Toxoplasma gondii é um protozoário coccídio intracelular obrigatório conhecido por sua habilidade em parasitar uma ampla gama de espécies hospedeiras. A região apical do parasito é rica em organelas que, em função dos produtos liberados, estão envolvidas no processo de adesão e invasão da célula hospedeira. As proteínas liberadas por micronemas (MICs), solúveis e transmembrana, possuem domínios adesivos essenciais para a virulência do parasita. Algumas dessas proteínas são encontradas associadas entre si na superfície do taquizoíta, formando complexos como TgMIC1/MIC4/MIC6 e TgMIC3/MIC8. Em estudos anteriores demonstramos a que o subcomplexo TgMIC1/MIC4 (Fração LAC+) liga-se à lactose e estimula macrófagos a secretar IL-12. Verificamos, utilizando células HEK293 transfectadas, que um dos principais mecanismos responsáveis pela produção de IL-12 decorria do reconhecimento de N-glicanos do ectodomínio de TLR2 pelo domínio de reconhecimento de carboidrato de TgMIC1. O objetivo do presente estudo foi o de investigar a capacidade de TgMIC1 e TgMIC4 de ativar macrófagos murinos e qual o papel desempenhado por TLR2 e/ou TLR4 no desencadeamento dessa ativação. Mostramos que macrófagos derivados de medula óssea de camundongos C57Bl/6, estimulados com TgMIC1 e TgMIC4, utilizadas isoladamente ou em combinação, secretam altos níveis de citocinas pró-inflamatórias, como TNF-?, IL-6, IL-12 e IL-1?, produzem altos níveis de óxido nítrico, e têm aumentadas suas capacidades migratória e fagocítica. Os ensaios que utilizaram macrófagos de camundongos C57Bl/6 nocauteados revelaram que a ausência de expressão de TLR2 ou de TLR4 prejudicou os efeitos ativadores exercidos por TgMIC1 e TgMIC4. Macrófagos TLR2-/- tiveram as manifestações de ativação celular significantemente reduzidas em relação aos macrófagos selvagens. Por outro lado, esses efeitos foram mais afetados pela ausência de TLR4, uma vez que as respostas obtidas frente ao estímulo com TgMIC1 ou TgMIC4 eram similares às verificadas em células não estimuladas (controle negativo). Concluímos que TgMIC1 e TgMIC4 interagem com os receptores do tipo toll 2 e 4 expressos por macrófagos, levando à ativação celular manifesta por alta produção de citocinas e outros mediadores inflamatórios, e aumento das capacidades migratória e fagocítica. A interação com TLR4 é preponderante em relação à estabelecida com TLR2 no desencadeamento de ativação celular / Toxoplasma gondii is an obligate intracellular coccidian protozoan known for its ability to parasitize a wide range of host species. The parasite\'s apical region is rich in organelles that, because of the products it releases, are involved in the processes of adhesion and invasion of the host cell. The soluble and transmembrane proteins released by the micronemes (MICs), have adhesive domains that are essential for the parasite virulence. Some of these proteins can be found associated with each other on the taquizoite surface, as it is the case of TgMIC1/MIC4/MIC6 or TgMIC3/TgMIC8 complexes. Our group has demonstrated in previous studies that the TgMIC1/TgMIC4 subcomplex (LAC+ fraction) binds to lactose and stimulates macrophages to release IL-12. Using HEK293 transfected cells, we showed that one of the main mechanisms leading to IL-12 release by macrophages, was the recognition of N-glycans of the TLR2 ectodomain by the carbohydrate recognition domain of TgMIC1. Thus, the objective of this study was to address whether TgMIC1 and TgMIC4 activate murine macrophages and what is the role of TLR2 and TLR4 in macrophage activation. Our results show that the stimulation of C57BL/6 mice bone marrow derived macrophages with TgMIC1 and TgMIC4, alone or in combination, induce the release of high levels of proinflammatory cytokines such as TNF-?, IL-6, IL-12 and IL-1?, and also nitric oxide; and increases phagocytic activity and cell migration activity. The assays using knockout C57Bl/6 macrophages showed that the absence of TLR2 or TLR4 expression impaired the activating effects of TgMIC1 e TgMIC4. TLR2-/- macrophages presented significantly reduced cell activation manifestations compared to WT macrophages. On the other hand, these effects were more affected in the absence of TLR4, once the responses obtained with TgMIC1 or TgMIC4 stimulation were similar to those observed in non stimulated cells (negative control). We conclude that TgMIC1 and TgMIC4 interact with the receptors Toll like 2 and 4 expressed in macrophages, leading to cell activation, resulting in high cytokine and inflammatory mediators production, and increased migratory and phagocytic capacity. The interaction with TLR4 is predominant over that established with TLR2 in the triggering of cell activation

Ao longo dos anos, a humanidade contribuiu para o surgimento de diversos patógenos, tornando-se vítimas de doenças transmitidas dos animais para o homem, as chamadas antropozoonoses. Dentre os animais, os tatus, mamíferos selvagens primitivos, apresentam características anatômicas e fisiológicas peculiares, que os tornam mais susceptíveis à diversas doenças e potenciais reservatórios de patógenos zoonóticos, relevantes para a saúde pública. O objetivo deste estudo foi avaliar a prevalência de cinco patógenos zoonóticos (Toxoplasma gondii, Trypanosoma cruzi, Leishmania spp, Mycobacterium leprae e Paracoccidioides brasiliensis) em quatro espécies de tatus; P. maximus, E. sexcinctus, D. novemcinctus e C. unicinctus do Pantanal e Cerrado do Mato-Grosso-do-Sul. Um total de 50 tatus foram analisados. Sendo, 43 amostras de soros de indivíduos de vida livre (16 Priodontes maximus; 17 Euphractus sexcinctus; 02 Dasypus novemcinctus e 08 Cabassous unicinctus) provenientes do Pantanal e 07 conjuntos de fragmentos de tecidos (pulmão, fígado e baço), de 06 E. sexcinctus e 01 (D. novemcinctus) atropelados em três rodovias do Cerrado do Mato-Grosso-do-Sul. Dos 43 indivíduos amostrados no Pantanal, 13/43 ou 30,23% apresentaram anticorpos anti-T. gondii; 01/43 ou 2,32% anti-T. cruzi e 4/43 ou 9,30% anti-Leishmania (L.) infantum chagasi. Amostras de fragmentos de orelha dos 43 indivíduos do Pantanal, e fragmentos de tecido (pulmão, fígado e baço) dos tatus do Cerrado, também foram analisadas para M. leprae e Leishmania spp através de biologia molecular, nas quais foram negativas. Dos tatus provenientes do Cerrado, analisados para P. brasiliensis, 07 ou 100% dos indivíduos foram positivos. Baseado nestes resultados, pode-se afirmar que os tatus apresentam uma relação e histórico de exposição a estes patógenos, seja através do contato com outras espécies, seres humanos ou condições ambientais onde ocorrem. Contudo, confirma-se a importância destas espécies para o entendimento dos ciclos de transmissão de patógenos e de forma estratégica, como indicadoras da saúde de um ecossistema em programas de investigação e monitoramento de doenças, especialmente as zoonoses. / Over the years mankind has contributed to the emergence of several diseases, while becoming victims of those passed among humans and other animals, known as zoonosis. Armadillos are primitive wild mammals who present peculiar anatomical and physiological characteristics which make them susceptible and potential reservoirs of zoonotic pathogens relevant to public health. The goal of the present study was to evaluate the prevalence of five zoonotic pathogens (Toxoplasma gondii, Trypanosoma cruzi, Leishmania spp., Mycobacterium leprae and Paracoccidioides brasiliensis) in four armadillo species (Priodontes maximus, Euphractus sexcinctus, Dasypus novemcinctus and Cabassous unicinctus) found in Pantanal and Mato-Grosso-do-Sul tropical savanna ecoregion, the Cerrado. A total of 50 armadillos were sampled: 43 free living individuals from Pantanal (16 P. maximus; 17 E. sexcinctus; 02 D. novemcinctus and 08 C. unicinctus) and 07 individuals found dead in three different roads of the Cerrado. Of the 43 individuals sampled in Pantanal, 13 (30.23%) presented T. gondii antibodies; 01 (2.32%) showed antibodies anti-T. cruzi; and 04 (9.30%) showed anti-Leishmania (L.) infantum chagasi antibodies. All 50 samples were also analyzed by molecular biology based on the polymerase chain reaction (PCR). None of the samples tested positive for M. leprae and Leishmania spp. And all seven or (100%) individuals from the Cerrado were tested positive for P. brasiliensis. The results suggest that the armadillos have been exposed to these pathogens, either by contact with other species, including humans or by their own environmental conditions in certain ecosystems. The armadillo species studied may have a greater susceptibility to these pathogens in the natural environment where they occur. Armadillos are key species in understanding diseases transmission cycle, especially regarding zoonotic pathogens and they can strategically act as an indicator of ecosystem health for research programs and zoonotic diseases monitoring.

Toxoplasma gondii ist ein weit verbreiteter einzelliger Parasit, der fast alle warmblütigen Organismen infizieren kann. Asexuelle Fortpflanzung des Parasiten in seiner Wirtszelle wird durch aufeinanderfolgende lytische Zyklen erreicht, was die Bereitstellung einer signifikanten Menge an Energie und Biomasse erforderlich macht. Diese Arbeit zeigt, dass Glukose und Glutamin die beiden wichtigsten physiologischen Nährstoffe für die Synthese von Makromolekülen (ATP, Nukleinsäure, Proteine und Lipide) in T. gondii sind. Die Verfügbarkeit einer der beiden Kohlenstoffquellen reicht aus, um das Überleben des Parasiten sicherzustellen. Der Parasit kann durch Erhöhen des Flusses von Glutamin-abstammendem Kohlenstoff durch den TCA-Zyklus und durch gleichzeitige Aktivierung der Gluconeogenese, eine stetige Biogenese von ATP und Biomasse zur Wirtszellinvasion und Replikation gewährleisten, bzw. der genetischen Deletion des Glukosetransporters entgegenwirken. Der Wachstumsdefekt in der Glykolyse-Mutante wird durch eine kompromittierte Synthese von Lipiden verursacht, die durch Glutamin nicht ausgeglichen werden kann. Die Zugabe von exogenem Acetat kann diesen Wachstumsdefekt allerdings kompensieren. In dieser Arbeit konnten darüber hinaus zwei unterschiedliche Phosphoenolpyruvat-Carboxykinase (PEPCK) Enzyme im Parasiten identifiziert werden, von denen eines im Mitochondrium lokalisiert ist (TgPEPCKmt), während das andere Protein nicht in Tachyzoiten (TgPEPCKnet) exprimiert wird. Parasiten mit intakter Glykolyse können die Deletion von TgPEPCKnet, als auch die genetische Deletion von TgPEPCKmt tolerieren, was ihre Redundanz für das Überleben der Tachyzoiten zeigt. TgPEPCKnet kann auch in der Glykolyse-defizienten Mutante deletiert werden, während TgPEPCKmt für das Überleben des Parasiten in dieser Mutante essentiell ist. Dies zeigte sich durch ein konditionelles Knockdown von TgPEPCKmt, das zu einer Inhibierung des Wachstums des Parasiten führte. / Toxoplasma gondii is a widespread protozoan parasite, infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. This work shows that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the TCA cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. Growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine, but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Furthermore, this work revealed two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in the mitochondrion (TgPEPCKmt), whereas the other protein is not expressed in tachyzoites (TgPEPCKnet). Parasites with an intact glycolysis can tolerate genetic deletions of TgPEPCKmt as well as of TgPEPCKnet, indicating their nonessential roles for the tachyzoite survival. TgPEPCKnet can also be ablated in glycolysis-deficient mutant, whereas TgPEPCKmt is refractory to deletion. In accord, the lytic cycle of a conditional mutant of TgPEPCKmt in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of tachyzoites.

Der obligat intrazelluläre Parasit Toxoplasma gondii ist der Erreger der Toxoplasmose, und dient zugleich als wichtiger Modellorganismus für weitere Human- und Tierpathogene, wie z.B. Plasmodium oder Eimeria. Die Vermehrung von T. gondii erfordert eine effiziente Biosynthese von Phospholipiden für die Herstellung neuer Membranen, was durch die de novo Synthese durch den Parasiten, und/oder den Import von Lipiden aus der umgebenden Wirtszelle gewährleistet werden kann. Während der Parasit zahlreiche Möglichkeiten für Synthese oder Import von PtdEtn und PtdSer verwendet, scheint die Biosynthese des abundantesten Membranlipids PtdCho auschließlich über den CDP-Cholin Weg zu erfolgen. Dieser erstreckt sich in T. gondii über 3 zelluläre Kompartimente, mit einer cytosolischen Cholin-Kinase (TgCK), einer im Zellkern lokalisierenden Cholin-Cytidylyltransferase (TgCCT) und einer Cholin-Phosphotransferase (TgCPT) im ER. Anders als die substrat-spezifische Ethanolamin-Kinase (TgEK), kann TgCK neben Cholin außerdem Ethanolamin phosphorylieren. TgCK zeigt eine geringe Affinität zu Cholin (Km ~0.77 mM), während eine verkürzte TgCK (TgCKS), welcher eine als Signalpeptid vorhergesagte N-terminale Sequenz (20 Aminosäuren) fehlt, eine etwa 3-fach höhere Aktivität aufweist (Km ~0.26 mM). Während jedoch die Wildtyp-TgCK cytosolische Cluster in Toxoplasma bildet, zeigt die verkürzte TgCK eine gleichmäßigere cytosolische Lokalisierung. Wir schlussfolgern daraus, dass der hydrophobe N-Terminus nicht notwendig ist für eine funktionale TgCK, sondern eine strukturelle Funktion bei der Protein-Lokalisierung hat. Eine konitionelle Mutante, in welcher der TgCK Promoter gegen den Tetracyclin-regulierbaren Promoter pTetO7Sag4 ausgetauscht wurde (Deltatgcki), zeigt erstaunlicherweise normales Wachstum und PtdCho Biosynthese. Die TgCK Aktivität und die daraus resultierende PtdCho Synthese sind nur zu ~30% regulierbar. Unsere Ergebnisse deuten auf die Verwendung eines alternativen Startcodons bzw. Promoters hin, welcher zur Expression einer verkürzten (~53-kDa) aber vermutlich aktiven Cholin Kinase führt, wodurch der Verlust der TgCK (~70-kDa) kompensiert wird. Der konditionelle Knockout von TgCCT, dem regulatorischen Enzym des CDP-Cholin Wegs, hatte einen 50%igen Wachstumsdefekt zur Folge. Diese Studie zeigt eine erstaunliche Flexibilität des Parasiten bezüglich seiner Membranzusammensetzung, und bestätigt zugleich die Annahme, dass PtdCho nicht von der Wirtszelle importiert werden kann. Diese Anpassungsfähigkeit stellt einen möglichen Faktor dar, der es T. gondii erlaubt sich in einem breiten Spektrum von Wirten zu vermehren. / Toxoplasma gondii is an obligate intracellular apicomplexan parasite that causes life-threatening disease in neonates and in immunocompromised people. Successful replication of Toxoplasma requires substantial membrane biogenesis, which must be satisfied irrespective of the host-cell milieu. Like in other eukaryotes, the two most abundant phospholipids in the T. gondii membrane are phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). Bioinformatics and precursor labeling analyses confirm their synthesis via the CDP-choline and CDP-ethanolamine pathway, respectively. This work shows that the 3-step CDP-choline pathway, involving the activities of TgCK, TgCCT and TgCPT, localizes to the cytosol, nucleus and ER membrane, respectively. The initial reaction is catalyzed by a dual-specificity choline kinase (TgCK, ~70-kDa), capable of phosphorylating choline as well as ethanolamine. The purified full-length TgCK displayed a low affinity for choline (Km ~0.77 mM). TgCK harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. The displacement of the TgCK promoter in a conditional mutant of T. gondii (deltatgcki) attenuated the enzyme expression by ~80%. Unexpectedly, the ?tgcki mutant was not impaired in intracellular growth, and exhibited a normal PtdCho biogenesis. To recompense for the loss of full-length TgCK, the mutant appears to make use of an alternative promoter and/or start codon, resulting in the expression of a shorter but active TgCK isoform identified by the anti-TgCK antiserum, which correlated with its persistent choline kinase activity. Accordingly, the ?tgcki showed an expected incorporation of choline into PtdCho, and susceptibility to dimethylethanolamine (a choline analog). Interestingly, the conditional mutant displayed a regular growth in off state despite a 25% decline in PtdCho content, which suggests a compositional flexibility in T. gondii membranes and insignificant salvage of host-derived PtdCho. The two-step conditional mutagenesis of TgCCT, which caused a reduced growth rate to about 50%, further substantiated this finding. The enzymatic activity of TgCCT and its role in PtdCho synthesis remain to be proven, however. Taken together, the results demonstrate that the CDP-route is likely essential in T. gondii. The competitive inhibition of choline kinase to block the parasite replication appears a potential therapeutic application.The work also reveals a remarkably adaptable membrane biogenesis in T. gondii, which may underly the evolution of Toxoplasma as a promiscuous pathogen.

Toxoplasma gondii und Plasmodium Spezies sind obligat intrazelluläre Parasiten, die Zucker zur Energiehomöostase als auch für die Synthese lebenswichtiger Makromoleküle verwenden. Die hier vorgestellten Daten zeigen, dass der Glukosetransporter von T. gondii, TgGT1, und die homologen Transporter von P. falciparum und P. berghei, PfHT1 und PbHT1, neben Glukose auch Mannose, Fructose und Galactose transportieren. Toxoplasma Tachyzoiten exprimieren neben TgGT1 noch einen weiteren putative Zuckertransporter (TgST2) an der Parasitenoberfläche. Beide Proteine sind nicht essentiell, wie durch ihre individuelle und gleichzeitige Gendeletion belegt wird. Die Deletion von TgGT1 bewirkt einen geringen Wachstumsdefekt. Die Mutante ?tggt1 zeigt keine Glukoseaufnahmeaktivität und folglich eine verminderte glukoseabhängige Motilität. In ?tggt1 Parasiten wird ein verstärkter Glutaminstoffwechsel nachgewiesen, der ausreichend ist dessen Motilität und Replikationsaktivität zu erhalten. Die ?tggt1 Mutante gewährt Einblick in die Anpassungsfähigkeit von T. gondii an unterschiedliche Wirtszellen. Im Gegensatz zu T. gondii benötigen erythrozytäre Plasmodien Glukose und der Transporter PfHT1 wird derzeit als drug-target eingestuft. Hier wird gezeigt, dass das PfHT1-Homolog, PbHT1, essentiell in Blutstadien des Nagerparasiten Plasmodium berghei ist, jedoch auch während des gesamten Lebenszyklus des Parasiten exprimiert wird. Ein PfHT1- und PbHT1-spezifischer Inhibitor (Compound 3361) kann die Entwicklung von P. berghei Leberstadien und Okineten stark hemmen. Um zukünftig PfHT1-Inhibitoren im Hochdurchsatzverfahren zu identifizieren und testen zu können, wurden auf Saccharomyces cerevisiae und P. berghei basierende Expressionssysteme für PfHT1 entwickelt. Abschließend stellt diese Arbeit die Unterschiedlichkeit des zentralen Kohlenstoffwechsels von Toxoplasma und Plasmodium Parasiten durch bisher unbekannter Aspekte heraus. / Toxoplasma gondii and Plasmodium species are obligate intracellular pathogens that utilize host sugars for energy homeostasis and macro molecular synthesis. Here, we report that the T. gondii glucose transporter, TgGT1, and of its homologs of P. falciparum and P. berghei (PfHT1 and PbHT1) transport glucose, mannose, galactose and fructose. Besides TgGT1, Toxoplasma harbours one additional surface localized putative sugar transporter (TgST2). Surprisingly both Proteins are nonessential and only the deletion of TgGT1 inflicts a mild defect in the parasite replication. The ?tggt1 mutant is unable to import glucose and consequently displays an attenuated glucose-dependent motility, which is completely rescued by glutamine. ?tggt1 performs increased glutamine metabolism that is sufficient to sustain motility and replication. The ?tggt1 strain provides a model for further investigating its adaptation to disparate host cells. In contrast to T. gondii, erythrocytic stages of Plasmodium species critically depend on glucose uptake, and the PfHT1 transporter is considered as a drug target against human malaria. Here, we report that PbHT1 (a PfHT1 homolog) is also essential for blood stage development in the rodent malaria parasite P. berghei. PbHT1 is expressed throughout the life cycle. Moreover, a PfHT1- and PbHT1-specific sugar analogue, compound 3361, can inhibit the hepatic development and ookinete formation in P. berghei. These results signify that PbHT1 and exogenous glucose are also required during the ex-erythrocytic stages of P. berghei. To permit a high-throughput screening of selective PfHT1 inhibitors and their subsequent in vivo assessment, we have established a PfHT1-expressing Saccharomyces cerevisiae mutant and generated a PfHT1-dependent ?pbht1 of P. berghei strain. This thesis underscores various previously unknown aspects of sugar metabolism in Toxoplasma and Plasmodium, and unravel their metabolic differences.

Os principais marcadores sorológicos para o diagnóstico da toxoplasmose aguda ou recente são os anticorpos IgM específicos e anticorpos IgG de baixa avidez. Entretanto em alguns pacientes, anticorpos IgM e baixa avidez de anticorpos IgG podem persistir, ultrapassando o período da fase recente aguda contribuindo para erros de interpretação diagnóstica. No presente estudo, a eficiência diagnóstica do ensaio imunoenzimático foi avaliada, com o uso de frações antigênicas ou peptídeos sintéticos originados do antígeno ESA de T.gondii, denominados de SAG-1, GRA-1 e GRA-7. Foram estudadas frações isoladas e combinadas em múltiplos peptídeos antigênicos (MAP), visando estabelecer um perfil confiável para definição sorológica de toxoplasmose recente aguda em amostra única de soro. A melhor eficiência diagnóstica do ensaio foi encontrada com o uso da combinação de peptídeos SAG- 1,GRA-1 e GRA-7, denominada MAP1. A detecção de anticorpos IgG e IgM anti- MAP1 apresentou a melhor definição entre a fase recente aguda da fase recente não aguda na toxoplasmose. Nossos resultados mostraram que IgM anti-MAP1 poderá se constituir um marcador sorológico importante no aumento da eficiência diagnóstica da toxoplasmose recente aguda / The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis

A prevalência de Toxoplasma gondii em mamíferos silvestres, quando próximos a áreas urbanizadas, deve ser analisada para diagnosticar riscos à saúde humana e animal. Em Natal-RN, duas Unidades de Conservação (UC), Parque da Cidade e Parque das Dunas, se destacam pela importância ecológica. Nestas UCs a presença de gatos domésticos errantes é constante. O objetivo foi avaliar e comparar a presença de T. gondii através de teste sorológico (MAT) e molecular (gene B1) em gatos e pequenos mamíferos silvestres. Houve associação entre o número de animais silvestres soropositivos em cada parque (p = 0,018), sendo que o Parque da Cidade demonstrou um quadro mais alarmante. A positividade para T. gondii através da amplificação do gene B1 foi positiva em 16,3% do total de animais silvestres analizados e 8,1% do total de gatos avaliados nas duas Ucs. Não houve associação entre o número de animais silvestres positivos para o gene B1 em cada parque (p = 1,00). Existe a participação de ambos os grupos de animais (silvestres e domésticos) no ciclo local do T. gondii. / The prevalence of Toxoplasma gondii in wild mammals close to urbanized areas should be analyzed to diagnose risks to human and animal health. In Natal-RN State, two protected areas, Parque da Cidade and Parque das Dunas are distinguished by ecological importance. In these protected areas, the presence of domestic cats is constant. The aim was to evaluate and compare the presence of T. gondii by serological testing (MAT) and molecular (B1 gene) in cats and small wild mammals. There was an association between the number of wild seropositive animals in each park (p = 0.018). The Parque da Cidade showed a more alarming situation. The positivity for T. gondii by B1 gene amplification was positive in 16.3% on wild animals analyzed and 8.1% on the cats evaluated in the two protected areas. There was no association between the number of wild animals positive for the B1 gene in each park (p = 1,00). There is a participation of both groups of animals (wild and domestic) in the local cycle of T. gondii.

Mais de 2 bilhões de pessoas em todo o mundo encontram-se infectadas com Toxoplasma gondii. Na região endêmica de Erechim, RS, cerca de 90% da população é soropositiva e cerca de 18% destes indivíduos apresentam lesões oculares com manifestações clínicas. A estrutura genética das populações do T. gondii tem sido bastante investigada, a despeito da infecção ter se espalhado pelo mundo, do grande número de hospedeiros intermediários e da capacidade do parasita de se reproduzir sexualmente. Linhagens de T. gondii com atípica ou nova combinação de alelos têm sido isoladas de animais não domésticos ou em outros continentes, como América do Sul e África, e de pacientes com apresentações clínicas incomuns. Em modelos murinos, as linhagens com o genótipo tipo I são altamente virulentas, em contraste às cepas tipo II e tipo III que são menos virulentas. Este trabalho propõe a caracterização fenotípica da resposta imune do hospedeiro frente a infecção por diferentes cepas de T. gondii, bem como o isolamento e a caracterização genotípica das linhagens de T. gondii que infectam indivíduos de Erechim no Rio Grande do Sul. Para a caracterização fenotípica utilizamos duas cepas de T. gondii já bem estabelecidas, a cepa RH (tipo I) e a ME49 (tipo II), e uma cepa isolada a partir de gatos domésticos do Brasil, chamada TgCatBr71. Sendo assim, através da fenotipagem das células dendríticas de camundongos C57Bl/6 infectados com as cepas citadas, foi possível observar que essas cepas induzem expressão das moléculas de superfície CD40, CD80, CD86 e MHC classe II em DCs CD11c+, porém sem significativa diferença entre as cepas. Com relação as células CD4+ e células CD8+, observamos o aumento das células CD8+ no decorrer da infecção pelas cepas RH e ME49, indicando a importância deste tipo celular na resposta protetora contra T. gondii. Avaliamos também a produção de citocinas IL-12, IFN-g e IL-10 em células esplênicas de camundongos infectados pelas três cepas no decorrer da infecção e detectamos que camundongos infectados pela cepa tipo II (ME49) apresentam síntese maior dessas citocinas do que camundongos infectados pela cepa tipo I (RH) e pela cepa TgCatBr71. Assim, concluímos que esta cepa TgCatBr71 se assemelha bastante a cepa do tipo I (RH), tanto em relação a evolução da doença no camundongos como nos padrões da resposta imune do hospedeiro. E que apesar dessas duas cepas diferirem da cepa tipo II (ME49), resultando em graus diferentes de patologia em camundongos C57Bl/6, todas a três cepas parecem produzir semelhante resposta imune protetora do hospedeiro. / More than 2 billion people are infected with Toxoplasma gondii around the world. In the endemic region of Erechim, RS, Brazil, about 90% of the population is soropositive and about 18% of these individuals have ocular lesions with clinical manifestations. The genetic structure of strains of T. gondii has been investigated, despite the infection has spread throughout the world, the large number of intermediate hosts and the ability to reproduce sexually. Strains of T. gondii with atypical or new combination of alleles have been isolated from wild animals and other continents, such as South America and Africa, and also from patients with unusual clinical presentations. In murine models, the type I genetic lineage are highly virulent, in contrast to strains type II and type III.Our work proposes the phenotypic characterization of the host immune response against the infection by different strains of T. gondii, and the isolation and genetic strains characterization of T. gondii that infect individuals of Erechim in RS, Brazil. In the phenotypic characterization were used two strains of T. gondii already well established, the strain RH (type I) and ME49 (type II), and a strain isolated from domestic cats from Brazil, called TgCatBr71 (type BrI). Thus, by phenotyping dendritic cells of C57BL/ 6 mice infected with the strains mentioned, we observed that these strains upregulated the expression of surface molecules such as CD40, CD80, CD86 and MHC class II in DC CD11c+ although with no significant difference between the strains. With respect to the CD4+ and CD8+ cells, the observed increase in CD8+ T cells during the infection by strains RH and ME49, indicating the importance of this cell type in the protective response against T. gondii. We also evaluated the production of cytokines IL-12, IFN-g and IL-10 from spleen cells and found that mice infected by the strain type II (ME49) have increased synthesis of these cytokines than mice infected by the strain type I (RH) and the strain type BrI (TgCatBr71). Thus, we concluded that type BrI (TgCatBr71) strain is similar to type I (RH) strain, both for the evolution of the disease and also concerning the immunological parameters evaluated. Besides, despite these two strains differ from the strain type II (ME49), resulting in different degrees of pathology in mice C57BL / 6, all three strains seem to produce similar protective immune response of the host.

O presente estudo teve como objetivo avaliar a ocorrência de anticorpos anti-Toxoplasma gondii e anti-Leptospira spp. em peixes-bois (Trichechus inunguis) da Amazônia Brasileira. Amostras sanguineas de 74 peixes-bois foram colhidas. Para a pesquisa de anticorpos anti-T. gondii, os soros foram diluídos a 1:25, 1:50 e 1:500 e a presença de anticorpos foi determinada pela técnica de aglutinação modificada (MAT). Para Leptospira spp. a presença de anticorpos foi determinada pela técnica de soroaglutinação microscópica (SAM), com ponto de corte na diluição de 1:100. Dos 74 peixes-bois 29 (39,2%) foram positivos para T. gondii apresentando título de 25. Para Leptospira spp. 23 (31,1%) foram reagentes para quatro sorovares com títulos variando de 100 a 1600. Os sorovares foram: Patoc, Castellonis, Icterohaemorrhagiae e Butembo. Houve coaglutinação entre os sorovares Patoc, Castellonis e Icterohaemorrhagiae (títulos 100 e 200) em um animal (n°51). Este foi o primeiro relato da ocorrência de T. gondii e Leptospira spp. em peixes-bois da Amazônia brasileira. / This study aimed to evaluate the occurrence of anti-T. gondii and Leptospira spp. in manatees (Trichechus inunguis) from two preservation centers located in the Brazilian Amazon. Blood samples from 74 manatees were taken. For detecting antibodies to T. gondii, sera were diluted 1:25, 1:50 and 1:500 and precense of antibodies was determined by the modified agglutination test (MAT). To Leptospira spp. sera were diluted 1:100 and tested against a collection of 24 antigenic serovars and presence of antibodies was determined by microscopic agglutination test (MAT). Of the 74 manatees 29 (39.2%) were positive to T. gondii with titre at 25. For Leptospira spp. 23 (31.1%) samples were kacted with four serotypes with titers ranging from 100 to 1600. The serovars reagents were Patoc, Castellonis, Icterohaemorrhagiae and Butembo. In one sample, coaglutination of serovars Patoc, Castellonis and Icterohaemorrhagiae (titres 100 and 200) was observed. This is the first report of the occurrence of T. gondii and Leptospira spp. in manatees in the Brazilian Amazon.

O trabalho teve por objetivo diagnosticar a infecção experimental e natural por Toxoplasma gondii em pombos (Columba livia), por técnicas sorológicas, biológicas e moleculares. Pombos foram infectados com oocistos esporulados de T. gondii e acompanhados por 60 dias com coleta de soro semanal para o acompanhamento da curva de anticorpos séricos e eutanásia quinzenal para avaliar a presença do parasito em diferentes tecidos. Observou-se concordância em todas as técnicas utilizadas, indicando serem eficazes no diagnóstico da infecção nessa espécie. Pombos de vida livre foram capturados nos municípios de São Paulo, Sorocaba e Ibiúna e anticorpos anti-T. gondii não foram observados. Nestas aves o bioensaio em camundongos foi realizado, independente da ausência de anticorpos e em nenhuma foi possível o isolamento do parasito. / The study aimed to diagnose the experimental and natural infection by Toxoplasma gondii in pigeons (Columba livia), by serological, biological and molecular techniques. Pigeons were infected with sporulated oocysts of T. gondii and monitored for 60 days with weekly serum collection for monitoring the curve of serum antibodies and euthanasia two weeks to assess the presence of parasites in different tissues. Agreement was observed in all the techniques used, indicating to be effective in the diagnosis of infection in this species. Free-living pigeons were captured in the municipalities of São Paulo, Sorocaba and Ibiúna and anti-T. gondii antibodies were not observed. In birds the bioassay was conducted in mice, regardless of the absence of antibodies and none was possible to isolate the parasite.