Determination of a methodology for detection of Giardia spp. cysts and helminth eggs, from soil

Orientador: Regina Maura Bueno FrancoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: 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 parasitosesAbstract: 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 transmissionMestradoParasitologiaMestre em Parasitologi

Characterisation of protease activity in extracellular products secreted by Giardia duodenalis trophozoites treated with propolis

Results from our laboratory revealed propolis activity on Giardia trophozoites proliferation. Since therapeutic agents can inhibit the activity of proteases related to relevant biologic and physiologic processes of parasites, this study was undertaken to characterise the proteolytic activity of excretory/secretory products (ESP) of trophozoites treated with propolis. ESP was obtained from culture supernatants of trophozoites exposed to 250 and 500 mu g mL(-1) of propolis. ESP were tested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the protein profiles and the protease activity was assayed in gelatin-containing gels. Synthetic inhibitors were used to characterise the protease classes. Treated and non-treated ESP showed a similar protein and hydrolysis pattern. A simple pattern of protein composed by five evident bands of approximately 167, 132, 79, 61 and 51 kDa was found, and the zymograms comprised hydrolysis zones distributed from > 170 to 23 kDa. No inhibition was seen on protease activity of propolis-treated trophozoites, whose hydrolysis pattern was similar to control. One may conclude that both ESP degraded gelatin and the activity was predominantly due to cysteine proteases. Although propolis had no effect on the proteolytic activity, further studies could identify the active constituents responsible for propolis antigiardial activity and their mechanisms of action