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Isolamento e caracterização molecular e biológica de Toxoplasma gondii e pesquisa de Neospora caninum em roedores urbanos da Grande São Paulo (SP) / Isolation, molecular and biological characterization of Toxoplasma gondii and survey for Neospora caninum in urban rodents from Great São Paulo (SP)Vanessa Muradian 08 May 2009 (has links)
Com o objetivo de identificar Toxoplasma gondii e Neospora caninum em roedores urbanos da Grande São Paulo (SP) 217 roedores (quatro camundongos -Mus musculus, 20 ratazanas - Rattus norvegicus e 193 ratos de telhado - Rattus rattus) foram capturados entre abril de 2005 e fevereiro de 2008, para realização do diagnóstico biológico e molecular. Das 20 ratazanas (Rattus norvegicus) capturadas apenas uma foi positiva para T. gondii pelo bioensaio, correspondendo a 5% de positividade entre as ratazanas e 0,46% entre todos os roedores capturados. O isolado obtido dessa ratazana foi caracterizado como genótipo recombinante I, III e u-1 por PCR-RFLP, similar ao isolado previamente descrito e obtido de duas ovelhas e um gato, todos do Estado de São Paulo. Quatro amostras de roedores negativos ao bioensaio resultaram como positivas ao T. gondii pela nested PCR B1. Entretanto, quando submetidas à nested PCR ITS1 e à restrição enzimática (RFLP) não houve confirmação desse resultado em três delas. Em relação ao N. caninum, amostras de tecido cerebral e cardíaco de 121 roedores foram examinadas por nested PCR Nc5, tendo sido encontradas 12 amostras positivas, provenientes de 10 roedores. Quando submetidas à nested PCR ITS1 e à restrição enzimática (RFLP) N. caninum não foi confirmado em nenhuma das amostras. Este estudo conclui que a ocorrência de T. gondii e N. caninum em roedores urbanos da Grande São Paulo (SP) é baixa, sugerindo que estes animais não possuem papel importante na cadeia epidemiológica como reservatórios desses agentes para predadores como os cães e gatos urbanos. / In order to identify Toxoplasma gondii and Neospora caninum in urban rodents from the Great São Paulo (SP) 217 rodents (four Mus musculus, 20 Rattus norvegicus and 193 Rattus rattus) were captured between April 2005 and February 2008 to biological and molecular diagnosis. One out of the 20 Rattus norvegicus was considered positive by bioassay in mice, corresponding to a 5% positivity among Rattus norvegicus and a 0.46% positivity considering all captured rodents. The isolate from this rat was characterized as a recombinant I, III and u-1 genotype by RFLP-PCR. This characterization had been previously described from isolates obtained from two sheep and one cat also from São Paulo state. Four samples from rodents with negative results by bioassay were positive to T. gondii by nested PCR B1. However, when tested with nested PCR ITS1 and RFLP these results were not confirmed in three of these samples. Regarding N. caninum, brain and heart samples of 121 rodents were examined by nested PCR Nc5 and 12 samples from 10 rodents were positive. When tested with nested PCR ITS1 and RFLP all of these samples turned out to be negative. This study concludes that T. gondii and N. caninum have low occurrence in urban rodents in the Great São Paulo area, and they are not important as T. gondii and/or N. caninum reservoirs to predators like urban cats and dogs.
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"Toxoplasma gondii vs radiação ionizante: Estudo da imunidade intestinal em camundongos C57Bl/6j experimentalmente vacinados com taquizoítos irradiados" / Toxoplasma gondii vs ionizing radiation: Intestinal immunity induced in C57bl/6j mice by irradiated tachyzoitesAndrés Jimenez Galisteo Junior 20 April 2004 (has links)
Em nosso estudo pretendemos estudar a via oral para o desenvolvimento de vacina para toxoplasmose utilizando parasitas irradiados por Cobalto-60, como uma alternativa para a prevenção desta importane parasitose. Para tanto, avaliamos o desenvolvimento da imunidade sistêmica e intestinal e a resistência à infecção, em camundongos imunizados por esta via com taquizoítos irradiados a 255Gy, e desafiados com cistos da cepa ME-49. Camundongos isogênicos C57Bl/6j foram imunizados com 107 taquizoitos de T. gondii irradiados a 255Gy em diferentes esquemas e utilizando adjuvantes leite (anti-péptico) e hidróxido de alumínio (anti-ácido). As preparações de taquizoítos irradiados por via oral induziram produção de imunoglobulinas IgG e IgA no soro de camundongos, sendo predominante a subclasse de IgG2a, similar a infecção crônica, determinadas por ELISA. Seu uso com adjuvantes anti-pépticos ou anti-ácidos induziu produção de IgA fecal e menos significativamente de IgG fecal. Existem indícios de indução de tolerância em nível intestinal, com queda da produção de anticorpos e da proliferação celular antígeno dirigida, especialmente quando o leite foi utilizado como adjuvante, em ensaios de produção in vitro de anticorpos ou proliferação estimulada por antígenos de T.gondii, por esplenócitos e linfócitos intestinais. As preparações orais induziram proteção quantitativa ao desafio dos animais imunizados por cepa cistogênica, que foi semelhante a imunização parenteral, quando o hidróxido de alumínio foi usado como adjuvante. Todos estes dados mostram a possibilidade de desenvolvimento de uma vacina oral para toxoplasmose utilizando taquizoítos irradiados, com aplicação prática num futuro próximo para uso em campo, utilizando iscas atrativas para imunizar felinos domésticos e selvagens. / We study the oral route for the development of a vaccine for toxoplasmosis, using parasites irradiated with 60 Cobalt, as an alternative for vaccine development to this worldwide parasitic infection. We evaluated the development of immunity at serum or mucosal levels, and their efficiency in protect the mice against challenge with oral cysts of the ME-49 strain. C57Bl/6j isogenic mice were immunized by oral route with 107 255 Gy irradiated tachyzoites from RH strain, at several protocols using milk as anti-peptic adjuvant and alum hydroxide as antacid. The preparations of irradiated tachyzoites induced production of serum IgG and IgA in immunized mice, as determined by ELISA, with IgG2a as the dominant subclass, similar to chronic infection. Their use with adjuvant allowed the excretion of significant amounts of IgA in stools also IgG, despite a lesser extent. There are suggestion of tolerance induction at mucosal level, with lower antigen induced proliferation and lower in vitro antibody production by spleen and gut lymphocytes, with the latter doses, specially when milk was used as adjuvant. All oral preparations induced some quantitative protection against challenge, which was similar to the parenteral route only isolated alum hydroxide was used as adjuvant. All these data support the possibility of the development of an oral vaccine against toxoplasmosis, using irradiated tachyzoites, which would be possible tool in near future for use in field baits, for immunizing either domestic or wild felids.
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Untersuchungen zur Prävalenz von Toxoplasma-gondii-Antikörpern bei Schlachtschweinen aus verschiedenen Haltungsformen und in handelsüblichen Hackfleischproben in der Region Halle/WittenbergHintersdorf, Petra 15 May 2013 (has links) (PDF)
Das Ziel der Untersuchungen in der vorliegenden Arbeit bestand in der Erarbeitung von Datenmaterial zur Bestimmung der Prävalenz von Toxoplasma-gondii-Antikörpern bei Schlachtschweinen und in handelsüblichen Hackfleischproben vom Schwein.
Um den Einfluss unterschiedlicher Haltungsbedingungen zu ermitteln, wurde jeder Betrieb anhand einer Checkliste im Rahmen einer Feldstudie überprüft.
Die Einteilung der Bestände erfolgte in 5 Kategorien (Kategorie 1: >1000 Tiere, Kategorie 2: >500-1000 Tiere, Kategorie 3: 100-500 Tiere, Kategorie 4: Tiere aus Ökobetrieben, Kategorie 5: Tiere aus einzelbäuerlicher Haltung).
Im Zeitraum von Februar 2001 bis August 2002 wurden 1028 Schlachtschweine aus 104 Beständen (958 Mastschweine, 66 Sauen, 4 Spanferkel) schwerpunktmäßig aus dem Landkreis Wittenberg mit Ausnahme der Tiere aus Ökobetrieben in den Schlachtbetrieben
beprobt. Als Probematerial dienten Blutserum und aus der Zwerchfellpfeilermuskulatur gewonnener Fleischsaft der Schlachtschweine. Des Weiteren wurden 240 Hackfleischprodukte
aus dem Handel beprobt, von denen ebenfalls der Fleischsaft untersucht wurde.
Eine weitere Aufgabenstellung der Arbeit bestand darin zu prüfen, ob der als serologischer Test verwendete ELISA neben der Eignung für Blutseren auch zur Untersuchung des Fleischsaftes verwendet werden kann. Es wurde ein ELISA nach SEINEKE (1996) auf der Grundlage eines
Tachyzoitenlysates als Antigen verwendet. Die Detektion der gebundenen Antikörper erfolgte durch POD (Meerettichperoxidase)-markierte Anti-Schwein-Antikörper von der Ziege. Zur Bewertung der Messergebnisse wurde ein Indexverfahren herangezogen. Blutserum wurde 1:400, Fleischsaft 1:40 verdünnt.
Die Ergebnisse der Studien lassen sich wie folgt zusammenfassen:
1. Die Untersuchungen mit dem verwendeten ELISA bieten eine akzeptable Sicherheit (Korrelationskoeffizient von rs = 0,841) bei hoher Signifikanz, wie aus den Doppelbestimmungen bei 711 Blutproben ermittelt werden konnte.
2. Fleischsaft ist grundsätzlich für die Anwendung des ELISA bei Ermittlung des Toxoplasmagondii- Antikörperstatus geeignet (Korrelationskoeffizient rs = 0,472; p < 0,01).
3. Die Gesamtprävalenz der Serumproben beträgt 20,6% mit dem Cut-off-Wert bei einem Index > 20. Diese Ergebnisse weisen aus, dass ein beachtliches Toxoplasmoserisiko besteht, wenn Schweinefleisch roh verzehrt wird.
4. Bei Betrachtung der einzelnen Bestandskategorien wurden folgende Seroprävalenzen bei Mastschweinen ermittelt: Kategorie 1: 15,4%, Kategorie 2: 7,3%, Kategorie 3: 16,4%, Kategorie 4: 15,1%. Höchste Prävalenzen ergaben sich bei unter individuellen, einzelbäuerlichen Bedingungen gehaltenen Schweinen der Kategorie 5: 52,0%.
5. Bei Sauen muss auf Grund des Alters generell von höheren Prävalenzen ausgegangen werden. Es wurde ein Wert von 27,2% für alle überprüften Sauen ermittelt.
6. Ein reelles Bild über das Vorkommen von Toxoplasma-gondii-Antikörpern kann nur durch die Betrachtung der Einzelbestände ermittelt werden. So findet man in der Gruppe der Ökotiere
(Kategorie 4) Prävalenzschwankungen von 1,4% bis 34,6%, sowie zwischen den Mastschweinebeständen der Kategorien 1 bis 3 von 1,6% bis 20,5%. Insofern kann allein aus der Kenntnis der Haltungsform nicht sicher auf ein geringes oder hohes Vorkommen
des Parasiten geschlossen werden.
7. Die Feldstudien in den einzelnen Beständen belegen eindeutig den Einfluss der Haltungsbedingungen der Schweine auf die ermittelten Prävalenzen für Toxoplasma gondii.
Grundsätzlich konnte festgestellt werden, dass unabhängig von Bestandsgröße und Haltungskategorie der direkte oder indirekte Einfluss von Katzen (z.B. über Ausläufe, Futter, Einstreu) als wesentliche Ursache für die Präsenz des Parasiten angesehen werden muss.
8. Beim Hackfleisch wurde eine Gesamtprävalenz für Toxoplasma-gondii-Antikörper von 5,3% nachgewiesen. Dies belegt das Risiko für Infektionen mit Toxoplasma gondii insbesondere beim Verzehr von rohen bzw. unzureichend erhitzten Schweinefleischerzeugnissen.
Die vorliegenden Untersuchungen liefern für ein begrenztes Territorium Datenmaterial zu Prävalenzen von Toxoplasma gondii sowohl für Proben von Schlachtschweinen als auch für Hackfleischproben aus dem Handel. Zur Bewertung des deutschlandweit zu erwartenden
Risikos sind jedoch weitere flächendeckende Untersuchungen erforderlich.
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Untersuchungen zur Verteilung von Toxoplasma gondii-Stadien in Geweben von Puten nach experimenteller InfektionZöller, Birte 02 December 2015 (has links) (PDF)
Einleitung: Toxoplasma (T.) gondii zählt zu den häufigsten intrazellulären Parasiten weltweit. Alleinige Endwirte im fakultativ heteroxenen Lebenszyklus sind die Feliden. Als Zwischenwirte können jedoch zahlreiche Säugetier- und Vogelarten dienen, in denen sich parasitäre Gewebezysten entwickeln. Einer der Hauptübertragungswege auf den Menschen stellt der Verzehr von T. gondii-haltigem Fleisch infizierter Nutztiere dar. Inwieweit Putenfleisch ein potentielles Infektionsrisiko birgt und welche Bedeutung Puten in der Epidemiologie der humanen Toxoplasmose besitzen ist nicht ausreichend geklärt.
Ziel der Untersuchungen: Ziel der vorliegenden Arbeit war es, ein reproduzierbares Infektionsmodell bei Puten für T. gondii zu entwickeln, um die Verteilung und Persistenz des Parasiten im Gewebe zu ermitteln. Es wurden verschiedene Parameter, wie Infektionsstadium, Infektionsdosis, Applikationsmodus und Untersuchungszeitpunkt hinsichtlich ihres Einflusses auf die Entwicklung parasitärer Gewebestadien verglichen.
Material und Methoden: Insgesamt wurden 74 Puten nach einer Aufzuchtperiode von 4 bis 8 Wochen experimentell mit T. gondii-Tachyzoiten oder Oozysten infiziert. Je nach Versuchsgruppe wurden Tachyzoiten vom Stamm ME49 intravenös und/oder intramuskulär appliziert oder Oozysten vom Stamm ME49, DX oder Hannover 1 oral verabreicht. Die Verifikation der Infektion erfolgte über den Nachweis T. gondii-spezifischer Antikörper mit Hilfe eines kinetischen ELISA. Drei bis acht Puten jeder Versuchsgruppe wurden 6 bis 8 oder 10 bis 12 Wochen nach der Infektion getötet.
Von jedem Tier wurden folgende Gewebeproben entnommen: Brust-, Oberschenkel- und Unterschenkelmuskulatur, Herz, Leber, Muskel- und Drüsenmagen, Gehirn, Lunge, Milz, Nieren, Darm, Pankreas und Hoden (sofern vorhanden). Die Organe wurden getrennt vollständig homogenisiert. Bei den Muskeln wurden Proben von verschiedenen Lokalisationen entnommen und ebenfalls einzeln homogenisiert. Der Nachweis von T. gondii-DNA in den Gewebeproben erfolgte mittels konventioneller PCR, basierend auf der Amplifizierung eines 469 bp Fragments des B1-Gens, und anschließender nested PCR (Länge Zielfragment: 375 bp). Zusätzlich wurden zu Beginn der Studie lichtmikroskopische Untersuchungen einzelner Organe in Form nativer Quetschpräparate (400fache Vergrößerung) auf T. gondii-Zysten durchgeführt.
Ergebnisse: Ungeachtet der Infektionsdosis und des inokulierten Parasitenstadiums konnten bei keinem der Versuchstiere klinische Symptome einer Toxoplasmose beobachtet werden. Die unterschiedlich hohen Infektionsdosen hatten im Allgemeinen keinen signifikanten Einfluss auf die Anzahl positiv getesteter Puten oder Organproben. Lediglich die Anzahl positiver Gehirnproben nahm mit ansteigender Oozystendosis signifikant zu. Bei der Betrachtung aller Versuchsgruppen fiel auf, dass die Befallshäufigkeit der Organe sowohl zwischen den Tieren verschiedener Infektionsgruppen als auch innerhalb einer Infektionsgruppe stark schwankte. So variierte die Anzahl positiv getesteter Organe bei den Tachyzoiten-infizierten Puten zwischen 0 und 7, bei den Oozysten-infizierten Puten zwischen 0 und 9 Organen pro Tier. Die Untersuchungsergebnisse zeigen, dass sich T. gondii heterogen in der Pute verteilt und mindestens 12 Wochen persistieren kann. Bezogen auf alle Versuchstiere gab es kein Organ, dass durchgängig negativ blieb. Nach der Tachyzoiteninfektion waren am häufigsten Leber (43,3%), gefolgt von Brustmuskel (26,7%) und Herz (20,0%) infiziert, während bei den Oozysten-infizierten Tieren der Erreger am häufigsten im Gehirn (47,2%), gefolgt von Oberschenkelmuskulatur (25,0%) und Herz und Unterschenkelmuskulatur (je 22,2%) nachgewiesen werden konnte.
Schlussfolgerungen: Tachyzoiten und Oozysten erwiesen sich als gleichermaßen geeignete Infektionsmedien und führten hinsichtlich der systemischen Verteilung des Parasiten in der Pute zu vergleichbaren Ergebnissen. Ein spezifischer Organtropismus des Erregers konnte nicht festgestellt werden. Aus Sicht der Lebensmittelhygiene und des Verbraucherschutzes bedeuten die Ergebnisse, dass im Fall einer T. gondii-Infektion ein potentielles Infektionsrisiko für den Menschen durch infiziertes Putenfleisch nicht ausgeschlossen werden kann.
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Etude de la synthèse des précurseurs majeurs à la synthèse des lipides membraniares : l'acide lysophosphatidique et l'acide phosphatidique chez Toxoplasma gondii / L'auteur n'a pas fourni de titre anglaisAmiar, Souad 01 December 2016 (has links)
Les Apicomplexa sont des parasites intracellulaires obligatoires. Ils peuvent être responsables d’importantes maladies infectieuses. Toxoplasma gondii par exemple, se développe au sein de la cellule hôte, dans une niche protectrice « la vacuole parasitophore » jusqu’à épuisement des ressources de la cellule hôte ou il provoque sa sortie pour ré envahir à nouveau, c’est la phase aigüe de la toxoplasmose. Afin de répondre à leur besoins nutritifs nécessaires à cette expansion rapide, le parasite combine de manière intéressante et très complexe les voies de synthèse de novo et d’import des nutriments depuis la cellule hôte. Dans le cas des lipides, le parasite en a besoin en une importante quantité pour assurer la ségrégation des organites, la formation des nouvelles membranes filles et l’expansion de la membrane de la vacuole parasitophore pendant la division. La synthèse de novo des lipides a été reportée essentielles pour le parasite tout comme la synthèse de novo des acides gras via la voie procaryote de synthèse des acides gras FASII dans l’apicoplaste.Dans cette étude nous apportant des éléments intéressants qui relient la voie de synthèse FASII et la synthèse des lipides. Nous avons pu démontrer que l’apicoplaste possède une voie de synthèse des précurseurs important voire essentielles à la synthèse de tous les lipides membranaires, qui est principalement le LPA dans le cas de T. gondii. Les enzymes acyltransférase impliquées dans la synthèse de ces précurseurs sont TgATS1 et TgATS2 pour former le LPA et le PA respectivement. Elles sont ortologues aux enzymes précédemment caractérisées chez les bactéries et le chloroplaste des plantes et algues. Les modifications de ces enzymes et les analyses de lipidique et de spectrométrie de masse, ont révélé le rôle l’implication de ces enzymes dans la synthèse des phospholipides membranaires à partir des acides gras néo synthétisés de novo (le C14:0). Cette étude présente aussi des résultats préliminaires sur une voie de synthèse du PA dans le réticulum endoplasmique. La être de TgATS2 n’est pas létale et elle est compensée par augmentation de l’abondance des acides gras C16 :0 et C18 :0 dans la fraction des phospholipides extraits. Ces informations suggèrent une importante collaboration entre l’apicoplaste et le réticulum endoplasmique pour la synthèse des lipides nécessaires pour le développement intracellulaire du parasite. / Apicomplexa phylum includes a large number of obligate intracellular parasites responsible for important human and animal diseases, especially malaria and toxoplasmosis. There is current no efficient vaccine against these parasites. Severe toxoplasmosis caused by Toxoplasma gondii, occurs in immunocompromised individuals and during congenital infection. T. gondii is dependent on large amounts of lipids for its intracellular development within the host cell. These lipids are acquired by a combination of host lipid scavenging and de novo biosynthetic pathways. T. gondii is able to de novo synthesis of fatty acid via a prokaryotic FASII pathway in the apicoplast, a relict non-photosynthetic plastid. Genome mining suggests that the apicoplast can generate phosphatidic acid, central phospholipid precursor. Our recent work confirmed that the apicoplast harbors the first step of PA synthesis via a glycerol-3-phosphate acyltransferase enzyme called ATS1 by homology to chloroplast enzyme, which generates lysophosphatidic acid (LPA). This essential LPA can be exported from the apicoplast for the de novo bulk synthesis of phospholipids sustaining parasite membrane biogenesis (Amiar et al. Plos Path. 2016). T. gondii genome encodes for two other acyltransferases named sn-acylglycerol 3-Phosphate acyltransferases (AGPAT). AGPATs ensure the second step of PA synthesis using LPA. In this work we showed that these enzymes are localized in the Endoplasmic Reticulum and the apicoplast (named AGPAT and ATS2, respectively). A genetic disruption of ATS2 using CRISPR-Cas9 strategy affects parasite growth and normal cytokinesis. Lipidomic analysis using mass spectrometry combined to stable isotope labelling of ATS2-KO reveals an important reduction of lipids containing apicoplast-generated fatty acid C14:0. However, an increase of lipids containing C16 and C18 fatty acids was observed, suggesting a compensation of ATS2 loss by AGPAT activity in ER. These data indicated an important collaboration between apicoplast and ER for lipid synthesis that involves massive lipid trafficking between the two organelles.
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Caractérisation d'un effecteur chez Toxoplasma gondii : découverte d'une voie alternative d'inflammation régulée par β-caténine / Parasite and host-cell interactions : Characterization of new effector proteins used by Toxoplasma gondii to interfere with host signaling pathwaysHe, Huan 27 September 2017 (has links)
Toxoplasma gondii est un parasite intracellulaire et obligatoire. Ce protozoa est un des parasites les plus successifs qui infectent tous les animaux à sang chaud, y compris l’humain. Ce succès est probablement à cause de la sécrétion d’une des séries de protéines effectrices, qui sont impliquées dans la modulation des voies de signalisation de la cellule hôte. Cette modulation permet aux parasites d’établir une infection chronique qui dure un long terme, et qui favorise leur transmission à un nouvel hôte. Dans cette étude, nous avons identifié un nouvel effecteur dérivé par la granule dense, appelé GRA18, qui est sécrété dans le cytoplasme de cellule hôte par les tachyzoites intracellulaires. La mutation de gra18 résulte une diminution de virulence chez les parasites de type II, qui suggère l’importance de GRA18 dans la pathogénicité de ce parasite. Afin d’étudier le mécanisme d’action de GRA18, nous avons effectué un criblage à haut-débit d’une librairie humaine chez la levure. Ce criblage nous permet d’identifier β-catenin, GSK3α/β, and PP2A-B56, ce qui sont tous les régulateurs bien connus dans la voie de signalisation canonicale de Wnt. Nous avons confirmé l’intéractome de GRA18 par l’approche biochimique. La surexpression de GRA18 induit l’accumulation de β-catenin dans le noyau de la cellule hôte, aussi que l’induition de gènes régulés par la signalisation de Wnt. Ces effets indiquent GRA18 joue un rôle de régulateur positif de β-catenin. A part de son rôle dans la prolifération, polarisation et la différentiation de cellule, β-catenin est également un facteur de transcription connu pour contrôler la réponse immunitaire et l’inflammation. L’analyse transcriptomique en comparant les macrophages dérivés par la moelle osseuse (BMDM) infectés par le sauvage (WT) et le gra18 mutant parasites confirme un rôle possible de GRA18, la modulation d’expression génique de cellule hôte, surtout ceux qui codent pour les chemokines. Cette régulation est ensuite confirmée par l’ELISA. L’hypothèse possible est que Toxoplasma sécrète GRA18 dans la cellule hôte afin de réguler positivement la production de chemokine reliée à la réponse de Th2, qui par contre atténue la réponse inflammatoire de l’hôte. Cette modulation augmente la chance de dissémination et la persistance de ce parasite par la formation de kyste. / Toxoplasma gondii, the obligate intracellular protozoan parasite, is one of the most successful pathogen that infects virtually all warm-blooded animals including humans. This success of the infection is likely due to its perfect ability to modulate numbers of host signaling pathways through the effector proteins, including those involved in immune responses. This modulation allows the parasite to establish a long-term chronic infection without causing severe symptom in the hosts, which facilitates its transmission to the new hosts. In this study, we identified GRA18, as a novel dense granule derived effector protein that is secreted into the cytoplasm of the host cell by the intracellular tachyzoite. GRA18 deficiency in type II strains attenuated the parasite virulence in mice model, suggesting the importance of GRA18 in the parasite pathogenesis. In order to investigate the mechanism of action of GRA18, we first performed a high-throughput two-hybrid screen of a human library in yeast that led to the identification of β-catenin, GSK3α/β, and PP2A-B56, all which are well known regulators of the canonical Wnt signaling pathway. We then validate the GRA18 interactome by biochemistry approach. The overexpression of GRA18 triggers the accumulation of β-catenin in the host cell nuclei as well as the induction of known canonical β-catenin target genes indicating that GRA18 is acting as a positive regulator of β-catenin. Besides its role in cell proliferation, polarization and differentiation, β-catenin is also a well-known co-transcription factor with important function in the control of inflammation and other immune responses. Transcriptomic analysis comparing mouse bone marrow–derived macrophages infected by wild type and GRA18-dificient parasite confirmed a possible role of GRA18 towards host gene expression and likely those encoding chemokines, which is further confirmed by ELISA experiments. An attractive hypothesis is that Toxoplasma delivers GRA18 to the host cell in order to regulate Th2-related chemoattractant chemokines, which in turn, dampens host inflammatory response leaving more chance for the parasites to disseminate and to cause the long-term persistence by forming the cyst.
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Vaccin nanoparticulaire muqueux contre la toxoplasmose chronique et congénitale / Mucosal nanoparticle vaccine against chronic and congenital toxoplasmosisNguyen, Thi Thanh Loi 25 April 2016 (has links)
La toxoplasmose est une anthropozoonose cosmopolite due à un protozoaire parasite intracellulaire obligatoire : Toxoplasma gondii. Cette maladie infectieuse est la plus souvent bénigne chez les personnes immunocompétentes mais revêt un caractère de gravité si l’atteinte concerne les femmes enceintes séronégatives ou les personnes immunodéprimées. En plus de cette incidence forte en médecine humaine, la toxoplasmose représente un important problème de santé vétérinaire. A l’heure actuelle, les seuls moyens de lutte contre ce parasite demeurent la chimiothérapie car il n’existe aucune stratégie prophylactique efficace. Le développement d’un vaccin efficace est d’une réelle nécessité et repose sur l’observation qu’une primo-infection par ce parasite confère à l’hôte immunocompétent une réponse immunitaire protectrice efficace à long terme et qui protège lors d’une réinfection et en particulier contre le risque d’une infection congénitale. / Toxoplasmosis is a cosmopolitan anthropozoonosis due to the obligate intracellular protozoan parasite Toxoplasma gondii. This infectious disease is most often benign in immunocompetent individuals but is particularly severe for pregnant women or immunocompromised patients. In addition to its significant impact on human medicine, toxoplasmosis is a major veterinary health problem. Currently, the only means to fight this parasite remain chemotherapy because there is no effective prophylactic strategy. The development of an effective vaccine is a real challenge and is based on the observation that a primary infection of immunocompetent hosts induces a effective and long-term protective immune response and protects during reinfection and in particular against the risk of congenital infection.
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Cistogênese e estágios esquizontes em células epiteliais de felinos infectadas pelo Toxoplasma gondii, in vitroMuno, Renata Morley de January 2011 (has links)
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Previous issue date: 2011 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / O Toxoplasma gondiié um protozoário parasito intracelular, agente causador
da toxoplasmose que é uma das zoonoses mais endêmicas do mundo. O T. gondii
possui dois tipos de reprodução: assexuada, que origina dois estágios evolutivos
infectivos, os bradizoítos e taquizoítos e a sexuada que ocorre no tecido epitelial
intestinal, exclusivamente de felídeos, hospedeirosdefinitivos, resultando na
formação de oocistos imaturos que são eliminados nas suas fezes. A disseminação
de oocistos no ambiente é o principal fator que explica a distribuição mundial do
parasito, associada à formação de cistos teciduais que aumentam a capacidade de
transmissão do parasito, através do consumo de carne crua ou mal cozida. Dada a
especificidade de o ciclo sexuado ocorrer no tecidoepitelial, o principal objetivo do
presente trabalho foi investigar, in vitro,a interação do T. gondiicom células
epiteliais oriundas do intestino de ratos e de rim de felídeos, a fim de estabelecer
possíveis correlações entre a origem do tecido (epitelial) e a fonte (rato e felídeos)
que possam determinar o destino intracelular do parasito. Assim, culturas de
linhagens celulares CRFK e IEC-6 foram infectadas com bradizoítos e taquizoítos de
T. gondiicom diferentes cargas infectivas. Após os desenhosexperimentais as
culturas foram processadas como de rotina para microscopia óptica de luz e
fluorescência e para microscopia eletrônica de transmissão e varredura. As análises
quantitativas mostraram que a linhagem CRFK foi mais susceptível à infecção frente
aos dois estágios infectivos do que a linhagem IEC-6 e que essa diferença foi
independente da carga parasitária e do estágio infectivo utilizados. As análises
qualitativas mostraram que a linhagem CRFK foi maissusceptível à infecção do que
a linhagem IEC-6 e esse evento foi independente da carga infectiva utilizada. As
análises qualitativas mostraram que a cistogênese se estabeleceu de forma
espontânea na CRFK quando infectada com bradizoítosda cepa ME49 ao se utilizar
a carga infectiva de 1:10 (parasito-célula hospedeira). Além disso, esta linhagem
celular apontou estágios infectivos morfologicamente distintos de taquizoítos e
bradizoítos, similares aos estágios esquizontes de T. gondii. O presente estudo
mostrou que existem diferenças no destino intracelular do parasito de acordo com o
tipo celular utilizado. O emprego de células epiteliais de felinos como modelo celular
da toxoplasmose experimental mostrou que pode contribuir com novos subsídios
para o estudo da biologia celular do parasito, assim como contribuir como
metodologia alternativa para o melhor entendimento do ciclo enteroepitelial doT.
gondii. Este modelo celular abre um novo campo de investigação para aspectos
moleculares desta interação que possam contribuir, por exemplo, para introdução de
estratégias que venham a interferir em umas das principais vias de disseminação da
toxoplasmose. Além disso, a CRFK se mostrou um modelo celular em potencial para
a produção de cistos de T. gondii in vitroem larga escala abrindo perspectivas na
redução do uso de animais experimentais para isolamento de cistos e inserção na
área de inovação e desenvolvimento tecnológico. / Toxoplasma gondiiis an intracellular protozoan parasite, the causative agent of
toxoplasmosis which is one of the most endemic zoonoses in the world. T. gondiihas
two types of reproduction: the asexual, which leadstwo evolutive infective stages,
the bradyzoites and tachyzoites and the sexual thatoccurs in the intestinal epithelial
tissue, exclusively in cats, the definitive hosts, resulting in the formation of immature
oocysts that are shed in their feces. The spread ofoocysts in the environment is the
main factor that explains the global distribution and dissemination of toxoplasmosis,
leading to the formation of tissue cysts increasingthe parasite capacity of
transmission, through ingestion of raw or undercooked meat. Once the specificity of
the sexual cycle occurs in epithelial tissue, the main objective of this study was to
investigate the in vitrointeraction of T. gondii epithelial cells derived from the
intestine of rats and kidney of cats, in order to establish possible correlations
between the origin of tissue (epithelial) and the source (rats and cats) that may
determine the intracellular fate of the parasite. Thus CRFK and IEC-6 cell lines were
infected with T. gondiibradyzoites and tachyzoites with different infective loads. The
kinetic study of the infective capacity of both stages of the parasites was performed
by fixing cell cultures at different times of interaction, aiming the quantitative analysis
of infection of both cell types fronts of two strains and two infective stages of the
parasite. After the experimental designs cultures were processed as routine for light
microscopy and fluorescence and transmission and scanning electron microscopy.
Quantitative analysis showed that CRFK line was more susceptible to infection than
the IEC-6 line, and that this event was independentof the infective stage or the
infective load used. The qualitative analysis showed that cystogenesis occurred
spontaneously in the CRFK line when infected with the ME49 strain bradyzoites, by
using the load of 1:10. In addition, this cell linepointed to morphologically distinct
stages of tachyzoites and bradyzoites, similar to schizonts stages of T. gondii. The
present study showed that there are differences in the intracellular fate of the
parasite according to the cell type used. The use of feline epithelial cells as cellular
model of experimental toxoplasmosis showed that it can contribute with new insights
for studying the cell biology of the parasite as well as contribute as an alternative
methodology for better understanding T. gondiienteroepithelial cycle. This cellular
model opens a new field of investigation for molecular aspects of this interaction that
can contribute, for example, to introduce strategies that will interfere in one of the
main routes of toxoplasmosis spread. In addition, the CRFK line presented itself as a
potential cellular model for cysts production in vitro in large scale opening
perspectives on reduction in the use of animals forcysts isolation and insertion in
inovation an technological development area.
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Estudo da expressão e participação de osteopontina durante a interação taquizoíto de Toxoplasma gondii célula hospedeira. / Differential expression of osteopontin and participation during interaction of Toxoplasma gondii tachyzoite - host cell.Erika Afonso Costa Cortez Marques 28 June 2007 (has links)
Toxoplasma gondii é um parasito do filo Apicomplexa que infecta uma grande variedade de hospedeiros, incluindo os humanos. O parasito invade a célula hospedeira por penetração ativa, com a participação das proteínas de suas organelas secretoras durante esse processo. Até o momento, somente um número limitado de proteínas secretoras tem sido descoberto, além disso, as moléculas efetoras envolvidas na invasão e sobrevivência do parasito não estão completamente compreendidas. A osteopontina (OPN) é uma glicofosfoproteína adesiva secretada, multifuncional, que contém o domínio arginina-glicina-ácido aspártico (RGD) de ligação à integrina, que está envolvida em uma variedade de eventos fisiológicos e patológicos, incluindo sinalização e sobrevivência celular. Pela primeira vez, nós demonstramos pelas técnicas de imunofluorescência e imunocitoquímica ultraestrutural que há uma intensa marcação para uma proteína OPN-like nos grânulos densos de taquizoítos de T. gondii extracelulares. O western blotting e o RT-PRC confirmaram a expressão de OPN-like nos taquizoítos. Nossos resultados também mostram que após a invasão dos macrófagos, a proteína OPN-like está localizada na membrana do vacúolo parasitóforo. Esses dados sugerem que os grânulos densos secretam uma proteína OPN-like, e nós podemos especular que essa proteína participa durante o processo de interação do parasito com as células hospedeiras.
. / Toxoplasma gondii is an apicomplexan parasite infecting a broad host range, including humans. The parasite invades host cell by active penetration with the participation of its secretory organelles proteins during this process. Until now, only a limited number of secretory proteins have been discovered, and the effectors molecules involved in parasite invasion and survival are not well understood. Osteopontin (OPN) is a multifunctional secreted adhesive glycophosphoprotein containing the arginine-glycine-aspartic acid (RGD) integrin-binding domain, which is involved in various physiological and pathological events including cell signaling and survival. For the first time we demonstrated by immunofluorescence and immunoelectron microscopy approaches that there is an intense labeling for an OPN-like protein in the dense granules of extracellular T. gondii tachyzoites. Western blotting and RTPCR confirmed this protein expression in tachyzoites. Our results also showed that after macrophage invasion the OPN-like protein is localized at the parasitophorous vacuole membrane. These data suggest that dense granules secrete an OPN-like protein, and we can speculate that this protein participates during the parasite interaction process with host cells.
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Fenótipo Lewis negativo: potencial fator de risco para infecção por cepa RH de Toxoplasma gondii em gestantesNakashima, Fabiana [UNESP] 23 February 2010 (has links) (PDF)
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nakashima_f_me_sjrp.pdf: 829285 bytes, checksum: 72d9338767526ff29fcf4612c5437bff (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Introdução: A infecção por Toxoplasma gondii em gestantes associa-se aos riscos de transmissão congênita. Este protozoário infecta os humanos utilizando como rota de infecção o trato gastrintestinal, onde se dá a síntese dos antígenos fucosilados Lea e Leb que determinam os fenótipos do sistema histo-sanguíneo Lewis [Le(a+b-), Le(a+b+) e Le(a-b-)]. A expressão destes fenótipos resulta de interações epistáticas entre os genes FUT2 (Secretor) e FUT3 (Lewis) que codificam as fucosiltransferases FUTII e FUTIII, respectivamente. Mutações no gene FUT3 determinam a ausência de fucosilação dos oligossacarídeos precursores do tipo 1 resultando na expressão do fenótipo Le(a-b-). A infecção por T. gondii e a expressão dos antígenos Lewis ocorrem no mesmo órgão e, embora aparentemente independentes, podem estar relacionadas entre si. Objetivo: Investigar a associação o sistema histo-sanguíneo Lewis e a infecção por T. gondii da cepa RH. Material e Métodos: Foram selecionadas 209 amostras de soro e de DNA genômico estocadas no Laboratório de Imunogenética do Departamento de Biologia Molecular da Faculdade de Medicina de São José do Rio Preto - FAMERP, coletadas de gestantes atendidas no Ambulatório de Gestação de Alto Risco do Hospital de Base da Fundação Faculdade Regional de Medicina de São José do Rio Preto – FUNFARME. Uma triagem para detecção dos anticorpos anti-T. gondii foi realizada nas amostras de soro pelo método hemaglutinação indireta (HAI). O ensaio imunoenzimático (ELISA) foi empregado para identificar os anticorpos específicos da classe IgG contra a cepa RH. Cento e noventa e cinco amostras de soro que apresentaram resultados concordantes entre os dois testes foram selecionadas para compor os grupos “reagente” e “não reagente”. Para inferir os fenótipos do sistema histo-sanguíneo Lewis, as amostras de DNA... / Toxoplasma gondii Infection in pregnant women is associated with risks of congenital transmission. This protozoa infects humans using as infection rout the gastro intestinal tract, where occur the synthesis of fucosylated Lea and Leb antigens which determine Lewis histo-blood group phenotypes [Le(a+b-), Le(a+b+), Le(a-b-)]. The expression of these phenotypes results from epistatic interactions between FUT2 (Secretor) and FUT3 (Lewis) genes which codes both the FUTII and FUTIII fucosyltransferases, respectively. Single nucleotide polymorphisms affecting FUT3 gene determine the absence of type 1 oligosaccharide precursor fucosylation and the expression of Le(a-b-) phenotype. The entrance of T. gondii and the expression of Lewis histo-blood group system occur in the same organ, probably there is some link between them. Objective: The aim of this study was to test the hypothesis that the Lewis histo-blood group system is associated with infection by T. gondii. Material e Method: A total of 209 serum sample and genomic DNA sample stored at the Immunogenetics Laboratory from the Molecular Biology Department of Medicine School in São José do Rio Preto – FAMERP, obtained from pregnant women who attended in the High-Risk Pregnancy Clinic of Hospital de Base were enrolled in this study. Anti-T. gondii antibodies were screened by indirect hemagglutination test. ELISA assays were used to identify specific IgG antibodies to RH strain. One hundred and ninety-five serum samples, with identical results between both tests were selected to compose the groups “reagent’ and “non reagent”. To infer the Lewis histo-blood group system phenotypes, genomic DNA samples corresponding to the serum sample were genotyped for the G428A mutation of the FUT2 gene and T202C and C314T of FUT3 gene by PCR-RFLP and PCR-SSP assays, respectively. The data were analyzed by Fisher’s exact test... (Complete abstract click electronic access below)
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