O objetivo deste trabalho foi avaliar o desempenho da remoção de cistos deGiardia spp. e oocistos de Cryptosporidium parvum, em águas de abastecimento com turbidez elevada, em experimentos em escala de bancada (coagulação, floculação, decantação e filtração). Para tanto, empregou-se o coagulante cloreto de polialumínio – PAC. O método de filtração em membranas foi adotado para a concentração de protozoários, seguido ou não da etapa de purificação por separação imunomagnética – IMS. Os métodos foram avaliados em experimentos de controle de qualidade analítica e o método sem IMS apresentou as seguintes porcentagens de recuperação, 80% ±16,32% para cistos de Giardia spp. e 5% ±10,00% para oocistos de C. parvum. O método com IMS apresentou 31,5%±7,55% de recuperação para cistos de Giardia spp. e 5,75%±3,20% de recuperação para oocistos de C. parvum. Os experimentos demonstraram que não houve melhora na remoção de ambos os protozoários na condição de maior dosagem de coagulante (65 mg.L-1 de PAC e pH 7,29). A condição de menor dosagem de coagulante (25 mg.L-1 de PAC e pH 6,76) apresentou um desempenho melhor, ao contrário de uma expectativa de que a maior dosagem de coagulante pudesse favorecer a remoção destes microrganismos. A condição de menor dosagem apresentou, na água filtrada, 50 e 75% de ausência de identificação de cistos de Giardia e oocistos de C. parvum, respectivamente. A condição de maior dosagem apresentou (oo)cistos na água filtrada de todas amostras analisadas. Estes resultados indicam a importância do controle da coagulação na remoção de protozoários. / The aim of this study was to evaluate the performance of Giardia spp. cysts and Cryptosporidium parvum oocysts removal in a bench scale experiment. The coagulant polyaluminium chloride – PACl was used in this research. The protozoa concentration tests were performed by applying the Membrane Filtration method, with and without Immunomagnetic Separation assay-IMS. The methods were evaluated using control experiments and the method without IMS had the following percentage recovery, 80% ± 16.32% and 5% ±10.00% for Giardia cysts and C. parvum oocysts, respectively. The method with IMS presented 31.5% ± 7.55% and 5.75% ± 3.20% of percentage recovery for Giardia cysts and C. parvum oocysts, respectively. Bench scale experimental results have clearly shown that there was no improvement in protozoa removal using the superior dosage of coagulant. The inferior dosage condition (25 mg.L-1 of PACl and pH 6,76) performed better, which was contrary to what was expected in which a superior dosage of coagulant could favour when removing microorganisms. The inferior dosage condition presented 50% and 75% of absence of Giardia cysts and C. parvum oocysts in the final water, respectively. The second coagulation condition (65 mg.L-1 of PACl and pH 7,29) presented protozoa (oo)cysts in the final water of all the samples examined. These results indicate the importance of coagulation control in protozoa removal.

Made available in DSpace on 2014-08-20T14:31:31Z (GMT). No. of bitstreams: 1 dissertacao_ana_berne.pdf: 2199093 bytes, checksum: 8e27f9fa1c22e061e919175c81aa8bb3 (MD5) Previous issue date: 2007-03-30 / The enteroparasitosis remains as an important public health problem in children in the Brazil, showing variable prevalence, according to the State and evaluated population. Studies with day-care center children are scarce, however, already knows that the exposure of the children in these places increased the susceptibility to parasitosis. Among the parasitosis the protozoary Giardia lamblia is responsible for severe diarrhea cases in children and the routine diagnosis methods presents many false negative results. The aim of this study was investigate the enteroparasitosis prevalence in children from a day-care public center of Rio Grande county, Rio Grande do Sul State and compare diagnosis techniques in samples of their fecal material to Giardia lamblia , the ELISA immunoassay and the centrifugal-sedimentation methods. 165 fecal samples where evaluated and processed by centrifugal-sedimentation and centrifugal-flotation methods, stained by trichromium and Kinyoun after the concentration by centrifugal-sedimentation. The general prevalence of enteroparasitosis was 64,2% (106/165). The most prevalent nematods species founded was Trichuris trichiura (24,2%) and Ascaris lumbricoides (22,4%) and the the most prevalent protozoary specie was Giardia lamblia (30,3%). The presence of opportunists coccids where also registered Cryptosporidium spp. (2,4%) and Isospora belli (0,6%). Among the positives 56,6% (60 samples) showed simple infection and 43,4% (46 samples) showed associated infection. The presence of non pathogenic protozoary like Entamoeba coli (15,2%), Endolimax nana (3,6%) and Enteromonas hominis (4,8%), indicated environmental fecal source contamination. The higher prevalence of nematods and protozoary in the studied population suggests the necessity of implementation of educational measures to prevent these enteroparasites. In the evaluation of comparative diagnosis of G. lamblia a higher positivity was verified in the ELISA technique 57% (90/158), followed by the centrifugal-sedimentation method 27,8% (44/158). The obtained results in this study suggests that is higher the prevalence of nematods and protozoary in the evaluated children and the ELISA technique to detect antigen in fecal sample showed higher efficiency to giardiasis diagnosis. / As enteroparasitoses ainda constituem um importante problema de saúde pública em crianças no Brasil, com prevalências bastante variáveis, conforme a região e população avaliada. Estudos com crianças que freqüentam creches são escassos, entretanto, sabe-se que nestes ambientes, as crianças estão mais expostas as parasitoses, dentre as quais o protozoário Giardia lamblia que é responsável por quadros graves de diarréia em crianças e os métodos de rotina utilizados no diagnóstico levam a muitos casos de falso-negativos. O objetivo deste trabalho foi investigar a prevalência de enteroparasitos em crianças de uma creche pública do Rio Grande, cidade portuária, localizada na região sul do estado do Rio Grande do Sul e comparar a técnica de ELISA (kit comercial Giardia II) com os métodos de centrífugo-flutuação e centrífugo- sedimentação para o diagnóstico de G. lamblia em fezes de crianças. Primeiramente foram avaliadas 165 amostras de fezes de processadas pelo método de centrífugo-sedimentação e centrífugo-flutuação e pelas colorações de tricrômio e de Kinyoun. A prevalência geral de enteroparasitos foi de 64,2% (106/165). Os nematódeos mais prevalentes foram Trichuris trichiura (24,2%) e Ascaris lumbricoides (22,4%) e o protozoário mais prevalente foi G. lamblia (30,3%). Também foram registradas as presenças dos coccídeos oportunistas Cryptosporidium (2,4%) e Isospora belli (0,6%). Dentre os positivos, 56,6% (60) apresentaram infecção simples e 43,4% (46) associadas. Constatou-se também a presença de protozoários não patogênicos, como Entamoeba coli (15,2%), Endolimax nana (3,6%) e Enteromonas hominis (4,8%), que indicou contaminação de origem fecal do ambiente. A alta prevalência de nematódeos e protozoários na população estudada sugere a necessidade de implementação de medidas educacionais, visando a prevenção destes enteroparasitos. Na avaliação do diagnóstico comparativo de G. lamblia foi verificado maior positividade para a técnica de ELISA, 57% (90/158), seguido do método de centrifugo-flutuação, 30,3% (48/158) e centrífugo-sedimentação, 27,8% (44/158). A partir dos resultados obtidos no presente estudo pode-se concluir que é alta a prevalência de nematódeos e protozoários nas crianças avaliadas e que a técnica de ELISA para detectar antígenos nas fezes é mais eficiente que os métodos de centrífugo-flutuação e centrifugo-sedimentação, podendo, portanto, ser utilizada, tanto no diagnóstico individual como em estudos epidemiológicos da giardíase.

Parasiten durchlaufen in ihrem Lebenszyklus morphologisch verschiedene Stadien. Die Kontrolle des Übergangs zwischen den Stadien kann die Transmission in einen neuen Wirt begünstigen. Bei vielen Parasiten ist unbekannt, welche Faktoren die Progression des Lebenszyklus beeinflussen. Der Ablauf kann genetisch prädeterminiert sein (kanalisiert) oder von äußeren Einflüssen abhängen (phänotypische Plastizität). Hier wurde die Progression des Lebenszyklus zweier Darmparasiten in Mäusen untersucht. Die Oozysten von Eimeria falciformis wurden quantifiziert und die Transkriptome von Parasit und Wirt wurden in Mäusen unterschiedlicher Immunkompetenz analysiert. Wenngleich erwartet wurde, dass die Immunantwort einen Stressor für das Pathogen darstellt, hatte die Immunkompetenz des Wirts keine Auswirkungen auf den Zeitpunkt der Oozystenausscheidung und das Transkriptomprofil des Parasiten. E. falciformis konnte nicht von der Immunschwäche des Wirtes profitieren; ist also hinsichtlich der Immunantwort des Wirts genetisch kanalisiert. In G. duodenalis wurde untersucht, inwiefern die Progression des Lebenszyklus, d.h die Trophozoitenreplikation bzw. die Zystenausscheidung, von Arginin abhängt. Die Replikation der Trophozoiten war nicht von Arginin aus der Nahrung abhängig; die Ausscheidung infektiöser Zysten war unter argininarmen Bedingungen jedoch verringert. Dies lässt vermuten, dass der Ablauf des Lebenszyklus von G. duodenalis, insbesondere die Enzystierung, an die Argininzufuhr gekoppelt ist. Die Umstellung des Metabolismus von G. duodenalis hin zur Produktion eines wichtigen Zystenwandbestandteils wird hier als mechanistische Verbindung zwischen ATP-Erzeugung aus Arginin in Nichtsäugetieren (Arginindihydrolase-Stoffwechselweg), verringerter Glykolyse und der Zystenwandsynthese erörtert. Somit könnte Arginin als Stimulus für phänotypische Plastizität bei der Enzystierung von G. duodenalis dienen. / Eukaryotic parasites have life cycles with morphologically distinct stages. Accurate timing of the conversion from one stage into another can be beneficial for transmission into a new host. Often little is known about determinants for such life cycle progression or the genes involved. Timing can be genetically pre-determined (canalized) or depend on exposure to a stimulus (phenotypic plasticity). Here, life cycle progression of two unicellular intestinal parasites was investigated in mice. For Eimeria falciformis, oocyst stage parasites were quantified, and parasite and host transcriptomes analyzed in differently immune competent hosts. Host immune response stimuli are expected to induce stress on the pathogen, but different host immune competences did not change the timing of oocyst shedding or influence parasite transcriptome profiles. E. falciformis was unable to benefit from hosts with weakened immune responses. It is therefore an example of a genetically canalized parasite with regards to host immune stimulus. In Giardia duodenalis, dependence on arginine for life cycle progression was investigated. The in vivo relevance for parasite replication is unknown. Trophozoite stage replication and cyst shedding were assessed in hosts fed normal and arginine-free diets. G. duodenalis did not depend on dietary arginine for trophozoite replication, but infective cysts were reduced in number under arginine-poor conditions. Dependence on arginine for life stage switching suggests that G. duodenalis could time progression by encysting upon arginine exposure. G. duodenalis metabolic reprograming to generate a major cyst wall component is discussed as a strategy to mechanistically link 1) non-mammalian ATP generation (arginine dihydrolase pathway) from arginine with 2) decreased glycolytic flux and 3) cyst wall generation. Therefore, arginine may be an external stimulus for phenotypic plasticity of encystation in G. duodenalis.

L’augmentation des interactions entre humains et animaux sauvages en lisière des habitats naturels pourrait faciliter la transmission d’agents pathogènes entre les humains et les différentes espèces animales d’un écosystème et ainsi favoriser l’émergence de maladies. Nous avons effectué une étude transversale portant sur l’infection par Giardia et Cryptosporidium chez les humains, les animaux domestiques, les rongeurs et les lémuriens au sein de l’écosystème de Ranomafana, Madagascar. Des échantillons de fèces ont étés collectés de manière non invasive chez des personnes volontaires, des mammifères domestiques et des rongeurs introduits habitant trois villages situés en lisière du Parc National de Ranomafana (PNR) ainsi que quatre espèces de lémuriens (Propithecus edwardsii, Prolemur simus, Eulemur rubriventer et Microcebus rufus) du PNR. Des analyses coproscopiques par la technique d’immunofluorescence directe ont été réalisées afin de détecter la présence de Cryptosporidium et Giardia. Leur prévalence a été estimée et certaines variables reliées à l’infection par les parasites ont été identifiées. Cryptosporidium et Giardia ont été détectés avec une prévalence estimée à 22,9 % et 13,6 % respectivement chez les humains. La prévalence de ces deux parasites variait de 0 % à 60 % chez les animaux domestiques et les rongeurs au sein des villages. L’espèce hôte, l’âge ainsi que la co-infection par un autre protozoaire sont les seules variables associées à l’infection par Cryptosporidium et Giardia dans cet écosystème tandis qu’aucune association avec une coinfection par un ordre de nématode n’a été détecté. De plus, Cryptosporidium a été détecté chez 10,5 % des lémuriens du PNR. Cette étude documente pour la première fois la présence de Cryptosporidium chez deux espèces de lémuriens du PNR. Par contre, Giardia n’a pas été détecté dans les échantillons issus de lémuriens du PNR. / Increasing human activities in the vicinities of natural habitats may facilitate the emergence and transmission of diseases between humans and domestic animals and wildlife species. We conducted a cross-sectional study investigating the prevalence of Giardia and Cryptosporidium, two ubiquitous and potentially zoonotic protozoan parasites in various populations of humans and animals from the Ranomafana National Park ecosystem (RNP), Madagascar. Fecal samples were obtained non-invasively from human volunteers, domestic animals and introduced rodents inhabiting three villages in the vicinity of the national park and from four species of free-ranging lemurs (Propithecus edwardsi, Prolemur simus, Eulemur rubriventer and Microcebus rufus) from the RNP. Samples were analyzed using the direct immunofluorescence technique. Prevalences of Giardia and Cryptosporidium were estimated and variables associated with infections by the protozoa were identified. Cryptosporidium and Giardia were detected with a prevalence of 22.9 % and 13.6 % in humans respectively. The prevalences of these two parasites varied from 0 % to 60 % in domestic animals and introduced rodents from the villages. Species, age category and co-infection with the other protozoan were significantly associated with the infection by Cryptosporidium and Giardia in this ecosystem, whereas coinfections by different helminths order were not significantly associated with Cryptosporidium or Giardia. Moreover, Cryptosporidium was detected in 10.5 % of lemurs sampled from the RNP. This study reports for the first time the occurrence of Cryptosporidium in two species of lemurs from the RNP. Giardia was not detected in fecal samples from lemurs inhabiting the RNP.

Foodborne illnesses are a significant public health challenge in the United States, with an estimated 9.4 million illnesses annually attributed to the consumption of contaminated food, of which 59% are estimated to be caused by viruses, 39% by bacteria and 2% by parasites. Timely detection and identification of the pathogens causing foodborne outbreaks is vital for the implementation of outbreak control strategies, allowing public health officials to prevent additional illnesses and maintain confidence in the food supply. Public health laboratories employ a variety of traditional and molecular testing techniques to identify foodborne outbreak etiologic agents. One technology is the Luminex XMap® microsphere system, which is also marketed as the Bio-Plex™ 200. This platform has a multiplexing capability with the potential to simultaneously detect up to 100 targets in one reaction. The studies described here show that the combination of two Bio-Plex assays with real-time virus assays and one extraction method provides a flexible foodborne outbreak screening algorithm that potentially identifies an outbreak-associated pathogen on the first day of specimen submission and aids in focusing confirmatory laboratory testing. In these studies, two microsphere-based assays were designed for use on the Bio-Plex 200 system as screening assays for the detection of four enteric protozoa (Giardia intestinalis, Cyclospora cayetanensis, Cryptosporidium parvum, Entamoeba histolytica) and six virulence determinants of shiga toxin-producing Escherichia coli (STEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC) and Shigella spp. Precision and limits of detections were established for both assays. The sensitivity and specificity of the protozoan assay as compared to reference methods ranged from 81.25% to 100% for most targets, while sensitivity for the E. histolytica target was 42.86%. Sensitivity and specificity for the bacterial assay was 100% as compared to reference methods. However, cross-reactivity of the protozoan assay E. histolytica target with E. dispar and of the bacterial assay uidA target with enteropathogenic E. coli strains was noted. Additionally, real-time detection of norovirus and rotavirus nucleic acids extracted with the QIAamp DNA Stool Mini Kit was statistically comparable to detection when extracted with the Ambion® MagMAX™-96 Viral RNA Isolation Kit combined with the KingFisher® Magnetic Particle Processor.

Giardia duodenalis é um protozoário entérico de distribuição mundial responsável por causar a giardíase em uma grande variedade de mamíferos, incluindo os humanos. É considerada uma espécie complexa, no qual os isolados podem ser classificados em sete agrupamentos genéticos distintos apesar de serem morfologicamente indistinguíveis. O presente trabalho teve como objetivo avaliar a variabilidade genotípica de isolados de G. duodenalis provenientes de humanos naturalmente infectados, residentes em cidades do litoral de São Paulo, na Baixada Santista. A caracterização molecular de 43 isolados pelo seqüenciamento parcial de genes codificadores da enzima glutamato-desidrogenase (gdh) e da proteína beta-giardina (bg) mostrou que o assemblage B da G. duodenalis é o mais freqüente na região litorânea, ocorrendo em 53,5% (n=23) das amostras, sendo o assemblage A identificado em 34,8% (n=15). A maioria das seqüências obtidas pelo gene gdh se mostrou polimórfica, caracterizada por picos duplos de nucleotídeos em algumas posições em cromatograma e quando a análise dos dois genes foi combinada cinco isolados apresentaram identidades diferentes. A esses fenômenos duas explicações são atribuídas, infecção mista ou heterozigose de seqüência alélica. As análises filogenéticas mostraram que o gene bg é mais conservado que o gene gdh, não sendo capaz de discriminar os sub-agrupamentos que constituem os Assemblages do parasita. Com base nos resultados apresentados podemos concluir que a participação de genótipos zoonóticos é relevante na epidemiologia das giardíases nos indivíduos residentes das cidades litorâneas do estado de São Paulo e que estudos de caracterização molecular da G. duodenalis são indispensáveis para melhor conhecimento da epidemiologia desta infecção. / Giardia duodenalis is an enteric protozoa of global distribution responsible for causing giardiasis in a wide range of mammals including humans. Is considered complex specie in which the isolates can be classified in different genetic groups despite to be morphologically indistinguishable. The purpose of this study was evaluating the genetic variability of G. duodenalis isolates from humans naturally infected residents in the coast cities of Sao Paulo, in Baixada Santista. The molecular characterization of 43 isolates using the gene encoding glutamate-desidrogenasi enzyme (gdh) and beta-giardin protein (bg) showed that the assemblage B is the most common in the coast region, occurring in 53.5% (n=23) samples, the assemblage A was identified in 34.8% (n=15). Most sequences obtained by the gdh gene showed polymorphism, characterized for double peaks of nucleotides in some chromatogram positions and when the analysis of two genes was combined five isolates were differently identified. For this phenomena can be attributed two explanations, mixed infections or heterozygosis allelic sequence. The phylogenetic analysis showed that bg gene is more conserved than gdh gene, not being able to discriminate the sub-groups of parasite assemblages. Based on the presented results we can conclude the participation of zoonotic genotypes is important in the epidemiology of giardiasis in residents of the coast cities of Sao Paulo state and molecular characterization studies of G. duodenalis are essential for better understanding the epidemiology of this infection.

Use of tubular anaerobic digesters to treat livestock waste in developing countries has energy, agricultural, health, social and environmental benefits. However, careful use of digester effluent as a soil amendment is required due to the potential presence of protozoan parasites Cryptosporidium parvum and Giardia lamblia. This research investigated the performance of four tubular digesters in the Monteverde region of Costa Rica. High (>75%) volatile solids and BOD5 removal efficiencies were observed, which was attributed to the formation of a biologically active floccular sludge layer. Computational fluid dynamics (CFD) and bioprocess models were developed to evaluate the transport and transformation mechanisms in the digesters. The CFD model estimated a mean liquid hydraulic residence time (HRT) of 23 days and the bioprocess model estimated an average mean cell residence time (MCRT) of 115 days. Cryptosporidium parvum and Giardia lamblia inactivation studies were performed in the laboratory under conditions similar to the environmental conditions observed in the field tubular digesters. The environmental conditions included: ambient temperatures (21-24°C), neutral pH and total ammonia nitrogen (TAN) concentrations below 250 mg NH4+-N/L. Inactivation rate constants for Cryptosporidium parvum and Giardia lamblia were 0.056 and 0.726 day-1, respectively. An (oo)cysts solid-liquid phase distribution study indicated that 70% of both (oo)cysts adhered to biosolids. A tubular digester model was used to estimate the concentration of viable (oo)cysts in the digester effluents. (Oo)cysts adhesion to solids, total solids concentration in the digester and HRT were the main factors contributing to the modeled effluent concentration of viable (oo)cysts. Since the model predicted presence of viable (oo)cysts in the tubular digester effluent, a quantitative microbial risk assessment (QMRA) model was developed to estimate the risk of infection from exposure to raw livestock waste and tubular digester effluents in two rural communities in Costa Rica. The risk of infection from Cryptosporidium parvum and Giardia lamblia was assessed for occupational and public exposure pathways; fomite and soil contamination and crop contamination from runoff. Results from the QMRA indicated that the concentration of (oo)cysts in the raw livestock waste, inactivation rates at the various exposure pathways and the treatment of livestock waste were the main contributing factors to the risk of infection. This research indicated that treatment of livestock waste in tubular digesters significantly decreased the risk of infection to below WHO’s acceptable individual annual risk of infection (10-4). This is the first study to combine mathematical modeling with field studies to determine the physical and biological processes in tubular digesters. This is also the first study to combine mathematical models with field and laboratory studies to determine the concentration of (oo)cysts in tubular digester effluents and to predict the risk of infection from Cryptosporidium parvum and Giardia lamblia if tubular digester effluent is used as a soil amendment.

Agriculture is key to New Zealand�s economy with land-use conversions in response to market forces occurring regularly. Recently, high-intensity dairy farming has replaced low-density livestock farming, often degrading surrounding waterways. Of particular concern is that dairy cattle can be a source of the parasite Giardia, which in humans is a common cause of gastrointestinal infection. Thus, this thesis evaluated whether dairy farm conversions posed significant consequences for public health. First I examined the prevalence of Giardia in calves in a rapidly intensifying dairying region of New Zealand. A total of 1190 faecal samples were collected from calves one to seven weeks old during two spring calving seasons and screened by direct immunofluorescent microscopy. Giardia cysts were detected in 31% of samples. To evaluate the potential risk that this environmental source of Giardia posed to the human population, molecular genotyping was used to compare forty Giardia strains isolated from calves with thirty isolates from humans collected in the same region and period. Sequencing the β-giardin gene, Giardia duodenalis assemblages A and B were identified from both hosts, with genotype comparisons revealing substantial overlap of identical genotypes for both assemblages, implying zoonotic transmission. Environmental agencies routinely promote the planting of streamside edges to decrease nonpoint pollution from dairy farms entering waterways. However, current methods for tracking pathogens across farmland and into waterways via surface runoff are limited and typically have been developed using artificially created landscapes. Furthermore, no studies have investigated how Giardia moves across the landscape in farm surface runoff. I developed a field-based tracking method specific for Giardia and used this technique to compare the ability of recently planted vegetation strips with bare soil strips cleared of vegetation at decreasing pathogen concentrations; a typical scenario when planting barriers to reduce waterway contamination. A spike containing a bromide tracer and inactivated Giardia cysts was applied in drip-irrigated surface runoff, with one-minute samples collected from the bottom of the plot. A significant treatment effect was identified for Giardia, with 26% fewer detected in runoff from the planted strip, highlighting the immediate benefit of vegetation planting in removing pathogens. Next I evaluated the effects of four riparian treatments on Giardia runoff: exotic pasture grass and weeds growing in the absence of cattle grazing due to fencing, in comparison to monocultural plantings of three New Zealand native grassland species. Runoff experiments were performed after planting, both prior to and following the main summer growing season. Bromide recovery was high from all four treatments (54 - 99%), with no significant treatment effects. By comparison, Giardia recovery was low (1 - 13%). Prior to summer, two native species reduced Giardia in runoff more than the pasture grass/weed treatment which was almost vegetation-free at this time. After summer, Giardia recoveries were uniformly lower in all treatments. These results demonstrate that after one growing season, fencing waterways produces riparian buffers, via the growth of exotic pasture plants released from grazing, that decrease pathogen concentrations in surface runoff to concentrations indistinguishable from native plantings. Given infectious organisms are known to be in the environment, it is important to assess the risk these pose to human populations. Findings from this research can be used to improve currently available risk-assessment models for Giardia transmission from infected dairy animals via water to humans.

Giardia lamblia is a eukaryotic parasite of the upper small intestine of humans and animals. The infecting trophozoite cells do not invade the epithelium lining of the intestine, but attach to the brush border surface in the intestinal lumen. The giardiasis disease in humans is highly variable. Prior to this study, the molecular mechanisms involved in establishment of infection or cause of disease were largely uncharacterized. In this thesis, the molecular relationship between Giardia and the human host is described. The interaction of the parasite with human epithelial cells was investigated in vitro. Changes in the transcriptome and proteome of the parasite and the host cells, and changes in the micro-environment of the infection have been identified using microarray technology, and 1- and 2-Dimensional SDS-PAGE protein mapping together with mass spectrometry identification. The first large-scale description of cellular activities within host epithelial cells during Giardia infection is included in this thesis (Paper I). We identified a unique activation of the host immune response and induction of apoptosis upon infection by Giardia. Four important virulence factors of the parasite, directly linked to the success of Giardia infection, were characterized and are presented in Papers II and III. The parasite was shown to have immune-modulating capacities, and to release proteins during host-interaction that facilitate the establishment of infection. Additional putative virulence factors were found among Giardia genes transcriptionally up-regulated during early infection (Paper IV). In summary, this thesis provides important insights into the molecular mechanisms of the host-parasite interaction.

As sequencing technologies advance genome studies are becoming a basic tool for studying an organism, and with more genomes available comparative genomics is maturing into a powerful tool for biological research. This thesis demonstrates the strength of a comparative genomics approach on a group of understudied eukaryotes, the diplomonads. Diplomonads are a group of single cell eukaryotic flagellates living in oxygen-poor environments. Most diplomonads are intestinal parasites, like the well-studied human parasite Giardia intestinalis. There are seven different G. intestinalis assemblages (genotypes) affecting different hosts, and it’s under debate whether these are one species. A genome-wide study of three G. intestinalis genomes from different assemblages reveals little inter-assemblage sexual recombination, supporting that the different G. intestinalis assemblages are genetically isolated and thus different species. A genomic comparison between the fish parasite S. salmonicida and G. intestinalis reveals genetic differences reflecting differences in their parasitic lifestyles. There is a tighter transcriptional regulation and a larger metabolic reservoir in S. salmonicida, likely adaptations to the fluctuating environments it encounters during its systemic infection compared to G. intestinalis which is a strict intestinal parasite. The S. salmonicida genome analysis also discovers genes involved in energy metabolism. Some of these are experimentally shown to localize to mitochondrion-related organelles in S. salmonicida, indicating that they possess energy-producing organelles that should be classified as hydrogenosomes, as opposed to the mitosomes in G. intestinalis. A transcriptome analysis of the free-living Trepomonas is compared with genomic data from the two parasitic diplomonads. The majority of the genes associated with a free-living lifestyle, like phagocytosis and a larger metabolic capacity, are of prokaryotic origin. This suggests that the ancestor of the free-living diplomonad was likely host-associated and that the free-living lifestyle is a secondary adaptation acquired through horizontal gene transfers.  In conclusion, this thesis uses different comparative genomics approaches to broaden the knowledge on diplomonad diversity and to provide more insight into how the lifestyle differences are reflected on the genetic level. The bioinformatics pipelines and expertise gained in these studies will be useful in other projects in diplomonads and other organismal groups.