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.

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.

During the transformation of the bacterial endosymbiont into current mitochondria the protein import apparatus had to be created de novo. The reduced mitochondria (mitosomes) of the parasitic protist Giardia intestinalis represent unique cellular model for the examination of these fundamental transport processes. As the main objective of this project I will try to characterize the motor complex, which propels the protein transport, and also the translocation channel in the inner mitosomal membrane. To this aim I will exploit the presence of two membrane components Pam16 and Pam18, which were discovered in our laboratory, and which constitute the functional core of the motor complex. Based on the information from the analogous systems of yeast and mammalian mitochondria, these two components should physically interact with so far unknown translocation channel. In all other eukaryotes this channel is formed by a conserved protein Tim23. The absence of this protein in the genome of G. intestinalis suggests presence of completely novel, or maybe the original-bacterial protein. Having in hand this simplified mitochodrial model the project has potential to bring not only new data in parasite biology but also generate new information on the function and evolution of mitochondrial protein import.

[Abstract not supplied]

The diplomonads are a diverse group of eukaryotic flagellates found in microaerophilic and anaerobic environments. The most studied diplomonad is the intestinal parasite Giardia intestinalis, which infects a variety of mammals and cause diarrheal disease. Less is known about Spironucleus salmonicida, a parasite of salmonid fish, known to cause systemic infections with high mortality. We created a transfection system for S. salmonicida to study cellular functions and virulence in detail (Paper I). The system was applied to explore the mitochondrion-related organelle (MRO) in S. salmonicida. We showed that S. salmonicida possesses a hydrogenosome (Paper II) with a higher metabolic capacity than the corresponding MRO of Giardia, the mitosome. Evolutionary analysis of key hydrogenosomal proteins showed ancient origin, indicating their presence in the ancestral diplomonad and subsequent loss in Giardia. Annexins are of evolutionary interest since these proteins are found across all kingdoms. Annexin-like proteins are intriguingly expanded into multigene families in Giardia and Spironucleus. The annexins of S. salmonicida were characterized (Paper III) with distinct localizations to various cellular structures, including a putative adhesion structure anterior in the cell. The disease-causing Giardia trophozoites differentiate into infectious cysts, a process essential for transmission and virulence of the parasite. Cysts are often spread via contaminated water and exposed to environmental stressors, such as UV irradiation. We studied the survival and transcriptional response to this stress factor (Paper IV) and results showed the importance of active DNA replication machinery for parasite survival after DNA damage. In addition, we studied transcriptional changes along the trajectory of encystation (Paper V), which revealed a coordinated cascade of gene regulation. This was observed for the entire transcriptome as well as putative regulators. Large transcriptional changes appeared late in the process with the majority of differentially regulated genes encoding hypothetical proteins. We studied the localizations of several of these to gain information of their possible function. To conclude, the diplomonads are complex eukaryotic microbes with cellular processes adjusted to match their life styles. The work in this thesis has provided insight of their adaptations, differences and similarities, but also new interesting leads for future studies of diplomonad biology and virulence.

In biennial following (spring 2005, autumn 2005, spring 2006, autumn 2006) we're rasitology examine 288 samples dropping from heifers and 288 samples dropping from bulls. Exhibits we're examined floatation in Sheather´s sugar solution. In examinate dropping we're proved present cysts Giardia intestinalis and oocysts Cryptosporidium andersoni and family Eimeria. In heifers was most frequent parasites coccidia Eimeria sp. (28,5 %). Flagellata Giardia intestinalis occur in 17 % of all designs. At least we're open up Cryptospordium andersoni (9,4 %). In bulls we're noted highest occurrence coccidia Eimeria sp. (19,1 %). Almost same occurrence we're found out near Giardia intestinalis (18,8 %). Very low prevalence we're have observed near Cryptosporidium andersoni (1,4 %).

Giardia intestinalis is a two-staged flagellated protozoan parasite which infects the lumen of the small intestine causing the gastrointestinal disease called giardiasis. This disease infects millions of people per year, commonly caused by inadequate water treatment and proximity to livestock. The main line of treatment against this disease has been metronidazole, a member of the nitroimidazole family of prodrugs, which when activated cause non-specific damage to the proteins and genetic material within anaerobic cells. Until recently, G. intestinalis has shown little resistance to metronidazole, but as resistance climbs it has become clear that genetic regulation is key. This study attempts to investigate a select number of key genes through the use of modified transfected plasmids for overexpression and with knockdown using morpholino oligos. Wild type (WB) G. intestinalis cells, transfected with plasmids modified to both overexpress specific genes and incorporate the human influenza hemagglutinin tag (HA-tag) were seen to change in survival rate when exposed to metronidazole. Localization of a select group of these genes was also found using the HA-tag. Two of these genes (quinone oxidoreductase and nitroreductase 1) had protein folding simulations run in order to be visualizee and compare their structures to closely matching equivalents. Finally, morpholino oligo nucleotides were used to knockdown expression in these two genes, leading to a significant decrease in survival for those targeted against quinone oxidoreductase when exposed to metronidazole.

Giardia intestinalis is a single-celled intestinal parasite infecting humans and animals. The species is divided into eight genetic groups, assemblages, with different host specificity. Stool samples from 99 dogs, 61 cats and 22 chinchillas were examined for the presence of Giardia using microscopy and PCR diagnostics. The found populations were assigned to assemblages using a multi-locus genotyping scheme, with the goal of mapping the occurrence of zoonotic assemblages A and B and evaluating the risk of transmission of Giardia from pets to humans. The Giardia prevalence in examined dogs was 36,4%. The majority of dog infections was caused by dog-specific assemblages D and C. Individual cases of infection with assemblage F, or a mix of assemblages A+D, A+F, B+D, C+D and D+F were also detected. The prevalence in cats was 14.8%, and the dog assemblages C and D prevailed as well. In individual cases, cats were infected with assemblages A or F, which is specific for cats. The highest prevalence, 85.7%, was detected in chinchillas. The majority of chinchilla infections was caused by the zoonotic assemblage B (88.9%). The found sequences were compared to those obtained from animals with clinical giardiasis, but no identical matches were found between these two pools. The nature of mixed infections was studied by...

Giardia intestinalis is a protozoan parasite causing the diarrheal disease called giardiasis that infects millions of people worldwide each year. The life cycle of Giardia intestinalis is characterized by two stages; the sturdy, infectious cyst and the vegetative, motile trophozoite. This project sought to investigate the regulation of the encystation process where trophozoites transform into cysts. Twelve genes with interesting transcriptomic profiles were chosen to study as putative transcription factors and regulators of encystation. These genes were cloned onto a plasmid with a Strep-Tag and transfected into Giardia intestinalis. The protein expression and localization was studied using immunofluorescence microscopy with antibodies against the Strep-Tag at different time points after inducing encystation. Although the project did not fully characterize these genes, protein expression was seen in all cases except two. Some proteins were seen localized to the nuclei and others had a localization pattern similar to the localization of cyst wall protein. In addition, a dramatic phenotype resembling cells going through programmed cell death was observed in one of the transfectants early in encystation and would be interesting to study further. The transfectant strains generated from this project remain interesting candidates to investigate as putative transcription factors.

Gastrointestinal parasites impose a great and often silent burden of morbidity and mortality on poor populations in developing countries. Veron, Dominican Republic (DR), is a rural city in the southeastern corner of the country where many Dominicans and Haitians migrate to for work in support and expansion of the tourist industry of Punta Cana. Few studies of the prevalence of gastrointestinal (GI) parasitic infections have been published in the DR. Presently, there is a high prevalence of gastrointestinal parasitic infections throughout the poorest areas of the DR and Haiti. This study investigated the prevalence of GI protozoan and helminth parasites from children at the Rural Clinic of Veron during 2008. Participants provided a fecal sample that was examined microscopically for protozoan and helminth parasites using the fecal flotation technique to concentrate and isolate helminth ova and protozoan cysts. Of 108 fecal samples examined, 107 were positive for one or more parasites. Participant ages ranged from 2 to 15 years; 52 were males and 56 were females. Percent infection rates were 48.2% for Ascaris lumbricoides, 13.9% for Enterobius vermicularis, 24.1% for Entamoeba histolytica, and 22.2% for Giardia intestinalis. 9.3% had double infections. A survey of subject characteristics and risk factors was completed by each parent/guardian. Any plan to reduce GI parasites in children of this region will require a determined effort between international, national, and local health authorities combined with improved education of schools, child care providers, food handlers, and agricultural workers. A special effort must be made to reach out to immigrants and those not part of the public education system and to address microbial water quality. / Ph. D.