As formas sintomáticas de toxoplasmose são um grave problema de saúde pública e ocorrem em cerca de 10 a 20% das pessoas infectadas. Com o objetivo de melhorar o diagnóstico molecular de toxoplasmose sintomática em pacientes brasileiros, este estudo avaliou o desempenho da PCR em tempo real (qPCR) na detecção do DNA de T. gondii testando dois conjuntos de iniciadores moleculares (B1 e REP-529) para diagnóstico molecular. A metodologia foi testada em 807 amostras clínicas com diagnóstico clínico conhecido, ELISA e PCR convencional (cPCR) em um período de 9 anos. Todas as amostras foram de pacientes com suspeita clínica de diferentes formas da toxoplasmose. De acordo com a curva mínima de limite de detecção (no CT), o REP-529 apresentou maior sensibilidade para detectar DNA de T. gondii do que B1. Ambos os conjuntos de iniciadores moleculares foram avaliados retrospectivamente usando o DNA de 515 amostras clínicas diferentes. Os 122 pacientes com resultado negativo na PCR em tempo real, provavelmente por terem infecção crônica, demonstraram alta especificidade (REP-529, 99,2% e B1, 100%). Das 393 amostras com ELISA positivo (IgG), 146 apresentaram diagnóstico clínico de toxoplasmose e cPCR positivo. As sensibilidades de REP-529 e B1 foram de 95,9% e 83,6%, respectivamente. A comparação dos desempenhos do REP-529 e B1 foi analisada prospectivamente em 292 amostras. Assim, a partir de um total de 807 amostras de DNA analisadas, 217 (26,89%) apresentaram PCR positivo, pelo menos em um conjunto de iniciadores e com toxoplasmose sintomática confirmada pelo diagnóstico clínico. O REP-529 foi positivo em 97,23%, enquanto o B1 amplificou apenas 78,80%. Depois de comparar várias amostras em um laboratório brasileiro de referência, este estudo concluiu que o conjunto de iniciadores REP-529 apresentou melhor desempenho do que B1. Essas observações foram baseadas após o uso de casos com diagnóstico clínico definido, ELISA e cPCR. / Symptomatic forms of toxoplasmosis are a serious public health problem and occur in around 10-20% of the infected people. Aiming to improve the molecular diagnosis of symptomatic toxoplasmosis in Brazilian patients, this study evaluated the performance of real time PCR (qPCR) in detecting T. gondii DNA testing two primer sets (B1 and REP-529) for molecular diagnosis. The methodology was assayed in 807 clinical samples with known clinical diagnosis, ELISA, and conventional PCR (cPCR) results in a 9-year period. All samples were from patients with clinical suspicion of several features of toxoplasmosis. According to the minimum detection limit curve (in CT), REP-529 had greater sensitivity to detect T. gondii DNA than B1. Both primer sets were retrospectively evaluated using 515 DNA from different clinical samples. The 122 patients without toxoplasmosis (with negative real-time PCR) provided high specificity (REP-529, 99.2% and B1, 100%). From the 393 samples with positive ELISA (IgG), 146 had clinical diagnosis for toxoplasmosis and positive cPCR. REP-529 and B1 sensitivities were 95.9% and 83.6%, respectively. Comparison of REP-529 and B1 performances was further analyzed prospectively in 292 samples. Thus, from a total of 807 DNA analyzed, 217 (26.89%) had positive PCR with, at least one primer set and with symptomatic toxoplasmosis confirmed by clinical diagnosis. REP-529 was positive in 97.23%, whereas B1 amplified only 78.80%. After comparing several samples in a Brazilian referral laboratory, this study concluded that REP-529 primer set had better perform than B1 one. These observations were based after using cases with defined clinical diagnosis, ELISA, and cPCR.

O objetivo deste estudo foi o isolamento de Toxoplasma gondii de ovinos e caprinos e posterior caracterização genotípica desses isolados. Amostras de soros ovinos (495) de 36 Municípios do Estado de São Paulo e de caprinos (143) de seis Municípios dos Estados da Bahia (10), Paraíba (12), Rio Grande do Norte (7) e São Paulo (114) foram testadas, através do Teste de Aglutinação Modificado (MAT&ge;25) à presença de anticorpos anti-T. gondii. Dos animais amostrados, 24,2% e 32,2% dos ovinos e caprinos respectivamente, apresentaram-se positivos com títulos que variaram de 25 a 3200 em ambas espécies. Dentre os ovinos houve associação (p<0,001) entre sexo, idade e sistema de produção com a presença de anticorpos anti-T. gondii. Os caprinos apresentaram associação entre idade, sistema de produção, e raça com a soropositividade. Para o isolamento do agente o bioensaio em camundongos foi realizado utilizando-se pool de tecidos de ovinos (cérebro, coração e diafragma) e caprinos soropositivos (cérebro, coração e diafragma e masseter). Dos 82 bioensaios realizados com amostras de ovinos, 16 isolados (19,5%) foram obtidos. Houve associação entre o título de anticorpos anti-T. gondii e o isolamento do agente (p<0,001) com maior quantidade de isolados obtidos de animais com títulos mais altos. Para a caracterização genotípica das amostras utilizou-se a análise de polimorfismo de comportamento dos fragmentos de DNA gerados por enzimas de restrição (RFLP) sobre produtos do locus SAG2 amplificados pela PCR, que as identifica em genótipos I, II e III. Amostras do tipo I (87,5% dos ovinos e 100 % dos caprinos) e do tipo III (12,5% dos isolados de ovinos) foram obtidas e não houve o encontro de isolados tipo II ou mistas. Nos tecidos dos animais com títulos de anticorpos superiores a 400, e dos quais não se obteve o isolamento do agente pelo bioensaio (21 amostras de ovinos), foi realizada a nested-PCR das amostras primárias (homogeneizado de tecidos), não sendo obtida nenhuma amplificação. Amostras do tipo III não causaram óbitos nos camundongos e as tipo I causaram óbitos em 51,0% e 83,4% dos isolados de ovinos e caprinos, respectivamente. Houve associação entre a sobrevida dos camundongos infectados com o genótipo I de caprinos e ovinos (p<0,001). / The aim of this study was to analyze the SAG2 locus of Toxoplasma gondii isolated from sheep and goats in order to determine the frequency of occurrence of the three different lineages (types I, II and III). Serum samples of sheep (495) from 36 counties in São Paulo State and goat (143) from six counties in States of Bahia (10), Paraíba (12), Rio Grande do Norte (7) and São Paulo (114) were analyzed by the modified agglutination test (MAT&ge;25) for the detection of antibodies anti-T. gondii. Occurrence of antibodies anti-T. gondii was 24.2% and 32.2% in sheep and goats, respectively. The titers varied from 25 to 3200 in both species. Among sheeps, association of seropositivity (p<0,001) with sex, age, production system and presence of antibodies anti-T. gondii was observed. Association was also observed between the frequency of seropositive goats and age, production system and breed. For T. gondii isolation, pool of tissues of each seropositive sheep (brain, heart and diaphragm) and goat (brain, heart, diaphragm and masseter) were bioassayed in mice. From a total of 82 bioassay performed with sheep samples, 16 isolates (19.5%) were obtained. Association between antibodies anti-T. gondii titers and isolation was observed (p<0.001). Considering the goats, 12 isolates (42.6%) were obtained from 26 bioassays. For the genotypic characterization (genotype I, II and III), the restriction fragment length polymorphism (RFLP) was performed on fragments of the locus SAG2 amplified by PCR. Samples of type I (87.5% from sheep and 100% from goats) and type III (12.5% from sheep) was observed. Neither type II nor mixed samples were observed. In tissues samples from animals with antibodies titers above 400 in which T. gondii was not isolated (21 sheep sample), nested-PCR was performed and no amplification was observed. Mortality was not observed in the mice infected by the isolates type III. The isolates of type I from sheep and goats, respectively, killed 51.0% and 83.4% of the infected mouse. Association was also observed between mortality rate in infected mice and genotype I of sheep and goat (p<0.001).

Apesar de o protozoário Toxoplasma gondii ser considerado a única espécie válida para o gênero, são reconhecidas três linhagens clonais, denominadas Tipo I, Tipo II e Tipo III, predominantes em países da Europa ocidental e Estados Unidos. Com a pesquisa de amostras deste parasito provenientes de outras regiões do mundo, como no caso do Brasil, várias outras configurações genotípicas diferentes das dos arquétipos mencionados acima vêm sendo encontradas. Neste trabalho, quarenta galinhas/galos (Gallus domesticus) de criação livre de oito propriedades rurais de áreas limítrofes ao Pantanal da Nhecolândia no estado do Mato Grosso do Sul foram eutanasiadas e amostras de sangue, cérebro e coração foram coletadas. O sorodiagnóstico foi realizado através do Teste de Aglutinação Modificado (MAT) e, com um pool de órgãos dos animais positivos, foi realizado bioensaio em camundongos. Foram obtidos, após o bioensaio, 11 isolados de T. gondii. O DNA foi extraído dos tecidos dos camundongos infectados e a tipificação dos isolados foi realizada utilizando 12 marcadores PCR-RFLP, genericamente denominados SAG1, 5´3´SAG2, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico e CS3. Vinte e sete galinhas (67,5%) foram sorologicamente positivas para T. gondii e 13 (32,5%) foram negativas. Das amostras soropositivas, 7 (25,9%) foram positivas na diluição 1:5, 3 (11,1%) em 1:10, 2 (7,4%) em 1:20, 3 (11,1%) em 1:320, 1 (3,7%) em 1:640, 3 (11,1%) em 1:1280, 2 (7,4%) em 1:2560, 4 (14,8%) em 1:5120 e 2 (7,4%) em 1:10240. Quanto à genotipagem, cinco genótipos foram revelados nos 11 isolados de galinhas de cinco propriedades do município de Aquidauana e uma propriedade do município de Rio Verde do Mato Grosso, incluindo um genótipo misto encontrado em um isolado (TgCkBr198) que demonstrou um complexo cujos padrões são uma combinação de 2 alelos observados em oito loci. Dois genótipos foram descritos pela primeira vez, considerando os 140 isolados de galinhas de diferentes regiões brasileiras avaliadas em estudos anteriores. Os resultados corroboram com estudos anteriores sobre os isolados de T. gondii no Brasil, confirmando sua diversidade e atipicidade. / Despite the protozoan Toxoplasma gondii is considered the only valid species for the genus, three clonal lineages are recognized, known as the Type I, Type II and Type III, which predominate in Western European countries and the USA. The search for samples of this parasite from other regions of the world, as in the case of Brazil, has revealed different genotypes other than the archetypes above mentioned. In this study, forty free range chickens (Gallus domesticus) of eight farms from areas belonging to the Pantanal Nhecolândia in the state of Mato Grosso do Sul were euthanized and samples of blood, brain and heart were collected. The serodiagnosis was performed using the technique of modified agglutination test (MAT) and a pool of organs of seropositive animals was used to perform the bioassay in mice. It was obtained 11 isolates of T. gondii. DNA was extracted from tissues of infected mice and characterization of isolates was performed using 12 PCR-RFLP markers, known generically SAG1, 5\'3\'SAG2, SAG2, SAG3, BTUB, GRA6, c22-8, C29-2, L358, PK1, Apico and CS3. Twenty-seven chickens (67.5%) were serologically positive for T. gondii and 13 (32.5%) were negative. Among the seropositive samples, 7 (25.9%) were positive at 1:5 dilution, 3 (11.1%) in 1:10, 2 (7.4%) in 1:20, 3 (11.1%) at 1:320, 1 (3.7%) at 1:640, 3 (11.1%) in 1:1280, 2 (7.4%) in 1:2560, 4 (14.8%) on 1:5120 and 2 (7.4%) at 1:10240. With regard to genotyping, five genotypes were found within 11 isolates from chickens from five properties in the municipality of Aquidauana and one property of the municipality of Rio Verde of Mato Grosso, including a mixed genotype found in one isolate (TgCkBr198) that showed a complex pattern which is a combination of 2 alleles observed at eight loci. Two genotypes have been described for the first time, considering the 140 isolates from chickens in different Brazilian regions evaluated in previous studies. The results corroborate with previous studies on the isolates of T. gondii in Brazil, confirming their diversity and atypicality.

Foi realizada a pesquisa de anticorpos anti-Toxoplasma gondii, através do teste de aglutinação modificado (MAT), em 68 amostras de soros de capivaras de seis municípios no estado de São Paulo. Anticorpos (MAT?25) foram encontrados em 51 (75%) capivaras examinadas. Dentre estas realizou-se o bioensaio em camundongos, com tecidos do cérebro, coração e língua, de 40 capivaras, sendo obtidos 36 isolados (90%). Não houve associação entre o número de isolados e idade das capivaras (p=0,21), sexo (p=0,58) ou tipo de criação (p=0,62), isto é, criadouros e vida livre, bem como a freqüência de isolamentos e os títulos de anticorpos (p=0,99). A análise de polimorfismo de comprimento dos fragmentos de DNA gerados por enzimas de restrição (RFLP) sobre produtos do locus SAG2 amplificados pela reação em cadeia pela polimerase (PCR) revelou que 20 isolados (55,5%) pertenciam ao genótipo I, 14 (38,9%) ao genótipo III e dois (5,6%) que apresentaram genótipo misto (tipos I e III). Não foi encontrado isolado tipo II. A proporção de isolados tipo I entre as capivaras de vida livre foi maior (p=0,049) do que entre as capivaras provenientes de criadouros. Por outro lado, entre as capivaras de criadouros, a proporção de isolados tipo III foi maior (p=0,041). A maioria dos isolados tipo I (12/20) causou óbito em todos os camundongos infectados e, em nenhum grupo com este isolado, 100% dos camundongos sobreviveram. A maioria dos isolados tipo III (8/14) não matou nenhum camundongo infectado. A freqüência de óbitos em camundongos com genótipo I (86%) foi maior do que o tipo III (44,9%) (p<0,001), enquanto a sobrevida dos camundongos com genótipo III foi significativamente maior que a dos camundongos com genótipo I (p<0,001). Foram encontrados cistos nos cérebros dos camundongos infectados em todos os 36 isolados. A análise genotípica também foi realizada diretamente dos tecidos de 35 das 36 capivaras (homogeneizados de tecidos) das quais houve isolamento pelo bioensaio, usando nestedPCR-RFLP no locus SAG2. Foram caracterizadas 22 amostras (62,8%), 21 delas idênticas aos dos isolados correspondentes. Em uma amostra genótipo misto foi obtido dos tecidos primários e tipo I no isolado. Os genótipos mistos foram confirmados pelo seqüenciamento de DNA dos produtos da nestedPCR obtidos das amostras primárias das capivaras. / Antibodies to Toxoplasma gondii were assayed by the modified agglutination test (MAT) in serum samples of 68 capybaras from six counties in São Paulo state, Brazil. Antibodies (MAT?25) were found in 51 (75%) capybaras examined. Tissues (brain, heart and tongue) of 40 of the seropositive capybaras were bioassayed in mice and 36 (90%) isolates were obtained. There was no statistical association between number of isolates and age (p=0.21), gender (p=0.58) and type of rearing (p=0.62), as well as no association with frequency of isolations and antibody titer distribution (p=0.99). Restriction fragment length polymorphism (RFLP) analysis in PCR-amplified SAG2 locus products revealed that 20 isolates (55.5%) were genotype I, 14 (38.9%) were genotype III and two (5.6%) were mixed genotypes (types I and III). Type II isolate was not found. The proportion of type I isolates in the group of wildlife capybaras was higher (p=0.049) than in the captive rearing group. On the other hand, the proportion of type III isolates was significantly higher in the captive rearing group (p=0.041). Most of the type I isolates (12/20) killed all infected mice and none of those groups had 100% of surviving mice. Most of the mice infected with genotype III isolate survived. The mortality rate in mice infected with genotype I (86%) was higher than the type III (44.9%) (p<0.001) and mice infected with type III isolates survived for longer periods than type I isolates (p<0.001). Tissue cysts were found in mice infected with all 36 isolates. Genotyping was also done directly from the tissue homogenates from the 35 of 36 capybaras using nested-PCR-RFLP analysis on the SAG2 locus. Twenty?two samples (62.8%) were characterized and in 21 the genotypes found were the same as those from the corresponding isolates. In one sample, mixed genotype was detected directly from the primary sample and type I from the mice isolate. The mixed genotype was confirmed by direct DNA sequencing of the nestedPCR products from the capybaras primary samples.

A toxoplasmose é uma zoonose de distribuição mundial. O Toxoplasma gondii infecta o Homem e a maioria dos animais homeotérmicos. A ingestão de oocistos esporulados é uma das formas de transmissão desse protozoário. Oocistos de T. gondii podem esporular na água do mar e manter a infectividade por até seis meses. Moluscos bivalves podem filtrar e reter oocistos de T. gondii da água do mar em condições experimentais. O objetivo deste trabalho foi investigar a presença de T. gondii em ostras (Crassostrea rhizophorae) e mariscos (Mytella guayanensis) em condições naturais. Um total de 300 ostras e 300 mariscos foram adquiridos no Mercado de Peixes do município de Santos no estado de São Paulo no período de 05/03/2008 a 29/08/08 e divididos em 60 grupos de cinco ostras e 20 grupos de 15 mariscos. Para o isolamento do parasita, cinco camundongos foram inoculados oralmente com homogenados dos tecidos de cada grupo de ostras ou mariscos. Para a detecção molecular, o DNA dos tecidos dos mariscos foi extraído pelo método de fenol-clorofórmio e o das ostras, pelo método de isotiocianato de guanidina, Em seguida, foi realizada a nested-PCR (Reação em Cadeia pela Polimerase) baseada na amplificação de um fragmento de 155pb do gene B1 de T. gondii. A genotipagem das amostras de T. gondii detectadas foi feita usando a PCR-RFLP (Polimorfismo de Comprimento de Fragmentos de DNA gerados por Enzimas de Restrição) usando os marcadores SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, CS3 e Apico. Não houve isolamento do parasita pelo bioensaio em camundongos. Nos grupos de mariscos, o T. gondii não foi detectado pela n-PCR e, nos grupos de ostras, houve detecção do T. gondii em dois grupos (3,3%). Não foi possível a genotipagem das amostras de T. gondii detectadas. O presente estudo permitiu concluir que ostras da espécie Crassostrea rhizophorae podem filtrar e reter oocistos de T. gondii e que o ambiente marinho do litoral do estado de São Paulo encontra-se contaminado com oocistos desse parasita. Assim, o consumo de ostras cruas pode representar uma potencial via de transmissão de T. gondii para o Homem e para os animais marinhos. / Toxoplasmosis is a worldwide zoonosis. Toxoplasma gondii is widely prevalent in humans and other mammals. This protozoan parasite can be transmitted by ingestion of sporulated oocysts. T. gondiioocysts can sporulate in seawater and retain their infectivity for at least six months. Experimentally, bivalve mollusks can filter and retain T. gondii oocysts from the seawater. The aim of this study was to investigate the presence of T. gondii in oysters (Crassostrea rhizophorae) and mussels (Mytella guayanensis) in natural conditions. A total of 300 oysters and 300 mussels were acquired from the Fish Market in Santos city, São Paulo state, from March 2008 to August 2008, and divided in 60 groups of five oysters and 20 groups of 15 mussels. To isolate the parasite, five mice were orally inoculated with tissue homogenates from each group of oysters or mussels. For molecular detection of T. gondii, DNA from mussels was extracted using a standard phenol-chloroform method and DNA from oysters was extracted using the guanidine isothiocianate method. A nested-PCR (Polymerase Chain Reaction) based on the amplification of a 155bp fragment from B1 gene of T. gondii was then performed. Eleven PCR-RFLP (Restriction Fragment Length Polymorphism) markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, CS3 e Apico were used to genotype positive samples. There was no isolation of the parasite by bioassay in mice. T. gondii was not detected in the groups of mussels by n-PCR. There was detection of T. gondii by n-PCR in two groups of oysters (3.3%). Genotyping of these two positive samples was not successful. The results indicate that oysters of the species Crassostrea rhizophorae, the commonest species from the coast of São Paulo, can filter and retain T. gondii oocysts and that the marine environment of the coast of São Paulo state is contaminated with oocysts of this parasite. The ingestion of raw oysters can represent a potential transmission source of T. gondii to humans and marine mammals.

Toxoplasma gondii est un parasite intracellulaire obligatoire. L'infection par T. gondii est largement répandue dans le monde entier. Néanmoins, elle est peu étudiée dans les pays d'Asie du Sud-est dont la Thaïlande.Nous avons réalisé 3 travaux sur le terrain en Thaïlande pour essayer de comprendre la circulation de ce parasite à travers une étude de séroprévalence chez des poulets en zone rurale et des essais d’isolement de souches chez les animaux en vue d’un génotypage. Lors des deux premières missions de terrain dans deux villages de la province de Kanchanaburi, nous avons cherché à déterminer la séroprévalence de l’infection chez des poulets (Gallus domesticus) en utilisant 2 tests sérologiques, Modified-Agglutination Test (MAT et immunofluorescence indirecte (IFAT) puis à isoler des souches de T.gondii à partir des animaux séropositifs. Lors de la troisième mission réalisée dans 3 autres provinces thaïlandaises(Nakhonratchasima, Lopburi et Saraburi), nous avons essayé d’isoler directement le parasite à partir de carcasses de poulets vendues sur les marchés ou d’autres animaux trouvés morts.La séroprévalence globale pour les 2 premières misions sur 600 poulets du Kanchanaburi était de 17,7% (IC 95% :14,6-20,7) et 33,0% (IC 95% : 29,2-36,8), par MAT et IFAT respectivement. Le calcul du coefficient κ montre une absence de concordance entre les deux tests.Au total, 162 essais d'isolement ont été effectués par inoculation à des souris, mais aucune souche viable de T. gondii n'a été isolée pendant ces 3 travaux sur le terrain. Cependant, nous avons détecté la présence d’ADN toxoplasmique en qPCR ciblant le gène 529 bp dans 13 culots de digestion d’organes de poulets, pigeon, caille et dans des cerveaux ou coeurs de souris inoculés par 16 autres poulets. Les Ct observés en qPCR étaient ≥33 indiquant une faible quantité d’ADN parasitaire dans nos échantillons qui n’a pas permis une caractérisation génétique par marqueurs microsatellites.Ce travail a démontré l'importance et les difficultés du travail de terrain pour l'étude de séroprévalence ainsi que l'étude d'isolement. L'isolement des souches de T. gondii a demandé un travail d'échantillonnage intensif, complexe dans l’environnement tropical et humide de la Thaïlande. Les différents paramètres ayant pu avoir un impact négatif sur nos résultats sont discutés. Ils expliquent l’absence d’isolement de souches chez des animaux séropositifs. / Toxoplasma gondii is an obligate intracellular parasite. Toxoplasma gondii infection is widespread throughout the world. Nevertheless, it is poorly studied in Southeast Asian countries including Thailand. We carried out 3 field works in Thailand to try to understand the circulation of T. gondii through a seroprevalence study in chickens in rural areas and strain isolation attempts in animals. During the two first field works, performed in Kanchanaburi province, we determined the seroprevalence in chickens (Gallus domesticus) using 2 serological tests, a Modified-Agglutination-Test (MAT) and an immunofluorescence assay (IFAT) and subsequently tried to isolate the strains of T. gondii from seropositive animals. During the third field work carried out in 3 other Thai provinces (Nakhonratchasima,Lopburi and Saraburi), we attempted to isolate strains directly from chicken carcasses sold in different markets or other dead animals.The overall seroprevalence for 600 chickens sampled over the two field works in Kanchanaburi was 17.7% (95%CI: 14.6% -20.7) and 33.0% (95% CI: 29.2-36.8), by MAT, and IFAT, respectively. The κ coefficient indicated an absence of concordance between the 2 serological tests.A total of 162 isolation attempts were performed by mouse bioassays, but no viable strain of T. gondii was isolated during these 3 field works. However, a qPCR targeting 529 bp T. gondii gene was positive for 13 digestion pellets of organs of chickens, pigeon, quail and in brains or hearts of mice inoculated with 16 other chickens. These qPCR were weaklly positive (Ct ≥33) indicating a low amount of parasite DNA in our samples that did not allow genotyping T. gondii with microsatellite markers.This work demonstrated the importance and difficulties of field work for the seroprevalence study as well as strain isolation. The isolation of T. gondii strains required intensive and complex sampling in the tropical and humid environment of Thailand. The diverse factors that could have a negative impact on our results are discussed. They might explain the absence of strain isolation from seropositive animals.

L'autophagie est un processus d'auto-dégradation conservé chez la plupart des eucaryotes. Généralement induit par un stress nutritif, il requiert la formation d'un compartiment à double membrane appelé l’autophagosome qui séquestre et transporte des composants intracellulaires dégradés et recyclés dans le lysosome. La protéine ATG8, qui occupe une position centrale dans ce processus, est recrutée aux membranes de l’autophagosome par un système de conjugaison très régulé. Toxoplasma gondii est un protozoaire parasite appartenant au phylum des Apicomplexes, qui contient une machinerie d'autophagie réduite. Suite à un stress nutritif, ce parasite intracellulaire obligatoire est néanmoins capable de générer des autophagosomes décorés par TgATG8. De façon surprenante, en condition normale de croissance intracellulaire, cette protéine se localise principalement à l’apicoplaste, un plaste non photosynthétique acquis par endosymbiose secondaire qui contient des voies métaboliques essentielles à la survie du parasite. Le but de ma thèse a été d’élucider les fonctions canoniques et non canoniques d‘ATG8 chez Toxoplasma. La première partie de cette étude porte sur la caractérisation fonctionnelle et spatio-temporelle de l'association de TgATG8 avec l’apicoplaste. Nous avons montré que TgATG8 est recrutée aux extrémités de l’apicoplaste en élongation, ce qui permet le maintien de l’organelle à travers les générations en le connectant aux centrosomes pour une répartition dans les deux cellules filles. La deuxième partie de ce travail vise à isoler et identifier par spectrométrie de masse des partenaires putatifs de TgATG8 qui seraient impliqués dans l’autophagie ou dans le rôle non-canonique à l’apicoplaste. Nous avons analysé la localisation subcellulaire de neuf candidats et des caractérisations fonctionnelles ont été entreprises pour trois protéines. Bien que nous n’ayons pas pu confirmer leurs interactions avec TgATG8, cela a permis l'identification de nouvelles protéines parasitaires: une phospholipase à l’apicoplaste essentielle à la survie du parasite, un régulateur potentiel du cycle cellulaire et un composant du cytosquelette du parasite. / Autophagy is a self-degradative process evolutionary conserved among eukaryotes. Typically induced by starvation, it involves the formation of a double membrane compartment called the autophagosome to sequester and deliver intracellular components for lysosomal degradation and recycling. The protein ATG8 occupies a central position in this process and is recruited to autophagosomal membranes by a highly regulated conjugation system. Toxoplasma gondii is a parasitic protist belonging to the Apicomplexa phylum, which possesses a reduced autophagy machinery. This obligate intracellular parasite is nevertheless able to generate TgATG8-decorated autophagosomes upon nutrient stress. Surprisingly, during normal intracellular parasite growth, TgATG8 mainly localizes to the apicoplast, a non-photosynthetic plastid acquired by secondary endosymbiosis which hosts essential metabolic pathways. My thesis aimed to elucidate the canonical and non-canonical roles of ATG8 in Toxoplasma. The first part of this study is the functional and spatio-temporal characterization of TgATG8 association with the apicoplast. We showed TgATG8 is recruited to both ends of the elongating plastid during parasite division, and allows the maintenance of the organelle across generations by permitting its centrosome-driven distribution into the two daughter cells. The second part of this work is the isolation and mass spectrometry-based identification of putative TgATG8-interacting proteins that would be involved in autophagy-related or non-canonical functions. We analyzed the subcellular localization of nine candidates and functional studies were conducted for three proteins. Although we were unable to confirm their interactions with TgATG8, this approach allowed the identification of novel and important parasite proteins: an essential apicoplast phospholipase, a potential regulator of the cell cycle, and a component of the parasite cytoskeleton.

Les infections d'origine alimentaire constituent un problème de santé publique majeur et présentent un impact économique important. Les agents pathogènes incriminés peuvent être d'origine bactérienne, virale ou parasitaire. Les conséquences de ces infections sur la santé des consommateurs peuvent être majeures, en fonction du statut immunitaire de chaque individu et de la provenance de l'agent infectieux, la mondialisation ayant accru les risques de dissémination de pathogènes viables virulents. Les denrées à risque sont principalement les aliments frais consommés crus ou peu cuits. Ceux-ci sont irrigués en continu durant leur croissance par des eaux pouvant être potentiellement contaminées par des agents infectieux.Au cours de ce travail, nous avons développé une stratégie d'extraction et de détection des parasites Toxoplasma gondii, Cryptosporidium et Giardia à partir de basilic et de framboise. Nous avons pour cela développé une technique d'immuno-capture des oocystes de Toxoplasma gondii grâce à un anticorps monoclonal dirigé contre la paroi des oocystes ainsi que des techniques de détection des trois parasites par qPCR. Ces protocoles ont été validés par des essais inter-laboratoires avec la participation de quatre laboratoires partenaires de ce projet. Nous avons par la suite cherché à mieux appréhender les risques réels pour les consommateurs en réalisant une étude mettant en parallèle une Reverse transcriptase PCR avec des essais in vivo, permettant ainsi de mettre en corrélation la viabilité des parasites avec leur infectiosité.Ce travail contribue à mieux maitriser les risques sanitaires liés à la présence de parasites dans lesdenrées alimentaires , par le biais d'une stratégiesusceptible d'être proposée aux industriels et normalisée. / Foodborne infections are a major public health problem and have a significant economic impact. Pathogens implicated in these outbreaks can be bacteria, viruses and parasites. Their impact on the consumer's health can be significant, depending on the immune status of the person and the pathogen source. Globalization has increased the risk of spreading of viable and virulent . The food at risk is mainly fresh food, eaten raw or undercooked, which has been widely irrigated by potentially contaminated waters during production. In this study, we have developed methods to extract and detect Toxoplasma gondii, Cryptosporidium and Giardia parasites from basil and raspberry with techniques able to detect the three parasites by qPCR. In this aim, we have developed a technique for the immuno-capture of Toxoplasma gondii oocysts using a monoclonal antibody directed against the oocyst wall. These protocols have been validated by interlaboratory trials with the participation of four laboratories involved in the same project. We have subsequently sought to better understand the real risks for consumers by undertaking a study of Reverse transcriptase PCR in parallel with in vivo, thus allowing to correlate the viability of parasites with their infectivity. This work contributes to a better control of health risks associated with food and presence of parasites, through a strategythat could be standardized and offered to industrial partners.

Plasmodium falciparum e Toxoplasma gondii são protozoários parasitas pertencentes ao filo Apicomplexa. Apirases são enzimas metabolizadoras de nucleotídeos extracelulares. Nesta tese mostramos pela primeira vez a presença de um membro desta família de enzimas em P. falciparum, o qual foi capaz de degradar ATP extracelular. Análises por RT-qPCR revelaram a expressão da apirase durante todo o ciclo intraeritrocítico. A adição de inibidores desta classe de enzimas foi capaz de prejudicar o desenvolvimento dos parasitas e a invasão de novas hemácias pelos merozoitos, sugerindo assim um papel da apirase nestes processos. A via de sinalização por Ca2+ é universal e vital para todas as células. Para melhor entender a fisiologia celular de P. falciparum construímos uma nova linhagem de parasitas transgênicos, PfGCaMP3, que nos tornam capazes de monitorar a dinâmica de Ca2+ sem o uso de protocolos invasivos de marcação. De modo semelhante utilizamos uma nova linhagem de T. gondii expressando de forma estável o indicador de Ca2+ GCaMP3 para estudar o papel deste íon na saída da célula. T. gondii possui o Ca2+ necessário para promover este processo, entretanto Ca2+ extracelular age como um fator intensificador neste passo essencial do ciclo lítico. / Plasmodium falciparum and Toxoplasma gondii are protozoan parasites that belong to phylum Apicomplexa. Apirases are metabolizing enzymes of extracellular nucleotides. In this work we show for the first time the presence of an apyrase in P. falciparum, which was able to degrade extracellular ATP. RTqPCR analysis revealed the expression of apyrase throughout the intraerythrocytic cycle. Addition of apyrase inhibitors was able to impair the development of the parasites and the invasion of new erythrocytes by merozoites, thus suggesting a role of apyrase in these processes. Calcium signaling is universal and vital to all cells. To better understand the cellular physiology of P. falciparum we construct a new strain of transgenic parasites, PfGCaMP3, which enable us to monitor the Ca2+ dynamics without using invasive protocols. Similarly we use a new strain of T. gondii that stably express the Ca2+ indicator GCaMP3 to study the role Ca2+ in parasite egress. T. gondii has the Ca2+ required to promote this process, however extracellular Ca2+ acts as an enhancer factor in this crucial step of the lytic cycle.

A toxoplasmose é uma zoonose de distribuição mundial. O Toxoplasma gondii infecta o Homem e a maioria dos animais homeotérmicos. A ingestão de oocistos esporulados é uma das formas de transmissão desse protozoário. Oocistos de T. gondii podem esporular na água do mar e manter a infectividade por até seis meses. Moluscos bivalves podem filtrar e reter oocistos de T. gondii da água do mar em condições experimentais. O objetivo deste trabalho foi investigar a presença de T. gondii em ostras (Crassostrea rhizophorae) e mariscos (Mytella guayanensis) em condições naturais. Um total de 300 ostras e 300 mariscos foram adquiridos no Mercado de Peixes do município de Santos no estado de São Paulo no período de 05/03/2008 a 29/08/08 e divididos em 60 grupos de cinco ostras e 20 grupos de 15 mariscos. Para o isolamento do parasita, cinco camundongos foram inoculados oralmente com homogenados dos tecidos de cada grupo de ostras ou mariscos. Para a detecção molecular, o DNA dos tecidos dos mariscos foi extraído pelo método de fenol-clorofórmio e o das ostras, pelo método de isotiocianato de guanidina, Em seguida, foi realizada a nested-PCR (Reação em Cadeia pela Polimerase) baseada na amplificação de um fragmento de 155pb do gene B1 de T. gondii. A genotipagem das amostras de T. gondii detectadas foi feita usando a PCR-RFLP (Polimorfismo de Comprimento de Fragmentos de DNA gerados por Enzimas de Restrição) usando os marcadores SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, CS3 e Apico. Não houve isolamento do parasita pelo bioensaio em camundongos. Nos grupos de mariscos, o T. gondii não foi detectado pela n-PCR e, nos grupos de ostras, houve detecção do T. gondii em dois grupos (3,3%). Não foi possível a genotipagem das amostras de T. gondii detectadas. O presente estudo permitiu concluir que ostras da espécie Crassostrea rhizophorae podem filtrar e reter oocistos de T. gondii e que o ambiente marinho do litoral do estado de São Paulo encontra-se contaminado com oocistos desse parasita. Assim, o consumo de ostras cruas pode representar uma potencial via de transmissão de T. gondii para o Homem e para os animais marinhos. / Toxoplasmosis is a worldwide zoonosis. Toxoplasma gondii is widely prevalent in humans and other mammals. This protozoan parasite can be transmitted by ingestion of sporulated oocysts. T. gondiioocysts can sporulate in seawater and retain their infectivity for at least six months. Experimentally, bivalve mollusks can filter and retain T. gondii oocysts from the seawater. The aim of this study was to investigate the presence of T. gondii in oysters (Crassostrea rhizophorae) and mussels (Mytella guayanensis) in natural conditions. A total of 300 oysters and 300 mussels were acquired from the Fish Market in Santos city, São Paulo state, from March 2008 to August 2008, and divided in 60 groups of five oysters and 20 groups of 15 mussels. To isolate the parasite, five mice were orally inoculated with tissue homogenates from each group of oysters or mussels. For molecular detection of T. gondii, DNA from mussels was extracted using a standard phenol-chloroform method and DNA from oysters was extracted using the guanidine isothiocianate method. A nested-PCR (Polymerase Chain Reaction) based on the amplification of a 155bp fragment from B1 gene of T. gondii was then performed. Eleven PCR-RFLP (Restriction Fragment Length Polymorphism) markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, CS3 e Apico were used to genotype positive samples. There was no isolation of the parasite by bioassay in mice. T. gondii was not detected in the groups of mussels by n-PCR. There was detection of T. gondii by n-PCR in two groups of oysters (3.3%). Genotyping of these two positive samples was not successful. The results indicate that oysters of the species Crassostrea rhizophorae, the commonest species from the coast of São Paulo, can filter and retain T. gondii oocysts and that the marine environment of the coast of São Paulo state is contaminated with oocysts of this parasite. The ingestion of raw oysters can represent a potential transmission source of T. gondii to humans and marine mammals.