Orientadores: Regina Maura Bueno Franco, Eliete Maria Silva, Jancarlo Ferreira Gomes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T03:30:34Z (GMT). No. of bitstreams: 1 Rebolla_MayraFrozoni_M.pdf: 5078074 bytes, checksum: 0e330cef23d1c66a5672e38240041608 (MD5) Previous issue date: 2012 / Resumo: Parasitoses intestinais são frequentes na população usuária do Sistema Único de Saúde de São Sebastião da Grama, município do interior do estado de São Paulo. Durante a ocorrência de um surto de gastroenterite em uma das creches do município, objetivou-se investigar a ocorrência de enteroparasitos nos trabalhadores e crianças das instituições municipais urbanas de educação infantil, avaliar o desempenho de técnicas diagnósticas na detecção de Giardia duodenalis em amostras fecais provenientes de creche, local de surto de gastroenterite, e implantar um programa de controle desta parasitose, o "Programa de Controle da Giardiose em Creches e Pré-escolas" (PCGCP) a fim de avaliar a adesão de gestores, trabalhadores e famílias das crianças às medidas higiênicas no ambiente escolar infantil. Para tanto se utilizou amostras fecais de 172 crianças e 33 trabalhadores, processadas pelos métodos Three Fecal Test Conventional® e Modified®, diagnosticando-se 89,53 % das crianças e 71,87 % dos trabalhadores positivos para parasitos e comensais. Blastocystis hominis foi o parasito intestinal de maior prevalência entre as crianças (86,62 %) e trabalhadores (65,62 %). Os casos de monoparasitismo foram mais frequentes que os de poliparasitismo entre as crianças e os trabalhadores estudados, bem como a prevalência de protozoários foi maior que a de helmintos. O modelo de regressão logística evidenciou associação significativa entre criança atendida na creche e a frequência de infecção por enteroparasitos (p = 0,01), por G. duodenalis (p = 0,00), por B. hominis (p = 0,02), e pelos protozoários intestinais (p = 0,01). Entre criança menor de um ano e a frequência de infecção por G. duodenalis (p = 0,00), e entre crianças cujos domicílios não possuíam coleta de lixo e a frequência de infecção por helmintos (p = 0,03). Os resultados obtidos utilizando-se os métodos de Faust et al. e ELISA foram concordantes substancialmente, contudo, a presença de resultados falsos positivos verificados no imunoensaio limitam sua aplicabilidade como teste diagnóstico em uma situação de surto. Como ferramenta de controle e prevenção da giardiose, foi implantado e avaliado o PCGCP nestes ambientes do estudo. A adesão e ativo engajamento dos gestores, trabalhadores e famílias das crianças ao PCGCP foi considerada satisfatória, e os resultados sugerem que o treinamento das práticas para se evitar a giardiose deva ser mantido de forma permanente a fim de se alcançar uma efetiva prevenção nos ambientes escolares infantis / Abstract: Intestinal parasites are common in the population using the Unified Health System of São Sebastião da Grama, a municipality in the state of São Paulo. The aims of this study to investigate the occurrence of intestinal parasites in children and workers of urban municipal institutions of early childhood education, to evaluate the performance of diagnostic techniques for detection of Giardia duodenalis in stool samples from daycare centers, where outbreaks of gastroenteritis occur. To implement a program to control this parasite, the "Program of Control of Giardiasis in Day Care Centers and Preschools" (PCGCP), in order to assess the commitment of managers, workers and children?s families to hygienic measures within these school environments. For this purpose we used fecal samples from 172 children and 33 workers, processed using the Conventional and Modified Three Fecal Test® method. As a result, 89,53 % of the children and 71,87 % of workers were diagnosed positive for pathogenic parasites and commensals. Blastocystis hominis was the most prevalent intestinal parasite among children (86,62 %) and employees (65,62 %). Monoparasitism cases were more frequent than those of multiple parasitic infections among children and workers. The prevalence of protozoa was greater than that of helminths. The logistic regression model revealed a significant association between child attended in the day care center and the frequency of infection with intestinal parasites (p = 0,01), by G. duodenalis (p = 0,00), by B. hominis (p = 0,02), and the intestinal protozoa (p = 0,01). Higher indices were found among children under one year of age (frequency of infection with G. duodenalis (p = 0,00)) and among children whose homes had no waste collection (frequency of helminth infection (p = 0,03)).The results obtained using the methods of Faust et al. and ELISA were substantially consistent. However, the presence of false positive results observed in the immunoassay limits its applicability as a diagnostic test in an outbreak situation. As a tool for control and prevention of giardiasis, the program PCGCP was implemented and its adherence was evaluated in the sites investigated, places considered at the scope of this study. The commitment and active engagement of managers, workers and families with children to PCGCP was considered satisfactory, and the results suggest that training practices to avoid giardiasis should be made permanent for an effective prevention in day care centers and preschools environments to be achieved / Mestrado / Parasitologia / Mestre em Parasitologia

Orientador: Regina Maura Bueno Franco / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T08:57:58Z (GMT). No. of bitstreams: 1 BonideOliveira_ClarisseMaria_M.pdf: 6327674 bytes, checksum: 05abbfdb0fdd48f248f326f48096fc33 (MD5) Previous issue date: 2012 / Resumo: O solo, principalmente quando melhorado com matéria orgânica proveniente de lodo de estação de tratamento de esgoto e/ou fezes humanas e de animais, pode se tornar importante fonte de veiculação de doenças por abrigar diversos parasitos. Estes, por serem amplamente dispersos no ambiente, podem gerar problemas de saúde pública. Como o solo é uma das principais rotas de transmissão desses organismos, este trabalho teve como objetivo - a partir dos protocolos descritos na literatura para a detecção de cistos de protozoários e ovos de helmintos em solos - avaliar as metodologias mais eficientes e escolher um método visando a padronização de um protocolo eficaz na detecção de cistos de Giardia e ovos de helmintos. Foram analisadas as variáveis: Homogeneização: vórtex por 2 minutos; agitador magnético por 10 minutos; homogeneização em mixer rotatório durante 30 minutos e homogeneização manual por 5 minutos; Soluções Dispersantes: 1% 7X ICN; glicina 1 M; 0,1M tetrassódio pirofosfato e 1% Tween 40; Filtração: ausência da etapa de filtração e , filtração em peneira, para os ensaios referentes aos ovos; Soluções de Purificação: sulfato de zinco (densidade 1,18), solução saturada de cloreto de sódio; sacarose (densidade 1,33 para protozoários e 1,3 para helmintos) e polietileno glicol 60%, além da separação imunomagnética (apenas para ensaios com cistos); Força Centrífuga Relativa: 650 x g durante 5 minutos (apenas para ovos); 1050 x g durante 10 minutos e 1050 x g durante 20 minutos. As amostras de solo foram contaminadas artificialmente com um número conhecido (5 x 10² ou 10³) de cistos de Giardia spp e de ovos de Ascaris suum. Comprovou-se, previamente aos experimentos, a negatividade do solo coletado quanto aos cistos de Giardia, mediante a realização da PCR, e flutuação em solução de sacarose, para ovos de helmintos. Verificou-se que, a homogeneização do solo com o ICN 7X em mixer rotatório é o processo mais adequado para cistos e ovos, a separação imunomagnética é a melhor forma de purificação para cistos e a solução de sacarose para ovos. As forças centrífugas relativas mais apropriadas são 650 x g durante 5 minutos para ovos e 1050 x g durante 10 minutos para cistos. Após a padronização da metodologia foi avaliada a aplicabilidade desta em solos irrigados por efluente de esgoto hospitalar tratado e em areia do filtro de tratamento deste esgoto. Foram encontrados cistos em ambas as matrizes. O uso desta metodologia efetiva permite o melhor controle da transmissão de parasitoses / Abstract: The soil, especially when enriched with organic matter from sludge sewage treatment plant or human and animal feces, may become an important source of diseases transmissions for holding several parasites. These parasites, for being widely spread in the environment, may cause public health problems. As the soil is one of the main routes for pathogens transmission, this study aimed to - from the protocols described in scientific literature for the detection of protozoan and helminth eggs in soil - evaluate the most effective methodologies and standardize a method to recover cysts and helminth eggs from soil. The following variables were analyzed: vortex Homogenization for 2 minutes, magnetic stirrer for 10 minutes, homogenization in rotary mixer for 30 minutes, and manual shaking for 5 minutes; Dispersion Solutions such as 1% ICN 7X, 1M glycine, 0.1M tetrasodiumPPi and1% tween 40; Filtration: the absence of filtration step, and filtering through a sieve, eggs testing; Purifying Solutions such as zinc sulphate (1,18density) saturated sodium chloride, sucrose (1,33 density for cysts and 1,3 density for eggs), polyethylene glycol 60% and immunomagnetic separation (just for cysts testing); Relative Centrifugal Force: 650 x g for 5 minutes (for eggs); 1050 x g for 10 minutes, 1050 x g for 20 minutes. Soil samples were artificially infected with a know number (5 x 10² or 10³) of Giardia spp cysts and Ascaris suum eggs. The negativity of the collected soil regarding the Giardia cysts was proved previously to the experiments, through PCR, and by flotation in sucrose solution for helminth eggs. It was verified that, the soil homogenization with ICN 7X in rotary mixer is the most efficient process for cysts and eggs, the immunomagnetic separation is the best purifying way for cysts and the sucrose solution for eggs. The most appropriated relative centrifugal force are 650 x g for 5 minutes for the eggs and 1050 x g for 10 minutes for the cysts. After the standardization of the methodology, its applicability was evaluated in soils irrigated with hospital treated wastewater and in the sand filter of this sewage treatment. Cysts were found in both matrices. The usage of this effective methodology allows better controlling of parasitosis transmission / Mestrado / Parasitologia / Mestre em Parasitologia

Les infections d'origine alimentaire constituent un problème de santé publique majeur et présentent un impact économique important. Les agents pathogènes incriminés peuvent être d'origine bactérienne, virale ou parasitaire. Les conséquences de ces infections sur la santé des consommateurs peuvent être majeures, en fonction du statut immunitaire de chaque individu et de la provenance de l'agent infectieux, la mondialisation ayant accru les risques de dissémination de pathogènes viables virulents. Les denrées à risque sont principalement les aliments frais consommés crus ou peu cuits. Ceux-ci sont irrigués en continu durant leur croissance par des eaux pouvant être potentiellement contaminées par des agents infectieux.Au cours de ce travail, nous avons développé une stratégie d'extraction et de détection des parasites Toxoplasma gondii, Cryptosporidium et Giardia à partir de basilic et de framboise. Nous avons pour cela développé une technique d'immuno-capture des oocystes de Toxoplasma gondii grâce à un anticorps monoclonal dirigé contre la paroi des oocystes ainsi que des techniques de détection des trois parasites par qPCR. Ces protocoles ont été validés par des essais inter-laboratoires avec la participation de quatre laboratoires partenaires de ce projet. Nous avons par la suite cherché à mieux appréhender les risques réels pour les consommateurs en réalisant une étude mettant en parallèle une Reverse transcriptase PCR avec des essais in vivo, permettant ainsi de mettre en corrélation la viabilité des parasites avec leur infectiosité.Ce travail contribue à mieux maitriser les risques sanitaires liés à la présence de parasites dans lesdenrées alimentaires , par le biais d'une stratégiesusceptible d'être proposée aux industriels et normalisée. / Foodborne infections are a major public health problem and have a significant economic impact. Pathogens implicated in these outbreaks can be bacteria, viruses and parasites. Their impact on the consumer's health can be significant, depending on the immune status of the person and the pathogen source. Globalization has increased the risk of spreading of viable and virulent . The food at risk is mainly fresh food, eaten raw or undercooked, which has been widely irrigated by potentially contaminated waters during production. In this study, we have developed methods to extract and detect Toxoplasma gondii, Cryptosporidium and Giardia parasites from basil and raspberry with techniques able to detect the three parasites by qPCR. In this aim, we have developed a technique for the immuno-capture of Toxoplasma gondii oocysts using a monoclonal antibody directed against the oocyst wall. These protocols have been validated by interlaboratory trials with the participation of four laboratories involved in the same project. We have subsequently sought to better understand the real risks for consumers by undertaking a study of Reverse transcriptase PCR in parallel with in vivo, thus allowing to correlate the viability of parasites with their infectivity. This work contributes to a better control of health risks associated with food and presence of parasites, through a strategythat could be standardized and offered to industrial partners.

Orientador: Regina Maura Bueno Franco / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T02:37:18Z (GMT). No. of bitstreams: 1 Leal_DiegoAveraldoGuiguet_D.pdf: 4157800 bytes, checksum: 6b17942eb61d5ff1784bbd719d71b15b (MD5) Previous issue date: 2013 / Resumo: Surtos de doenças gastrointestinais associados com a ingestão de moluscos bivalves crus ou mal cozidos têm sido reportados em todo o mundo. O procedimento mais utilizado para purificar os tecidos dos bivalves, consiste na utilização de água desinfetada com radiação UV aplicada em tanques de depuração, previamente à introdução dos animais no mercado. Os riscos de aquisição de infecções mediante o consumo de bivalves são maximizados quando estes são cultivados em áreas inapropriadas, ou quando são comercializados sem serem submetidos ao procedimento de depuração. Os objetivos deste estudo foram: avaliar a contaminação por cistos de Giardia duodenalis e oocistos de Cryptosporidium em ostras destinadas ao consumo humano, antes e após o procedimento de depuração com luz UV, e em águas salobras, nas etapas relacionadas com a cadeia produtiva de ostras no estuário de Cananéia, litoral sul de São Paulo; realizar a caracterização molecular de cistos de Giardia em diferentes etapas da produção de ostras para verificar os genótipos circulantes e aqueles relacionados com a infecção em humanos; enumerar a concentração de indicadores bacteriológicos de contaminação fecal, da área de cultivo até a etapa de depuração para verificar se atende à regulamentação do Conselho Nacional do Meio Ambiente (CONAMA). Quatro pontos foram selecionados para coleta de água e ostras e pesquisa de protozoários: ponto 1: região de cultivo e engorda das ostras; ponto 2: captação de água; ponto 3: etapa de filtração de água e ponto 4: tanque de depuração. As amostras de água foram analisadas por filtração em membranas seguida de separação imunomagnética (IMS) e reação de imunofluorescência direta (RID). A contaminação das ostras foi verificada mediante análise de conteúdo líquido interno e lavado branquial por IMS e RID. A reação em cadeia da polimerase e o sequenciamento foram realizados para pesquisa de Giardia duodenalis e caracterização de seus genótipos. Giardia foi o patógeno mais prevalente em todos os pontos de água, sendo detectada em: 50,0%, 25,0%, 33,3% e 50,0% das amostras dos pontos 1, 2, 3 e 4, respectivamente. O subgenótipo AII foi detectado em algumas amostras hídricas dos pontos 1, 2 e 4 e o genótipo C em duas amostras (pontos 3 e 4). Oocistos de Cryptosporidium foram detectados em 16,6% das amostras de água após a depuração. As ostras da região de cultivo continham cistos de Giardia duodenalis em 41,6% das amostras; o subgenótipo AII foi evidenciado em uma amostra. As ostras depuradas apresentaram a maior contaminação: 58,3% das amostras albergavam cistos de Giardia e 3 delas pertenciam ao subgenótipo AII. As análises bacteriológicas revelaram que o ponto de engorda (P1) estava adequado para o cultivo das ostras. Estes resultados refletem a ineficiência do procedimento de depuração com UV aplicado nesta planta de tratamento e a contaminação do produto - na etapa final que precede sua comercialização - por Giardia duodenalis pertencente ao subgenótipo AII, potencialmente infectante para seres humanos. Os resultados evidenciam a necessidade de monitoramento contínuo de patógenos e adequação da legislação do cultivo de moluscos bivalves no Brasil, a fim de garantir a segurança alimentar / Abstract: Many shellfish-borne associated outbreaks have been reported worldwide especially related to its ingestion raw or lightly cooked. In the shellfish industry, the most commonly utilized procedure to purify shellfish tissues consists in the utilization of UV disinfected water employed at depuration tanks, before they are placed on the market. The risks of acquiring gastroenteric diseases, through its consumption, are maximized when they are cultured on inappropriate growing areas or whenever they are sold without depuration procedure. The purposes of this study were: evaluate the contamination by Giardia duodenalis cysts and Cryptosporidium oocysts in oysters destined to human consumption, before and after UV depuration procedure, and in brackish waters at stages related to the food chain of edible oysters, from Cananéia estuary located on the south coast of São Paulo; perform molecular characterization of Giardia cysts at different stages of the production of oysters to verify the main circulating genotypes and those associated with human infection; enumerate bacteriological indicators of fecal contamination from growing site up to depuration site, to check if it complies with the National Environmental Council (CONAMA). Four sampling sites were selected for water and oysters harvesting and protozoa search: site 1- oysters growing area; site 2: catchment water; site 3: filtration stage of water treatment and site 4: oyster's depuration tank. Water samples were analyzed through membrane filtration technique followed by immunomagnetic separation (IMS) and direct immunofluorescence assay (IFA). Oysters' contamination was verified through the analysis of innerwater and gill wash using IMS and IFA. The polymerase chain reaction and sequencing reactions were performed to search for Giardia duodenalis and characterization of its genotypes. Giardia was the most prevalent pathogens in all water sites, where it was detected: 50.0%, 25.0%, 33.3% and 50.0% of water from sites 1, 2, 3 and 4 respectively. The subgenotype AII was detected in several water samples from sites 1, 2 and 4 and the genotype C in two samples (sites 3 and 4). Cryptosporidium oocysts were found in 16.6% of depuration water tank. Oysters from growing area harbored Giardia duodenalis cysts in 41.6% of the samples and subgenotype AII was evidenced once. Depurated oysters were the most contaminated: 58.3% were harboring Giardia duodenalis cysts and 3 belonged to subgenotype AII. Bacteriological analysis demonstrated that site 1 was adequate to growing oysters. These results indicate the ineffectiveness of the UV depuration applied in this shellfish treatment plant, and the contamination of the product - in the final stage which precedes its commercialization - by Giardia duodenalis belonging to subgenotype AII, potentially infectious to humans. The results highlight the need for continued monitoring of pathogens and suit the legislation on the cultivation of bivalve molluscs in Brazil in order to ensure food safety / Doutorado / Parasitologia / Doutor em Parasitologia

Orientador: Anete Pereira de Souza. / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T05:07:09Z (GMT). No. of bitstreams: 1 Durigan_Mauricio_D.pdf: 7600085 bytes, checksum: 74ae2337a73b6edfb14a403af4ffa590 (MD5) Previous issue date: 2015 / Resumo: Giardia duodenalis é um protozoário flagelado que parasita o homem e diversos animais domésticos e selvagens. Este parasito causa a doença giardiose que é uma das mais prevalentes doenças parasitárias de veiculação hídrica do mundo, responsável por aproximadamente 280 milhões de casos anualmente. Existe uma considerável variabilidade genética em G. duodenalis, de modo que seus isolados foram divididos em oito grupos genéticos (A-H), dois dos quais (A e B) são encontrados tanto em humanos quanto em animais. Os demais grupos (C-H) parasitam outros animais e apresentam maior especificidade a determinados hospedeiros não humanos. A contaminação ambiental por Giardia tem sido amplamente descrita embora esses estudos, em sua maioria, são realizados no nível de identificação de espécie. Há falta de estudos que correlacionam a contaminação ambiental e infecções clínicas na mesma região. O presente trabalho teve como objetivo principal contribuir para o conhecimento da diversidade genética da espécie Giardia duodenalis. Primeiramente, foi realizada a genotipagem multilocos dos principais grupos genéticos de G. duodenalis na região metropolitana de Campinas. Foram encontrados grupos genéticos associados principalmente a infecções humanas bem como isolados com potencial zoonótico em amostras ambientais e obtidas de outros animais. Foi encontrado um alto percentual (25%) de amostras com grupos genéticos mistos e um elevado número de haplótipos distintos, indicando grande diversidade genética do parasito nessa região. Na segunda parte deste trabalho, foi realizado um estudo populacional com amostras clínicas de Giardia provenientes de hospital, creche e centro de controle de zoonoses e amostras ambientais de esgoto hospitalar, efluente de estação de tratamento de esgoto e amostras hídricas de importantes rios e córregos urbanos. As análises populacionais, com exceção das amostras caninas, evidenciaram grande similaridade genética entre essas populações de Giardia. Na terceira parte do presente trabalho, foi realizada uma busca por repetições microssatélites (SSRs) nos genomas publicados de Giardia para desenvolvimento, caracterização e avaliação de polimorfismo de novos marcadores microssatélites. Foram encontrados 506, 438, 402 e 507 microssatélites correspondentes aos genomas AI, AII, B e E, respectivamente. Foram selecionados 80 SSRs específicos aos grupos genéticos A, B e E (40, 20 e 20, respectivamente), além de 36 SSRs compartilhados entre os três genomas. A análise de amplificação confirmou a existência de marcadores específicos aos grupos genéticos A, B e E, além de marcadores compartilhados entre os grupos. A caracterização dos SSRs permitiu a detecção de 12 locos SSRs polimórficos do grupo genético A e sete locos SSRs polimórficos do grupo genético B. Dentre os marcadores compartilhados, o loco GduABE01 apresentou polimorfismo. Os locos polimórficos podem servir para futuros estudos populacionais e os marcadores desenvolvidos podem ser utilizados para identificação dos principais grupos genéticos de G. duodenalis em amostras clínicas e ambientais. Os resultados apresentados contribuem para um melhor entendimento sobre a diversidade genética do parasito bem como sobre a presença de grupos com potencial zoonóticos inter-relacionados em diferentes regiões. Os novos marcadores moleculares disponibilizados podem contribuir para novos estudos populacionais, promovendo melhor discriminação entre os genótipos e possibilitando assim identificar a contaminação e promover o rastreamento da doença / Abstract: Giardia duodenalis is a flagellate protozoan that that parasites humans and several domestic and wild animals. This parasite causes giardiasis, one of the most common waterborne diseases in the world responsible for, approximately 280 million cases per year. There is a great genetic diversity in this species and its isolates have been grouped into eight distinct genetic assemblages (A-H). While groups A and B parasitize different hosts and have zoonotic potential, groups C, D, E, F, G and H usually found in animals and show greater specificity to the parasitized host. Environmental contamination for Giardia has been widely reported however, most of these studies have been performed only at species level. The present study aimed to contribute to the knowledge of the genetic diversity of the species Giardia duodenalis. In the first chapter of this document, multilocus sequence-based genotyping using three gene loci assigned most of the samples as belonging to human genotypes although isolates with zoonotic potential have also been identified in environmental and non-human clinical samples. A high percentage (25%) of mixed assemblages and a high number of different haplotypes were detected, which indicates high genetic diversity of this parasite in this region. In the second chapter, a population genetics study was performed with clinical samples from hospital, day-car center and a center for zoonosis control of the city and environmental samples from hospital sewage, effluent of a wastewater treatment plant and important water samples from rivers and urban streams. With the exception of the canine population, population genetic analysis showed consistent similarity between clinical and environmental populations. In the last chapter, we performed a search for microsatellites (SSRs) in the published genomes of Giardia to develop and characterize the polymorphism of new microsatellite markers. Our group identified 506, 438, 402 and 507 microsatellites of the genomes AI, AII, B and E, respectively. We have selected 80 markers specific to the genetic assemblages A, B and E (40, 20 and 20, respectively) and 36 shared SSRs between the three genomes. Analysis of amplification reactions confirmed the existence of specific loci of each genetic assemblage as well as shared loci among assemblages. Characterization of all loci allowed the detection of 12 polymorphic loci for group A and seven polymorphic loci for group B. Among the shared markers, GduABE01 presented polymorphism. The polymorphic markers can be used in future population genetic studies and the developed markers can contribute to the identification of the main genetic assemblages of G. duodenalis in clinical and environmental samples. The results presented here contribute to a better understanding of the genetic diversity of the parasite as well as the presence of zoonotic potential genotypes, related in different regions. The new molecular markers provided can contribute with population genetic studies in a high level of discrimination that allows identifying the source of contamination and molecular tracking of the disease / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Heat shock proteins (Hsps) are a class of molecular chaperones which were first discovered as proteins up-regulated in response to heat stress in Drosophila. Later, it was found that these set of proteins get up-regulated as a general stress response associated with destabilization of native protein structures. Over a period of time, intricate involvement of Hsps in various biological processes has been well established. Heat shock protein 90 (Hsp90) is one of the important representative of this class of proteins. Hsp90 is an essential molecular chaperone which is evolutionarily conserved. It has a selective set of proteins to chaperone called as clients, which majorly include transcription factors and protein kinases. Through its interaction with its clients it modulates cell cycle, signal transduction, differentiation, development and evolution. Previous studies from Candida, Leishmania and Plasmodium have implicated Hsp90 to be involved in stage transition and growth. It is also critically involved in regulating growth of other protozoans such as Dictyostelium, Entamoeba and Trypanosoma. Thus, selective inhibition of Hsp90 has been explored as an intervention strategy against important human diseases such as cancer, malaria and other protozoan diseases. In Plasmodium falciparum, Hsp90 plays a critical role in stage transition. The parasite inside the human RBC develops from ring to trophozoite to schizont stage and inhibition of Hsp90 using specific pharmacological inhibitor arrests the growth of parasite at ring stage. In Dictyostelium, it has been observed that Hsp90 function is required for development. Inhibition of Hsp90 causes mound arrest and stops the cells from entering to its next developmental stage, fruiting bodies. In parallel, Hsp90 in Candida has been shown to be involved in morphogenesis. In nature Candida exists as a single cell yeast form and upon entry into the human host these yeast forms undergo morphogenesis to form virulent filamentous fungi. Inhibition of Hsp90 mimics temperature mediated morphogenesis. All together, these studies suggest that Hsp90 functions in a context dependent manner and each biological system explored has given new insights into the Hsp90 biology. Giardia lamblia, a protozoan parasite of humans and animals, is an important cause of diarrheal disease causing significant morbidity and also mortality in tropical countries. In the present study we focus on the biology of Hsp90 from Giardia lamblia. Giardia has a biphasic life cycle with infective cyst stage and pathogenic trophozoite stage. These cysts are present in the environment and enter mammalian host through oral route. They undergo a process called as excystation in the intestine giving rise to trophozoites. The trophozoites so formed colonize the upper part of the small intestine which causes the symptoms of giardiasis. Some of the trophozoites escape from the nutrition rich milieu of the upper part of small intestine to the lower part. In this region, trophozoites undergo a process called as encystation, wherein each trophozoite forms a cyst which escapes through faeces back into the environment. As seen in the life cycle of Giardia there are two major biological transitions, excystation and encystation; and till date no definitive player or pathway is known to regulate these processes. With the knowledge of Hsp90 playing an important role in similar biological transitions in other organisms we were encouraged to study role of Hsp90 in Giardia lamblia. Trans-splicing based generation of a full length Hsp90 in Giardia lamblia To understand the role of Hsp90, we first carried out sequence alignment of Hsp90 predicted ORFs in Giardia genome with yeast Hsp90. On alignment we observed that Hsp90 in Giardia is discontinuous and is annotated to be encoded by two different ORFs. Hsp90 in most organisms is coded by a single ORF with none to many cis-spliced introns. In a relatively intron poor organism G. lamblia, cytosolic Hsp90 is coded by two different ORFs separated by 777 kb in the genome. On multiple sequence alignment, we noticed that these two ORFs correspond to two independent regions of the Hsp90 protein. The ORFs are designated as hspN and hspC, containing the N-terminal and the C-terminal region of the protein respectively. We began our study by sequencing whole genome of Giardia lamblia clinical strain. Our genome sequencing confirmed the split nature of hsp90 and showed high ‘synteny’ between the other sequenced isolates. Using PCR based approach we have ruled out the possibility of having a full length gene in the genome. In contradiction to the genome result, we have observed a higher molecular weight protein in the lysate on proteomic analysis which was further confirmed by western blotting. The protein was observed to have a molecular weight of 80 kDa which could be a resultant of combination of two ORFs, suggesting the presence of a full length mRNA for Hsp90. PCR amplification using primers against both the fragments resulted in amplification of 2.1 kb product from the RNA pool of Giardia. Sequencing of this product showed that hspN and hspC were stitched together to form a mature messenger for full length Hsp90. In total our results suggest a post transcriptional process, trans-splicing, to be involved in the construction of Hsp90. The transition marked by this fusion coincides with the canonical GU¬AG splice site transitions as observed in other eukaryotes. Interestingly, a 26 nt near-complementary region was observed inside and upstream of hspN and hspC ORFs respectively. Put together these results suggest that the 26 nt complementary region acts as the positioning element to bring these two precursors in spatial proximity. With efficient spliceosomal activity these two precursor forms are trans-spliced to generate a full length cytosolic Hsp90 in Giardia. There are only four genes which have cis-spliced introns in the Giardia genome and the core components of the spliceosomal machinery are also present. The presence of canonical splice site in both the transcripts suggests that these transcripts are fused together by the spliceosomal machinery by the phenomenon of trans-splicing. The formation of full length Hsp90 RNA by its fragmented gene is the first example of trans-splicing in Giardia. To understand, are there any other genes which are also similarly trans-spliced we have carried out shotgun proteomic analysis of the total cell lysate obtained from Giardia trophozoites. Using Hsp90 as template, in our proteomic datasets, we have designed an algorithm for identification of additional trans-spliced gene products at the protein level. We have identified a total of 476 proteins of which hypothetical proteins constitute the major class followed by metabolic enzymes. We have compared the theoretical molecular weights for the identified proteins with the experimentally determined mass. Any discrepancy in the molecular mass was further analyzed and we assigned a gene to be potentially trans-spliced based on three criteria: if they were encoded by two or more different ORFs (loci), absence of a single full length counterpart and presence of splice sites with branch point and positional elements. Using this algorithm we were able to identify dynein as a potential candidate of trans-splicing reaction which was confirmed by the nucleotide sequence analysis of the predicted ORFs. Interestingly, dynein gene fragments were observed to be scattered on different chromosomes with minor splice sites unlike hsp90 genes. In vivo Expression of Hsp90 sub-fragments, HspN and HspC In the mature Hsp90 mRNA formed upon trans-splicing, 33 additional codons are present right between hspN and hspC sequences and they were acquired from the upstream region of hspC ORF. The 33 codons encode for an important region of Hsp90 which harbours the conserved catalytic “Arg” residue; suggesting that the full length Giardia Hsp90 (GlHsp90) formed could be an active ATPase. To confirm the same we have carried out in vitro characterization of trans-spliced Hsp90. Towards this, we have cloned, expressed and purified His tag-GlHsp90. As a first step, highly purified protein was used to assess its efficiency in binding to it cognate ligand, ATP, and the known inhibitors. Our binding studies show that GlHsp90 binds to ATP with a dissociation constant of 628 M and to its inhibitors, GA and 17AAG with 1.5 μM and 17.5 μM respectively. The bound ATP will be subsequently cleaved by Hsp90 which is an essential step in the chaperone cycle. As determined in our ATPase assay we observed that GlHsp90 hydrolyzes bound ATP with the catalytic efficiency of 4.4 × 10-5μM-1.min-1which confirms that Hsp90 generated upon trans-splicing is an active ATPase. The uniqueness of the hsp90 gene arrangement in Giardia posed a new question. Do these gene fragments also get translated? Our results suggest that HspN and HspC are poly¬adenylated. In order to determine the levels of these transcripts we performed qRT-PCR using primers specific to HspN, HspC and GlHsp90. We have observed that, in comparison with HspN transcript level, HspC and GlHsp90 transcripts are 15 and 75 folds higher respectively. To check for the presence of translation products of these transcripts, we have re-analyzed our proteomic datasets wherein we could identify peptides corresponding to HspN and HspC in their respective molecular weight region, 45 to 35 kDa. To confirm the proteomic data, western blot analysis was performed for trophozoite lysate on both 1D and 2D gels using anti-HspN antibody. Two specific bands (1D) / spots (2D) corresponding to the full length Hsp90 and HspN were identified. Gel filtration analysis revealed that HspN co¬eluted with full length Hsp90 thereby suggesting that both the proteins are in a same complex. With the background that HspN and HspC are present at the protein level, we asked if these fragments in combination can hydrolyse ATP. We reconstituted recombinant HspN and HspC in equimolar amounts and scored for the hydrolysis of ATP. However, no Pi release was observed. To determine whether HspN and HspC could modulate Hsp90 function, ATPase activity was monitored in the presence of HspN or HspC, in vitro. It was observed that ATPase activity was inhibited by both the fragments thus suggesting that HspN and HspC negatively regulate Hsp90 ATPase activity. Role of Hsp90 in Giardia encystation Giardia has a biphasic life cycle with proliferative trophozoites and latent cyst stage. In Giardia, in vitro encystation was established nearly two decades back by modulating the medium conditions. However, the mechanism and triggers underlying this transition are not well characterized. To understand whether Hsp90 has any role in this transition, in vitro conversion of trophozoites to cysts was achieved. The cysts obtained showed all the characteristic features of mature Giardia cyst with cyst wall protein 1 (CWP1) on the cyst wall and four nuclei as determined by immunofluorescence analysis. Further, the levels of Hsp90 in trophozoites were compared with mature cysts at both transcript and protein levels and it was found that cysts show more than 50% reduction in the level of Hsp90 in comparison with normal trophozoites. In accordance, exogenous inhibition of Hsp90 using 17AAG promoted the formation of cysts in vitro by 60 folds in a dose dependent manner; however, the window period of Hsp90 function compromise plays an important role in this process. Higher numbers of cysts were obtained from the cells treated with inhibitors during pre-encystation condition but inhibition of Hsp90 during encystation did not affect the formation of cysts, suggesting that Hsp90 down-regulation plays an important role during commitment towards encystation. To further show that cyst formation is a specific response to Hsp90 inhibition we have carried out encystation in the presence of metranidazole and from heat shocked cells; however, in both the conditions we did not observe any significant change in cyst formation, thus confirming that Hsp90 plays an important role during encystation in Giardia lamblia. Summary In Conclusion, Our study throws light on a unique aspect of Hsp90 biology in Giardia Lamblia, wherein the formation of the full length protein is dependent on a unique trans splicing reaction of its gene components representing different domains. We have also shown that HsP90 fragments, HspN and HspC, are also expressed in Trophozoites. Our in vitro data suggests that these fragments possibly regulate the function of Hsp90. Furthermore, the full length of Hsp90 plays an important role in stage transition in Giardia wherein inhibition of Hsp90 induces encystations. The study has opened many new avenues for research. Understanding the exact role of HspN and HspC in vivo will provide better appreciation for the evolution of such a complex biogenesis of an essential protein.

Master of Veterinary Biomedical Science / Department of Diagnostic Medicine/Pathobiology / Michael W. Dryden / Patricia A. Payne / Giardiasis is a common intestinal protozal parasitic infection of the pet dog and cat population. Veterinarians often have difficulty correctly diagnosing this parasite. Studies were conducted to compare the zinc sulfate double centrifuge fecal flotation to the SNAP (registered trademark) Giardia fecal ELISA test manufactured by IDEXX laboratories Inc. in purpose bred beagles and shelter and commercial kennel dogs. In these evaluations the zinc sulfate double centrifuge fecal flotation and fecal ELISA test performed similarly. Both tests performed better in the shelter and commercial kennel dog population than the chronically infected purpose bred beagles. There was an increase in number of positive animals identified when 3 consecutive daily samples were evaluated as compared to any one individual day for either test method. Post treatment evaluation of the diagnostic tests was performed in 23 laboratory beagles. Each beagle was treated for 3 consecutive days with Drontal plus and then bathed on the last day of treatment and fecal samples were collected from the treated dogs every other day starting one day post treatment for 21 days. It was found that all beagles were negative on zinc sulfate double centrifugation fecal flotation, fecal ELISA and IFA within 24 hours of treatment and nineteen (82.6%) of the beagles did not re-shed cysts during the 21 day post-treatment evaluation period. Four beagles returned to shedding cysts (Flotation or IFA positive) between days 17 and 21. These findings suggest that a positive test within a week of treatment is likely the result of inappropriate treatment. After the prepatent period, positive results may occur due to a return to shedding, reinfection or inappropriate treatment. Chronically infected laboratory beagles may not be a good model for acute Giardia infections as these dogs are rarely clinically ill and detection is more difficult.

Giardia lamblia is a eukaryotic parasite that causes diarrhea in humans worldwide. Diarrheal diseases cause stunting and mental retardation in children in developing nations, therefore it is important to understand the molecular epidemiology of G. lamblia. Compounding this, it is not clear if companion animals such as dogs contribute to infections in humans through zoonotic transmission. The genotypes of G. lamblia that have been found in humans are A1, A2 and B, while those in dogs have been on rare occasions all three human genotypes, but largely C and D, which have only been reported in dogs and appear to be species-specific. The molecular epidemiology of G. lamblia in humans and dogs was assessed in an endemic region of Lima, Peru. With one exception, dogs were found to harbor the C and D dog genotypes of G. lamblia. A single family dog was found to harbor a human genotype of G. lamblia. A2 and B genotypes of G. lamblia, but not A1, were found in humans in the endemic region. Previous literature reported that A2 and B typing within genotype tools were available, however the A2 samples from the endemic region could not be distinguished from one another through nucleotide polymorphism sequence analysis. A molecular typing technique was developed to type A2 samples. The extensive sequence analysis performed on two chromosomes of G. lamblia, yielded different phylogenetic tree groupings for the same samples. This lead to algorithmic analysis, which demonstrated a significantly high probability that meiotic recombination is occurring in the A2 samples of G. lamblia. As G. lamblia is largely believed to be asexual, the conclusion of doctoral research performed in this study yielded controversial, yet significant evidence that sex in G. lamblia A2 genotype samples is indeed occurring.

Diarrhea caused by rotavirus and Giardia is a major health problem among children attending day-care centers because of inadequate personnel hygiene. Epidemiological evidence suggesting person-to-person transmission of enteric pathogens has long been recognized. This study was initiated to investigate the effectiveness of handwashing for the removal of rotavirus and Giardia from contaminated hands. The palms of participant hands were innoculated with approximately 103 Giardia cysts or 105 plaque forming units of rotavirus and the effect of washing using tap water alone, a liquid soap or a bar soap on their removal was assessed. Handwashing with liquid soap was found to be very effective in the removal of rotavirus and Giardia cysts as compared to washing with bar soap or tap water alone. The overall recovery of viruses in both bar soap and liquid soap was low (0.03-22.5%), probably due to virus inactivation by the detergent.

Giardia intestinalis is a parasitic protist that causes one of the most common diarrheal disease of parasite origin. The cell of Giardia contains two nuclei with unknown number of chromosomes until recently. Karyotype was determined five years ago using conventional cytogenetic method by Tůmová and collaborators. In my work, I assessed karyotype of four isolates, six lines and three clonal lines by the same method. It was confirmed, that two nuclei within one cell could differ in chromosome number, the differences found were 1, 2 or 6 chromosomes. Aneuploid number of chromosomes was found too. In case that both nuclei within single cell contained the same number of chromosomes, there were 10 chromosomes indentified in each nucleus. It was also revealed, that karyotype is not specific feature for different genetic groups (in this work assemblages A and E). Karyotype can be different even among lines and clonal populations derived from the same isolate. Changes in karyotype in the course of in vitro cultivation were detected within three populations. Results are discussed in relation to known facts.