Thesis (M.S.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Made available in DSpace on 2014-06-11T19:27:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-21Bitstream added on 2014-06-13T19:26:14Z : No. of bitstreams: 1 matos_dj_me_araca.pdf: 760484 bytes, checksum: c06dae3728a9ffd470f98e2c615bdd6d (MD5) / O presente trabalho teve como objetivo investigar a ocorrência dos protozoários Cryptosporidium spp. e Giardia spp. em leitões com 45 dias de idade. Amostras fecais de 107 leitões foram colhidas, em três dias alternados, em suinoculturas do Município de Araçatuba, São Paulo, Brasil. A ocorrência de oocistos de Cryptosporidium spp., foi observada em 4,7% (5/107) dos animais usando a técnica de Kinyoun, e a detecção de oocistos de Giardia spp. foi concomitantemente observada em 1,9% (2/107) dos animais pelo método de Faust.. Animais com a presença concomitante de ambos os parasitos apresentaram fezes com aspecto diarréico. A partir dos resultados obtidos é possível inferir que a ocorrência de Cryptosporidium e Giardia foi baixa, o que se atribuiu ao manejo e que a presença destes protozoários não estava associada a todos animais com sintomas diarréicos envolvidos no estudo. / The goal of this research was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in 45-days-old pigs. Fecal samples of 107 pigs were collected at three alternate days in piggeries in Araçatuba, São Paulo, Brazil. Cryptosporidium spp. oocysts were observed in 4.7% (5/107) of animals by means of Kinyoun acid-fast stain and cysts of Giardia spp. were simultaneously observed in 1.9% (2/107) of the animals by the method of Faust. Animals with positivity for the both parasites presented feces with diarrheal aspect. From these results it is possible to infer that the occurrence of Cryptosporidium and of Giardia was low because of the good management practices and both protozoa were not associated the presence of clinical symptoms.

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Das Protozoon Giardia duodenalis ist eine der Hauptursachen für infektiöse Magen-Darm-Erkrankungen. Die zugrundeliegenden Pathomechanismen sind jedoch nach wie vor unklar. Um die Pathogenität G. duodenalis‘ untersuchen zu können, wird ein Modellsystem benötigt, dass die Komplexität des Darmepithels widerspiegelt. Diese Arbeit zeigt die Etablierung eines Zellkultursystems auf der Basis von organoid-abgeleiteten Epithelien unter Verwendung von filter-basierten Zellkultureinsätzen. Wir haben Protokolle für die Etablierung von organoid-basierten Zellkulturen (ODMs) vier verschiedener Wirte zoonotischer Protozoen unter Verwendung eines einzigen Protokolls erstellt. Die Charakterisierung zeigte, dass das Modellsystem erfolgreich die Polarisierung des Darmepithels nachahmt, aus mehreren Zelltypen besteht und eine Infektion ermöglicht. Der Schwerpunkt der Arbeit lag auf der Analyse der durch G. duodenalis induzierten Barrierestörung in ODMs auf Transkriptions-, Protein- und Funktionsebene. Die Infektion von humanen duodenalen Zellen führte zu einem Verlust der epithelialen Barrierefunktion. Mit Hilfe des transepithelialen elektrischen Widerstandes und Dextran Flux wurde eine Erhöhung der Barrieredurchlässigkeit beobachtet. Die Hemmung von zuvor in immortalisierten Zellmodellen beschriebenen Reaktionswegen konnte die Barrierefunktion nicht wiederherstellen. Stattdessen konnten Veränderungen der Ionenhomöostase sowie den Zusammenbruch der zonula occludens nachgewiesen werden. Der beobachtete Phänotyp konnte auf die Aktivierung des cAMP/PKA/CREB-Signalwegs, als einen von mehreren kausalen Faktoren, zurückgeführt werden. Hier zeigen wir die Etablierung eines aus Organoiden abgeleiteten Modells, das die Untersuchung von G. duodenalis Infektionen in vitro ermöglicht. Mit unserem Modell konnten wir eine neue Reihenfolge von Ereignissen entschlüsseln, die einen der Faktoren während symptomatischer Giardiasis darstellt. / The protozoan Giardia duodenalis is a one of the major causes of gastrointestinal illness. Underlying pathomechanisms remain unclear. An in vitro model system that also mimics the complexity of intestinal epithelium is needed to allow pathogenicity studies. This thesis shows the establishment of a cell culture system based on organoid-derived epithelia using permeable cell culture inserts. We have provided guidelines on the establishment of organoid-derived monolayers (ODMs) of four different hosts of zoonotic protozoa using a single protocol. Characterization showed that the model system successfully mimics intestinal polarization, is composed of multiple cell types and allows for infection with multiple protozoan parasites. As the main focus of the thesis, analysis of G. duodenalis-induced barrier breakdown in ODMs was performed on transcriptional, protein and functional level. Infection of human duodenal, organoid-derived monolayers resulted in a time- and dose-dependent breakdown of epithelial barrier function. Barrier permeability increases were observed ranging from ions to macromolecules as measured by transepithelial electrical resistance and Dextran flux. Inhibition of previously proposed key pathogen-induced pathways observed in immortalized cell models did not rescue barrier dysfunction. We could instead show changes in ion homeostasis, and tight junctional breakdown. While none of the previously proposed effector pathways appeared to be responsible, we could pin-point the observed phenotype to activation of the cAMP/PKA/CREB signaling pathway, as one of the factors of the multifactorial barrier breakdown. The establishment of an organoid-derived infection model is shown, allowing the study of in vitro Giardia duodenalis infections. Using this model, we could decipher a new series of events that may be one of the factors causing the intestinal barrier breakdown observed in symptomatic Giardiasis.

Call number: LD2668 .T4 1985 S78 / Master of Science

Resumo: O presente trabalho teve como objetivo investigar a ocorrência dos protozoários Cryptosporidium spp. e Giardia spp. em leitões com 45 dias de idade. Amostras fecais de 107 leitões foram colhidas, em três dias alternados, em suinoculturas do Município de Araçatuba, São Paulo, Brasil. A ocorrência de oocistos de Cryptosporidium spp., foi observada em 4,7% (5/107) dos animais usando a técnica de Kinyoun, e a detecção de oocistos de Giardia spp. foi concomitantemente observada em 1,9% (2/107) dos animais pelo método de Faust.. Animais com a presença concomitante de ambos os parasitos apresentaram fezes com aspecto diarréico. A partir dos resultados obtidos é possível inferir que a ocorrência de Cryptosporidium e Giardia foi baixa, o que se atribuiu ao manejo e que a presença destes protozoários não estava associada a todos animais com sintomas diarréicos envolvidos no estudo. / Abstract: The goal of this research was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in 45-days-old pigs. Fecal samples of 107 pigs were collected at three alternate days in piggeries in Araçatuba, São Paulo, Brazil. Cryptosporidium spp. oocysts were observed in 4.7% (5/107) of animals by means of Kinyoun acid-fast stain and cysts of Giardia spp. were simultaneously observed in 1.9% (2/107) of the animals by the method of Faust. Animals with positivity for the both parasites presented feces with diarrheal aspect. From these results it is possible to infer that the occurrence of Cryptosporidium and of Giardia was low because of the good management practices and both protozoa were not associated the presence of clinical symptoms. / Orientador: Katia Denise Saraiva Bresciani / Coorientador: Marcelo Vasconcelos Meireles / Banca: Fernanda Paiva / Banca: Carlos Noriyuki Kaneto / Mestre

The concern of a Cryptosporidium or Giardia waterborne outbreak due to treated wastewater has had water treatment utilities using some of the highest water cleansing technologies available. Cryptosporidiosis and Giardiasis are severe diarrheal diseases which can lead to death, thus it is important that appropriate steps are taken to assure these parasites are not present in the effluent of treated wastewater. This study examined the results of 863 assays for Giardia and Cryptosporidium on the effluent of wastewater treatment facilities and found that county of collection, watershed of collection, and laboratory analyzing the sample have the most significant impact on the detection of Cryptosporidium oocysts and Giardia cysts in wastewater effluent and that there were minimal but significant differences in method of treatment and method of filtration. To date no other comprehensive analysis of this data has been done.

Studies using Mongolian gerbils found that during a primary infection with Giardia lamblia trophozoites, disaccharidase activities were decreased from day 10 post-infection (p.i.) until well past elimination of the parasite. However, during a challenge infection, enzyme deficiencies were short-lived. A challenge with a soluble extract of G. lamblia trophozoites also resulted in reductions in disaccharidase activity. The degree of these reductions in enzyme activity was dependent on the extract dose. Gel filtration of the trophozoite crude extract resulted in fractions F1, F2, and F3. However, only a challenge with F1 led to disaccharidase deficiencies. Further separation of F1 resulted in fractions F1a and F1b. Impairments of enzyme activity were obtained only in gerbils challenged with F1b. Protein analysis of F1b revealed several high and low molecular weight bands. When gerbils previously exposed to G. lamblia were challenged with an extract of Entamoeba histolytica trophozoites, disaccharidase activities remained comparable to controls. Moreover, enzyme levels in gerbils challenged with excretory/secretory G. lumblia products were affected in a manner which was inconsistent with the live parasitic challenge. Results suggest that the disaccharidase deficiencies in giardiasis are parasite-specific and are induced by a heat-stable constituent(s) of fraction F1b, possibly through an immune response to an antigenic component of this parasite fraction.