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Master's Dissertation
DOI
10.11606/D.60.2010.tde-29032012-085519
Document
Author
Full name
Mariana Bryan Augusto
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2010
Supervisor
Committee
Albuquerque, Sérgio de (President)
Abrahão, Ana Amelia Carraro
Allegretti, Silmara Marques
Title in Portuguese
Avaliação comparativa de diferentes métodos de quantificação de formas teciduais de Trypanosoma cruzi na infecção experimental
Keywords in Portuguese
atividade enzimática
CL B5
quantificação parasitária
Trypanosoma cruzi
vias de inoculação
Abstract in Portuguese
Completando 100 anos da descoberta da molestia, a Doenca de Chagas, causada pelo agente etiologico Trypanosoma cruzi, ainda e considerada nas Americas Central e do Sul um problema de saude publica, atingindo mais de 8 milhoes de individuos. Sua transmissao ocorre de distintas maneiras, como por vetores, transfusao sanguinea, transplante de orgaos, acidentes em laboratorios, via oral. O parasita e conhecido por sua heterogeneidade no genotipo e fenotipo, baseado em mutacoes cumulativas em diferentes sub-populacoes do parasita. A cepa CL Brener e uma cepa padrao para pesquisa com diversas peculiaridades interessantes como baixa infectividade em animais, resposta aos tratamentos existentes e seus marcadores geneticos serem estaveis. O clone CL B5 de T. cruzi e originado a partir da cepa CL Brener modificada geneticamente. A cepa possui um gene reporter, o LacZ de Escherichia coli que sintetiza uma enzima, a À-galactose que pode catalisar uma reacao colorimetrica com o substrato vermelho de clorofenol À-galactopiranosideo (CPRG). A atividade enzimatica e diretamente proporcional ao numero de parasita. No presente trabalho propomos a introducao de uma tecnica colorimetrica para caracterizacao de parametros para quantificacao do parasitismo tecidual e comparacao com a tecnica histologica e Real Time-PCR. Camundongos Balb/C machos foram divididos em grupos controle (nao infectado) e experimental variando a via de inoculacao, a saber, intraperitoneal (IP), subcutanea (SC) e oral (OR). As curvas parasitemicas foram realizadas por meio da coleta de sangue da cauda do animal. Apos morte dos mesmos, foram estudados os tecidos cardiaco, hepatico, esplenico, enterico e muscular (musculo esqueletico). Os orgaos foram retirados, divididos em tres porcoes equitativas para analise histologica, enzimatica e molecular. Na atividade enzimatica, comparando os tecidos em suas vias de inoculacao, o figado apresentou resultado significativo no grupo inoculado via IP. Tecido cardiaco apresentou diferenca estatistica (P<0,05) entre as vias de inoculacao OR e SC, apresentando maior atividade enzimatica na primeira, o que esta relacionado a uma maior quantidade de parasita tecidual. Nos cortes histologicos o grupo SC mostrou-se mais infectado com grande numero de ninhos de forma amastigota em praticamente todos os tecidos, enquanto nos demais grupos foram observados ninhos de amastigota em sua maioria no coracao e viii musculo esqueletico. A presenca do parasita nos demais tecidos foi confirmada pela eletroforese em gel de agarose 1,5% apos tecnica de PCR convencional. A Real Time-PCR nao se mostrou satisfatoria no trabalho devido a dificuldade na sua padronizacao. Concluindo, a metodologia enzimatica se mostrou favoravel e adequada na quantificacao parasitaria tecidual, podendo ser aperfeicoada para corroborar sua eficacia.
Title in English
Comparative evaluation of differents methodologies in tecidual types quantification of trypanosoma cruzi
Keywords in English
CL B5
enzymatic activity
quantifying parasites
route of infection
Trypanosoma cruzi
Abstract in English
One hundred years after the discovery of Chagas Disease, an illness caused by blood born protozoan Trypanosoma cruzi, is still consider a public health problem. It is a major cause of morbidity and mortality in Central and South Americas where more than 8 million individuals are already infected. This illness can be contracted by different forms as vector transmission, blood transfusion, tissue transplant, laboratory accidents and oral route. The parasite has a heterogeneous genotype and phenotype due to accumulating mutation in their sub-populations. CL Brener is considered as a pattern strain due to some intrinsic characteristics as low infectivity, good response to therapy drugs and a stable genetic heritage. The CL B5 clone of T. cruzi was originated by a genetic modification of the CL Brener strain which has a reporter gene LacZ of Escherichia coli, which induces the syntesis of â-galactosidase which is able to catalyze a colorimetric reaction using the substrat Chlorophenol red-â-D-galactopyranoside (CPRG). The enzymatic activity is directly related with the number of the parasites. In this study we proposed a new colorimetric assay to quantify T. cruzi load in different animal tissues by comparing with other methodologies such histological and molecular assay (Real Time-PCR). Male Balb/C mice were separated into control and experimental groups according to the route of infection (IP-intraperitoneal, SC-subcutaneous, OR-oral). Parasitemic curves were made by collecting blood samples from the tail of experimental animals. Fifteen days after, animals were euthanized and their tissues removed (heart, liver, spleen, intestine and skeletal muscles) and divided in three portions for the colorimetric analyses, histopathology and molecular assay. The enzymatic activity was performed comparing the number of parasites in different tissues according to the route of infection. Comparing them, it was observed that heart and liver displayed the highest number of parasites as compared with the other studied tissues. In the liver, IP route triggered the highest number of parasites while in the heart OR and SC routes displayed enhanced parasites. The histopathology analysis revealed that SC group presented the highest number of amastigote nests. For IP and OR groups, nests were mostly observed in heart and skeletal muscles. T. cruzi DNA were detected using electrophoreses 1,5 % agarose gel after conventional PCR technique, displaying characteristic bands of DNA. The Real Time-PCR was not a satisfactory x assay for this study due to difficulties on its patronization. To conclude, the enzymatic methodology was advantageous and appropriated on quantifying tissue parasites. Some further experiments will be needed to improve methodology and its efficacy.
 
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Publishing Date
2013-05-02
 
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