasale et sous-cutanée, réduit dramatiquement la charge parasitaire cérébrale par rapport à l’antigène non ciblé et est plus efficace que l’immunisation par la voie intranasale ou la voie sous-cutanée seule. Le ciblage des DCs potentialise la réponse immunitaire vers un profil Th1 par la production d’IFN-γ, d’IL-2, d’IgG2a sériques, tout en favorisant la production IgA muqueuses spécifiques. Parallèlement, nous avons montré qu’il était possible de conférer une reconnaissance par la protéine L à toutes les chaînes légères kappa, ce qui permettra dans l’avenir de purifier plus efficacement les antigènes ciblés par chromatographie d’affinité avec la protéine L. / Toxoplasmosis is a major public health problem and the development of a human vaccine is of high priority. Efficient vaccination against Toxoplasma gondii (T. gondii) requires both a mucosal and systemic Th1 immune response. Moreover, dendritic cells (DCs) play a critical role in orchestrating the innate immune functions and driving specific adaptive immunity to T. gondii. In this study, we explore an original vaccination strategy that combines administration via mucosal and systemic routes of fusion proteins able to target the major T. gondii surface antigen SAG1 to DCs using an antibody fragment scFv directed against DEC205 endocytic receptor. Our results show that SAG1 targeting to DCs by scFv via intranasal and subcutaneous administration improved protection against chronic T. gondii infection. A marked reduction in brain parasite burden is observed when compared with the intranasal or the subcutaneous route alone. DC targeting improved both local and systemic humoral and cellular immune responses and potentiated more specifically the Th1 response profile by more efficient production of IFN-γ, IL-2, IgG2a and nasal IgA. In parallel, we found a strategy to confer PpL recognition to all kappa chains. Therefore, affinity chromatography on protein L (PpL) matrix could be used to get easily highly purified targeted proteins.

Made available in DSpace on 2016-12-23T13:56:01Z (GMT). No. of bitstreams: 1 Tese_kelly_pdf.pdf: 1882941 bytes, checksum: dbb7b527acb39029842a16c46fd45c74 (MD5) Previous issue date: 2007-05-07 / Introduction: Toxoplasmosis is prevalent in Brazil. The infection during pregnancy can affect the fetus and cause sequelae in the child. Objectives: To determine seroprevalence of toxoplasmosis in pregnant women attending primary health cares in Vitória Municipality and evaluate correlate factors for the infection. Methods: A cross-sectional study, performed from January to December 2006, in 1153 pregnant women attending antenatal care in public clinics from the six health areas. A face-to-face interview with sócio-demographic, behaviors and clinics was performed; and serology for IgG, IgM and avidity of IgG by quimiluminescence (Diasorin) and eletroquimioluminescence (Biolab-Merrieux). Results: Prevalence of toxoplasmosis was 73.5% (CI 95% 70.95%-76.05%) and 1.3% (CI 95% 0.65%-1.95%) of acute infection. Considering avidity of IgG, prevalence of acute infection was 1.1% (CI 95% 0.5%-1.7%). A total of 26.5% (CI 95% 23.9%-29.0%) pregnant women were susceptive to toxoplasmosis and 72.2% (CI 95% 69.6%-74.8%) immunes. IgG antibodies were independent associated to buying meat in free markets [1.78 (CI 95% 1.02-3.13), and IgM antibodies during pregnancy were associated to lower education (up to four years) [5.30 (CI95% 1.67-16.83)]. Conclusions: These results corroborate the importance of earlier antenatal care and the inclusion of avidity test in toxoplasmosis diagnosis. It is requested a better sanitary control concerning the commerce of meat without registration. / Introdução: A toxoplasmose é de alta prevalência no Brasil. A infecção durante a gravidez pode resultar em doença fetal com graves seqüelas para a criança. Objetivo: Determinar a soroprevalência de toxoplasmose em gestantes atendidas nas Unidades de Saúde do Município de Vitória e avaliar fatores correlatos com a infecção. Métodos: Estudo de corte transversal, realizado de janeiro a dezembro de 2006, em 1153 gestantes atendidas nas Unidades de Saúde das seis regiões de saúde. Entrevista face-a-face contendo dados sócio-demográficos, epidemiológicos e clínicos foi realizada e sorologias para pesquisa de IgG, IgM e avidez de IgG pelo método de quimiluminescência (Diasorin) e eletroquimioluminescência (Biolab-Merrieux). Resultados: A prevalência da infecção foi de 73,5% (IC 95% 70,95%-6,05%) para IgG e 1,3% (IC 95% 0,65%-1,95%) para IgM. Quando se considerou a avidez de IgG, a prevalência de infecção aguda foi de 1,1% (IC 95% 0,5%-1,7%). Um total de 26,5% (IC 95% 23,9%-29,0%) gestante era susceptível à toxoplasmose e 72,2% (IC 95% 69,6%-74,8%) imunes. A presença de anticorpos IgG esteve independentemente associada à aquisição de carne em feiras livres [1,78 (IC95% 1,02-3,13). Já a presença de anticorpos IgM durante a gravidez apresentou associação com uma menor escolaridade (até quatro anos de estudo) [5,30 (IC95% 1,67-16,83)]. Conclusão: Estes resultados corroboram a importância da adesão precoce ao pré-natal com a inclusão do ensaio de avidez de IgG no diagnóstico da toxoplasmose. É necessário haver um maior rigor nas exigências sanitárias, no que diz respeito ao comércio de carnes sem registro.

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Made available in DSpace on 2014-12-17T14:10:27Z (GMT). No. of bitstreams: 1 MariaCFS_DISSERT.pdf: 790525 bytes, checksum: c565e1057a72e1ddf23dec1d882788e8 (MD5) Previous issue date: 2012-06-11 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, a protozoan that has a cosmopolitan geographic distribution and low host specificity. Usually a benign and selflimiting, infection can manifest itself in a severe systemic becoming overwhelming in fetuses and patients with immunosuppression. Domestic fowl are considered one of the most important hosts in the epidemiology of toxoplasmosis, since they are potential sources of infection for humans, in addition to playing the role of important indicators of environmental contamination by oocysts of T. gondii. We studied the prevalence of infection by the protozoan in chickens of different breeding systems mesoregions from the states of Rio Grande do Norte and Paraiba: broilers from commercial farms (200/PB) and free-range chickens of small farms (322/RN and PB). Were standardized IFAT and ELISA techniques for detecting specific antibodies in blood samples of birds, and commercial kit was used to determine the prevalence by IHAT. There was no seropositive reaction by T. gondii in the samples of broilers tested, indicating that the particularities of intensive management limit the chances of infection for these animals. Among the hens, the frequency of IgG anti-T. gondii diagnosed by the techniques of IHAT, IFAT and ELISA, respectively, were 3.73% (12/322), 37.88% (122/322) and 40.37% (130/322), for both young and adult animals. Amongst the seropositive samples by IFAT, 33 (27.05%) were positive at a dilution of 1:16, in 1:32, 31 (25.41%), in 1:64, 24 (19.67%), 15 (12.29%) in 1:128, and 19 presented titer greater than or equal to 1:256 (15.57%). The evaluation of the presence of anti-T. gondii should be careful, and reagents IHAT provided erratic results in this measure for the specie studied. This suggests the need for own standardization of the kit before the use in epidemiological studies in animal species. On the other hand, substantial agreement observed between IFAT and ELISA techniques (Kappa = 0.62) enables these methods as effective methodologies for the diagnosis of toxoplasmosis in chickens. The high prevalence of specific antibodies among poultry in the region studied attempts to the potential risk of transmission of toxoplasmosis to humans / A toxoplasmose ? uma zoonose causada pelo Toxoplasma gondii, protozo?rio que tem distribui??o geogr?fica cosmopolita e pouca especificidade parasit?ria. Comumente de curso benigno e autolimitante, a infec??o pode manifestar-se de forma sist?mica grave, tornando-se grav?ssima em fetos e pacientes com imunodepress?o. Galinhas dom?sticas s?o consideradas um dos mais importantes hospedeiros na epidemiologia da toxoplasmose, uma vez que s?o potenciais fontes de infec??o para humanos, al?m de desempenharem o papel de importantes indicadores da contamina??o ambiental por oocistos de T. gondii. Neste trabalho, estudou-se a frequ?ncia da infec??o pelo protozo?rio em galin?ceos de diferentes sistemas de cria??o provenientes de mesorregi?es dos estados do Rio Grande do Norte e Para?ba, tanto frangos de corte de granjas comerciais (200/PB), como galinhas caipira de pequenas propriedades rurais (322/RN e PB). Foram padronizadas t?cnicas de RIFI e ELISA para a detec??o de anticorpos s?ricos espec?ficos nas amostras sangu?neas das aves, e foi utilizado kit comercial para determina??o dessa preval?ncia pelo HAI. N?o foi observada infec??o por T. gondii em nenhuma das amostras de frango de corte analisada, indicando que particularidades do manejo intensivo limitam as chances de infec??o para esses animais. Entre as galinhas caipira, a frequ?ncia de anticorpos IgG anti-T. gondii diagnosticada pelas t?cnicas de HAI, RIFI e ELISA foi, respectivamente, 3,73% (12/322), 37,88% (122/322) e 40,37% (130/322), analisando animais jovens e adultos. Das amostras soropositivas pela RIFI, 33 (27,05%) foram reagentes na dilui??o 1:16; em 1:32, 31 (25,41%); em 1:64, 24 (19,67%); 15 (12,29%) em 1:128 e 19 apresentaram titula??o maior ou igual a 1:256 (15,57%). A avalia??o da presen?a de anticorpos anti-T. gondii deve ser criteriosa, sendo que os reagentes do HAI forneceram resultados err?ticos nesta medida, para a esp?cie estudada, sugerindo a necessidade de padroniza??o pr?pria dos kits para diagn?stico antes do uso em estudos epidemiol?gicos em esp?cies animais. Por outro lado, a concord?ncia substancial observada entre as t?cnicas RIFI e ELISA (Kappa = 0,62) capacita estas metodologias como t?cnicas eficazes no diagn?stico da infec??o pelo protozo?rio em galin?ceos. A alta frequ?ncia de anticorpos espec?ficos observada entre as aves da regi?o estudada atenta para o risco potencial de transmiss?o da toxoplasmose para o homem

Orientador: Gisele Fabrino Machado / Banca: Mary Marcondes / Banca: Rosemeri de Oliveira Vasconcelos / Resumo: A Leishmaniose visceral é uma enfermidade que possui uma grande variabilidade de manifestações clínicas, em humanos como em cães. Cães cronicamente infectados podem desenvolver desordens neurológicas, contudo, há poucos relatos que caracterizam as lesões e elucidam a patogenia da leishmaniose cerebral canina. Considerando a imunossupressão associada à leishmaniose visceral e que os patógenos oportunistas Toxoplasma gondii e Neospora caninum podem colaborar para a ocorrência de lesões no sistema nervoso central de cães naturalmente infectados por Leishmania chagasi, as populações de linfócitos B (CD79+) e T (CD3+) foram avaliadas no tecido nervoso de cães portadores de leishmaniose visceral e que possuem soropositividade para T. gondii e N. caninum. Lesões inflamatórias, caracterizadas por acúmulos de células mononucleares compostos principalmente por linfócitos T CD3+ predominaram em diversas regiões encefálicas dos cães infectados (P = 0,0012). Linfócitos B CD79+ foram detectados em pequena intensidade, não havendo diferença entre os grupos (P = 0,3604). Os resultados obtidos sugerem que a co-presença de leishmaniose visceral, toxoplasmose e neosporose é importante para o agravamento das lesões encefálicas, e que a imunossupressão gerada pela infecção por Leishmania não somente favorece a infecção por outros patógenos, mas colabora com esses, ocasionando lesões mais severas no tecido nervoso / Abstract: Visceral leishmaniasis is a disease with great variability regarding the clinical manifestations, in humans as in dogs. Chronically infected dogs may develop neurological disorders, however, there are few reports that characterise the lesions and make clear the pathogenesis of the canine cerebral leishmaniasis. Considering the immunossupression associated to visceral leishmaniasis and that the opportunist pathogens Toxoplasma gondii and Neospora caninum may collaborate to the occurrence of lesions in the central nervous system of dogs naturally infected by Leishmania chagasi, we evaluated the population of B (CD79+) and T (CD3+ ) lymphocytes in the nervous tissue of dogs with visceral leishmaniasis and with seropositivity to T. gondii and to N. caninum. Inflammatory lesions, characterised by mononuclear cellsaccumulation, composed mainly by CD3+ T lymphocytes predominated in several encephalic regions of the dogs from the infected groups (P=0.0012). CD79+ B lymphocytes were detected in very small intensity and presented no difference among groups (P=0.3604). The results presented herein suggest that the co-presence of visceral leishmaniasis, toxoplasmosis and neosporosis is important for the worsening of the encephalic lesions, and that the immunossupression caused by Leishmania infection not only propitiates the infection by other pathogens, but collaborate with them, causing more severe lesions in the brain / Mestre