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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Introdução Giardia intestinalis é um dos principais protozoários flagelados causadores de diarreia em humanos e animais, sendo um micro-organismo re-emergente, responsável por vários surtos de veiculação hídrica em nível mundial, razão pela qual tem merecido atenção das autoridades de Saúde Pública, e deve ser avaliado e monitorado, principalmente devido ao seu impacto negativo na qualidade do abastecimento público, em particular através do esgoto. Através de técnicas de biologia molecular é possível caracterizar e genotipar cistos presentes no esgoto, e identificar a circulação dos agrupamentos A e B, patogênicos para o homem. Neste estudo foram avaliadas amostras de esgoto bruto de cidades cosmopolitas da América Latina, no Estado de São Paulo, e em Lima, Peru. Objetivos Detectar através da técnica de reação em cadeia pela polimerase (PCR) a presença de G. intestinalis e os agrupamentos A e B, de importância para a saúde humana, em amostras de esgoto bruto provenientes do Estado de São Paulo, Brasil e de Lima, no Peru. Material e Métodos Um total de 18 amostras de esgoto bruto provenientes de portos, aeroportos e estações rodoviárias, com alto trânsito de pessoas de cinco municípios do estado de São Paulo foram coletadas pela técnica de Moore. Adicionalmente, 10 amostras provenientes de dois bairros (urbano e semi-urbano) na entrada da ETE de Carapongo, Lima, no Peru foram coletadas e concentradas por centrifugação. O DNA genômico foi extraído utilizando kit comercial (QIAamp DNA Stool Mini Kit® - Qiagen, USA). A amplificação do gene gdh (glutamato desidrogenase) de Giardia, foi realizada por nested PCR como descrito por CACCIÓ et al., 2008, e G. intestinalis e seus agrupamentos A e B, por meio de reações de reamplificação por PCR (rePCR). Resultados No Estado de São Paulo 61,1 por cento (11/18) das amostras coletadas foram consideradas positivas para G. intestinalis, e na cidade de Lima 60 por cento (6/10) das amostras foram positivas para G. intestinalis. Os agrupamentos A e B foram obtidos em ambos os países. Conclusões Os achados indicam que na cidade de Lima e no estado de São Paulo, cistos de Giardia associados à giardíase humana estão circulando no esgoto bruto, de modo que a descarga dessa matriz em águas superficiais pode representar perigo para a saúde. Além disso, o diagnóstico proposto possibilita a caracterização molecular de Giardia presente em amostras de esgoto bruto sem a necessidade de sequenciamento ou clonagem, favorecendo as rotinas laboratoriais / Introduction Giardia intestinalis is on of the major flagellated protozoans that cause diarrhea in humans and animals, and a re-emerging microorganism, responsible for several waterborne outbreaks worldwide, therefore it has received attention from public health authorities, and should be evaluated and monitored mainly due to their negative impact on the quality of public supply, in particular through the sewage. Through molecular biology techniques it is possible to characterize and to genotype cysts present in wastewater, and to identify the circulation of Assemblages A and B, that are pathogenic to humans. In this study were evaluated samples of raw wastewater from cosmopolitan cities in Latin America, in the State of São Paulo and in Lima, Peru. Objectives Detect, through the technique of polymerase chain reaction (PCR), the presence of G. intestinalis and its Assemblages A and B, of importance to human health, in samples of raw wastewater from the State of São Paulo, Brazil and Lima, Peru. Material and Methods A total of 18 samples of raw wastewater from harbors, airports and bus stations, with high traffic of people from five municipalities of the state of São Paulo were collected by the technique of Moore. In addition, 10 samples from two districts (urban and semi-urban) at the entrance of the WWTP Carapongo, Lima, Peru, were collected and subjected to centrifugation. Genomic DNA was extracted by using a commercial kit (QIAamp DNA Stool Mini Kit ® - Qiagen, USA). The amplification of the Giardia gdh gene (glutamate dehydrogenase) was performed by nested PCR as described by Cacció et al., 2008 and G. intestinalis and its Assemblages A and B, through PCR reamplification reactions (rePCR). Results In the State of São Paulo 61.1 per cent (11/18) of the collected samples were positive for G. intestinalis, and in the city of Lima 60 per cent (6/10) of the samples were positive for G. intestinalis. Assemblages A and B were obtained in both countries. Conclusions The findings indicated that in the city of Lima and in the state of São Paulo, Giardia cysts associated with human giardiasis are circulating in raw wastewater, so that the discharge of this matrix in surface waters may pose a health hazard. Furthermore, the proposed diagnostic enables the molecular characterization of Giardia present in raw wastewater samples without the need of sequencing or cloning, favoring laboratory routines.