Comparative evaluation of differents methodologies in tecidual types quantification of trypanosoma cruzi

Author(s)

Mariana Bryan Augusto

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2010-08-02

Examining board members

Sérgio de Albuquerque; Ana Amelia Carraro Abrahão; Silmara Marques Allegretti

• Advisor: Sérgio de Albuquerque

Abstract

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. (AU)

• FAPESP's process: 08/02003-9 - Comparative evaluation of differents methodologies in tecidual types quantification of Trypanosoma cruzi.
• Grantee: Mariana Bryan Augusto
• Support Opportunities: Scholarships in Brazil - Master