Giardia intestinalis is a protozoan parasite that causes diarrhea, and due to its 200 million human cases each year it has become important to try and reduce the infected. Giardia intestinalis expresses so called variant surface proteins (VSPs) on its surface. VSPs are surface antigens that the immune system can detect and react to. In order to hide from the immune system and protect itself Giardia is able to perform antigenic variation, where it switches the expressed VSP to another one of the 200 VSPs in its genomic repertoire. Understanding the regulation and expression of the VSPs is an important steppingstone towards being able to create a vaccine or a better treatment to try to reduce the negative health effects created by this parasite. The main components that regulate VSPs that are being expressed on the surface are Dicer and Argonaute. They are both involved in the RNA interference (RNAi) machinery that has been suggested to silence the VSPs that are not being expressed post-transcriptionally. The aim of this study is to create mutants of Giardia that will no longer express Argonaute or Dicer and thereby be able to analyze the VSP expression and to see if a difference in expression can be detected. This can lead to an understanding of the regulatory mechanism of antigenic variation. We were able to create mutants that most likely did not express Argonaute or Dicer, however since PCR verification of successful knockout did not work, except for mutant Dicer nr. 12, genome sequencing is needed for verification of all the knockout mutants. When analyzing the VSP expression using immunostaining the only surviving Argonaute mutant and the two Dicer mutants that had gone through an initial limiting dilution showed that there was a significant difference in VSP expression for the Argonaute mutants and one of the Dicer mutants. There was a higher fluorescence detected in the nr. 8 Argonaute mutant and the 12#2 Dicer mutant, which indicate a higher VSP expression for those two strains compared to the Cas9 expressing wild type. Furthermore, fluorescence dots could be detected around the parasites especially for Dicer 12#2 which could indicate that this mutant has a higher turnover of VSPs and is releasing more of them into the surrounding area. Due to time limitations many follow up experiments were not able to be performed, however the results obtained give an important insight into the regulation of VSPs in Giardia intestinalis.

The anaerobic unicellular eukaryotic organism Giardia intestinalis is a worldwide parasite. Giardiasis, the intestinal disease caused by Giardia, is one of the most common parasitic disease in the developed part of the world, that causes health problems not only to humans but also to animals. This organism is also interesting for its many unique cellular features. One of them is the presence of mitosomes - the organelles derived from mitochondria. Analogously to mitochondria, mitosome is limited by two membranes and shares the mode of the protein transport. However, mitosome does not have its own genome and as far as we know, there is only one pathway of the iron-sulfur cluster biosynthesis in this organelle. Using the in vivo enzymatic tagging technique, several novel mitosomal proteins were identified, including GL50803_16424. The protein GL50803_16424 attracted our attention by interacting with components of all mitosomal subcompartments: the outer membrane, the membrane and the matrix. In addition, the expression of HA-tagged GL50803_16424 resulted in the formation of peculiar structures near the mitosomes never seen before in G. intestinalis. Bioinformatic approaches revealed that the GL50803_16424 has domain similar to the myelodysplasia- myeloid leukemia factor 1-interacting protein. Our...

Made available in DSpace on 2016-01-26T12:51:41Z (GMT). No. of bitstreams: 1 eleniralvesmachadogodoy_tese.pdf: 3122950 bytes, checksum: 6689a740cf3688e175794a873791beb4 (MD5) Previous issue date: 2012-05-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Introduction: Giardia duodenalis is the protozoan frequently found in the intestinal infection causing gastroenteritis worldwide. The G. duodenalis is complex specie with at least seven different assemblages. The A and B assemblages have been associated with infections in human as well as in other mammals, while the other assemblages have demonstrated to prefer different animal species. Genotypes C and D was include isolated of dogs; E is related to production animals, such as, goats, pigs, sheeps and bovines and genotypes F and G are exclusive of domestic felines and rats, respectively. Molecular studies had also disclosed that the genotype presents two sub-groups; A-I and A-II and B; BIII and BIV. Objective: The present study objectified to study domestic, the epidemiology of the G. intestinalis genotypes in human beings, canines, domestic felines, ovines, bovines and caprines, specifically in relation to the standards of transmission in the Northwestern region of the São Paulo State, Brazil, and to evaluate the hypothesis of zoonotic infection. Methods: During the period of July 2009 to October 2010 they had been studied fecal samples of 61 animals and 154 human beings proceeding from the city of Araçatuba, State of São Paulo. The feaces samples of the animals had been gotten in the Control center of Zoonoses of the city and in the Veterinarian Hospital of Universidade Estadual Paulista Julio de Mesquita Filho . The human fecals specimens had been collected in day-care centers in the periphery of the city and laboratories of Clinical Analysis of the private net of the city. The parasitologic diagnosis was made by optic microscopy, by the techniques of Faust and Hoffmann, Pons & Janer. The genotypes of G. intestinalis had been characterized by PCR-RFLP and confirmed by sequencing analysis of the ß-giardin gene. Results: The human beings samples had shown a positivity of 25.3% (39/154), of these, the percentage in children was of 26.8% (36/134), and in adults got 15% of positive samples (3/20). The frequency of G. intestinalis between the studied animals was of 23% (14/61). A total of 32 isolated of G. intestinalis obtained from human beings feces and 6 of the dogs and cats had been characteristic only of the genotype A (AI and AII/AIII). Conclusions: Regarding the Giardíase frequency, our results are similar to the majority of described percentages in children and adults from São Paulo State and other States of Brazil. The prevalence observed in the animal population is in accordance with the described worldwide. The present study was made possible the detention of the considered genotypes zoonotics of G. intestinalis, circulating between the domestic and human animals of the city of Araçatuba, inferring the possibility of zoonotic transmission of the parasite in the northwest region of the State of São Paulo. The absence of these genotypes in production animals offers the perspective that those are not involved in the chain of transmission to the man in the same locality. / Introdução: Giardia intestinalis tem sido associada a episódios diarréicos em inquéritos epidemiológicos. Pesquisas moleculares evidenciaram que G. intestinalis apresenta sete genótipos: A, B, C, D, E, F e G. Somente os genótipos A e B foram detectados em humanos, mas também em outros hospedeiros mamíferos, sendo considerados, portanto zoonóticos. Genótipos C e D incluem isolados de cães; E é relacionado a animais de produção; porcos, ovelhas, bovinos e caprinos e os genótipos F e G são exclusivos de felinos domésticos e ratos, respectivamente. Estudos moleculares também revelaram que o genótipo A apresenta dois subgrupos; A-I e A-II e B; BIII e BIV. Objetivo: O presente trabalho objetivou estudar a distribuição dos genótipos de G. intestinalis em humanos, caninos, felinos domésticos, ovinos, bovinos e caprinos, especificamente em relação aos padrões de transmissão no noroeste do Estado de São Paulo, Brasil, e avaliar a hipótese de infecção zoonótica. Métodos: No período de julho de 2009 a outubro de 2010 foram estudadas amostras fecais de 61 animais e 154 humanos provenientes do município de Araçatuba, Estado de São Paulo. As amostras de fezes dos animais foram obtidas no Centro de Controle de Zoonoses do município e no Hospital Veterinárío da Universidade Estadual Paulista Julio de Mesquita Filho . Os espécimes fecais humanos foram coletados em creches na periferia da cidade e em laboratórios de Análises Clínicas da rede privada do município. O diagnóstico parasitológico foi feito por microscopia óptica, pelas técnicas de Faust e Hoffmann, Pons & Janer. Os genótipos de G. intestinalis foram caracterizados por PCR-RFLP e confirmado por seqüenciamento do gene ß-giardina. Resultados: As amostras humanas mostraram uma positividade de 25.3% (39/154), destas, a percentagem em crianças foi de 26.8% (36/134), já em adultos obteve-se 15% de amostras positivas (3/20). A frequência de G. intestinalis entre os animais estudados foi de 23% (14/61). Um total de 32 isolados de G. intestinalis obtidos a partir de fezes humanas e seis de cães e gatos foram característicos apenas do genótipo A (AI e AII/AIII). Conclusão: Com relação à frequencia da Giardíase, nossos resultados são semelhantes à maioria das porcentagens descritas em crianças e adultos do Estado de São Paulo e de outros Estados do Brasil. A prevalência observada na população animal está de acordo com as taxas de infecção mundiais descritas. Foram detectados genótipos considerados zoonóticos de G. intestinalis, circulando entre os animais domésticos e humanos da cidade de Araçatuba, inferindo a possibilidade de transmissão zoonótica do parasito na região noroeste do Estado de São Paulo. A ausência destes genótipos em animais de produção oferece a perspectiva de que os mesmos não estão envolvidos na cadeia de transmissão ao homem na mesma localidade.

Introducción: Giardia intestinalis es el protozoario intestinal más común a nivel mundial y su diagnóstico parasitológico está basado en el examen microscópico, sin embargo, debido al carácter intermitente de la excreción del parásito en las heces el método puede revelar baja sensibilidad, esto ha motivado la búsqueda de nuevas alternativas de diagnóstico entre las que destacan aquellas que tiene como base la biología molecular. Objetivos: Evaluar 3 primeros para la detección molecular de G. intestinalis en muestras fecales. Diseño: Se realizó un estudio observacional, de corte transversal. Lugar: Instituto de Medicina Tropical “Daniel A. Carrión”, UNMSM. Procedimiento: Se evaluaron primers que amplifican las regiones de la beta-giardina y de la proteína de choque térmico 70 del ADN de G. intestinalis. Principales medidas de resultados: Se recolectó muestras fecales positivas y negativas a G. intestinales y a otros parásitos, las cuales fueron concentradas por centrifugación, luego almacenadas a -20°C y posteriormente analizadas mediante la técnica de PCR convencional. Resultados: Se estableció una temperatura de hibridación de 60°C para los primers de la beta-giardina y la proteína de choque térmico 70. La mezcla de reacción se estandarizó con las siguientes condiciones: Cl 2 Mg 1.5 mM, primers 0.6 µM, dNTPmix 0.3 mM y taq polimerasa 0.75 U. El límite de detección de los primeros fue de 87.3 ng/µL para beta-giardina, 359.5 ng/µL para GHSP70-1 y 24.1 ng/µL para GHSP70-2. Conclusiones: Se estableció una temperatura de hibridación y concentración de cloruro de magnesio común para los primers. Se observó un mejor límite de detección para el primer GHSP70-1 identificándose bandas en 7 diluciones con una sensibilidad y especificidad mayor que para el primer de la beta-giardina. / Introducción: Giardia intestinalis is the most common intestinal protozoan worldwide and its parasitologic diagnosis is based in microscopic examination; nonetheless, due to the intermittent parasites excretion in the feces, this method could reveal low sensitivity, this has motivated the search of new diagnostic alternatives such as those based on molecular biology. Goals: To assess 3 primers for the molecular detection of G. intestinalis in stool samples. Design: an observational, cross-sectional study was implemented. Settings: Tropical Medicine Institute “Daniel A. Carrión”, UNMSM. Procedures: We assessed primer that amplifies beta-giardin and heat-shock protein 70 of the G. intestinalis. Main measures of: Positives and negatives stool samples for G. intestinalis and for other parasites were collected and then concentrated by centrifugation and stored at -20°C for further analysis using conventional PCR. Results: A 60°C hybridization temperature was established for the primers of beta-giardin and the heat-shock protein 70. The master mix was standardized with the following conditions: 1.5mM Cl2Mg, 0.6 uM primers, 0.3mM dNTPmix and 0.75U Taq polimerasa. Limit detections were 87.3 ng/µL for beta-giardin, 359.5 ng/µL for GHSP70-1 and 24.1 ng/µL for GHSP70-2. Conclusions: We established a common hibridization temperature and a magnesium chloride common for the primers. A better detection limit was established for the primer GHSP70-1, identifying bands in seven dilutions with sensitivity and specificity higher for the beta-giardine primer. keywords: Giardia intestinalis, PCR, beta-giardina, heat-shock protein 70.

On two farms in a two-year period (spring 2005,autumn 2005, spring 2006, autumn 2006) calves faecal samples from the rectum or from the floor were obtained for parasitologic examination. A total of 560 samples from 209 calves were examined using the floatation method in Sheather's sugar solution. In positive samples the presence of Giardia intestinalis cysts, Cryptosporidium parvum, C. andersoni and Eimeria spp. oocysts was found. The infection by C. parvum was the highest from the 1st to the 3rd week and the infection by C. andersoni was the highest from the 7th to the 8th week of the calves age. In both breedings Coccidia Eimeria spp. was the most diagnosed, followed by G. intestinalis. With calves bred in stables by two a higher probability of parasitic infection was detected (46,9 %) compared to breeding of calves in outer individual boxes (17,6). The excrements of the calves were mainly of pasty or even of mushy consistence and the prevalence of the parasites was in most cases diagnosed in single infections.

Intestinal infections with pathogenic protozoa may cause severe disease and remain a neglected problem in regions with inadequate sanitation and hygiene standards, especially in sub-Saharan Africa. Unfortunately, very little data about the prevalence of these infections in risk groups exist from the region. The present study was therefore conducted to assess the prevalence of intestinal protozoan infections in a representative population sample on Ijinga Island, north-western Tanzania. Methods: This was a cross-sectional study which was carried out in 2016 as part of the on-going SchistoControl pilot project on Ijinga Island, north-western Tanzania. A single stool sample was collected from 357 participants and examined microscopically for presence of trophozoites or cysts of intestinal protozoan parasites. In addition, real-time polymerase chain reaction (qPCR) was used to determine the species of intestinal protozoa. Results: Based on microscopy and qPCR, the prevalence of Giardia intestinalis infection was 12% and 15.1%, respectively. Based on microscopy, the prevalence of Entamoeba histolytica/Entamoeba dispar was 26.1%. However, through species identification using qPCR, 21.8% of the study participants were carrying non-pathogenic E. dispar and none of them was infected with E. histolytica. Conclusion: Intestinal protozoan infections are common among the population in the study area. The detection of these infections in different age groups indicates a poor hygienic standard in the community. Improvement in water, sanitation, hygiene and public health education on hand washing will help in controlling these infections. / Intestinale Infektionen mit pathogenen Protozoen können schwere Erkrankungsbilder verursachen und stellen eine oftmals vernachlässigte Problematik, vor allem in Regionen mit geringen Sanitär- und Hygienestandards, dar. Dies betrifft insbesondere südlich der Sahara gelegene Länder. Hier existieren leider keine oder nur unzureichende Prävalenzdaten, was zum Teil auch auf die inadäquaten diagnostischen Möglichkeiten zurückzuführen ist. Die Übertragung erfolgt fäkal-oral über verunreinigte Nahrungsmittel sowie kontaminiertes Trinkwasser und kann symptomatische und asymptomatische, akute und chronische Infektionen hervorrufen. Methoden: Es handelt sich um eine Querschnittsstudie, die 2016 im Rahmen des laufenden SchistoControl-Pilotprojekts auf der Insel Ijinga im Nordwesten Tansanias durchgeführt wurde. Es wurden Stuhlproben von 357 Teilnehmern gesammelt und mikroskopisch auf Trophozoiten oder Zysten von intestinalen Protozoen untersucht. Zusätzlich wurde die Realtime-Polymerasekettenreaktion (qPCR) zur Speziesdifferenzierung verwendet. Ergebnisse: Basierend auf Mikroskopie und qPCR betrug die Prävalenz von Giardia intestinalis 12% bzw. 15,1%. Basierend auf der Mikroskopie betrug die Prävalenz von Entamoeba histolytica / Entamoeba dispar 26,1%. Durch Speziesdifferenzierung mittels qPCR waren jedoch 21,8% der Studienteilnehmer positiv für E. dispar und keiner positiv für E. histolytica. Schlussfolgerung: Intestinale Protozoeninfektionen sind in der Bevölkerung des Studienortes häufig. Der Nachweis dieser Infektionen in verschiedenen Altersgruppen weist auf einen geringen Hygienestandard in der Gemeinde hin. Eine Verbesserung der Wasser-, Sanitär-, Hygienestruktur und des Gesundheitsbewusstseins wird zur Kontrolle dieser Infektionen beitragen.

Advances in DNA sequencing led to a technological breakthrough, that allowed analyzis of complete genomes including those of parasitic protists Trichomonas vaginalis and Giardia intestinalis . These organisms are studied not only for their clinical importance, but also from the evolutionary point of view for their adaptation to anaerobic environment. Genome sequencing and annotations of predicted proteins alone did not bring detail view into functioning of their mitochondrion related organelles ­ in G. intestinalis mitosomes, not­participating in energetic metabolism, in T. vaginalis hydrogenosomes, producing molecular hydrogen and ATP by means of substrate phosphorylation. Traditional methods based on a fractionation by ultracentrifuging in density gradient and subsequent biochemical and enzymological analyzes were extended by one­ and two­dimensional electrophoresis with subsequent identification of proteins by mass spectrometry. Methods of multidimensional separation of peptides produced by specific proteolysis of a complex mixture...

7 ABSTRACT Mitochondria of opisthokonts undergo permanent fusion and fission throughout the cell cycle. Keeping these two processes in balance is vital for various aspects of mitochondrial and cellular homeostasis. Both mitochondrial fusion and division mechanisms are controlled by highly conserved dynamin-related GTPases that are present in all kingdoms of life. The aspects of mitochondrial dynamics outside the opisthokonts is, however, almost completely unexplored phenomenon. In our work, we introduced a tool for live imaging of the reduced forms of mitochondria into model organisms Giardia intestinalis and Trichomonas vaginalis, anaerobic protist parasites from the Excavata supergroup of Eukaryotes. Using this technique, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, of G. intestinalis. The division of mitosomes is restricted to Giardia mitosis and is absolutely synchronized with the process. The synchrony of the nuclear and the mitosomal division persists also during the encystation of the parasite. Surprisingly, the sole dynamin-related protein of the parasite seems not to be involved in mitosomal division. However, throughout the cell cycle mitosomes associate with the...

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