Efficacy of thymol against Echinococcus granulosus cell line

ALBANI C. M.; GENDE LIESEL; EGUARAS MARTIN; DENEGRI G. M.; ELISSONDO M. C.

Buenos Aires

Congreso; I CONGRESO INTERNACIONAL .DE ZOONOSIS Y VII CONGRESO ARGENTINO DE ZOONOSIS1; 2011

Introduction   Human cystic echinococcosis, caused by the larval stage of the dog tapeworm Echinococcus granulosus, remains a major public-health problem on several countries. Until the 1980s, surgery was the only option for treatment of echinococcal cysts. Later, surgery was supplemented or even replaced by chemotherapy with benzimidazole compounds. However, the low affectivity of these synthetic anthelmintics and the appearance of resistance to these compounds stimulated the research of new chemotherapeutic alternatives, such as the use of medicinal plants. Thymol is one of the major components of the essential oils of Thymus sp. and is a widely known anti-microbial agent. Recently the in vitro effect against protoescoleces, microcysts and cysts was demonstrated. The aim of the present work was to determine the in vitro effect of thymol against an Echinococcus granulosus cell line. Materials and Methods The cell line was established from hydatid cyst germinative layer and maintained in culture for at least one year. The cells were cultured in medium 199 supplemented with antibiotics and reducing agents and 5 x 105 cells/well were seeded in 24-multiwell plates. Thymol was added to the medium resulting in final concentrations of 10, 5 and 1 μg/ml. Cell viability was assessed to the days 2, 5, 7, 14 and 21 using the methylene blue exclusion test. During the experiments, cultures were followed microscopically to determine possible changes. Samples from each of the dosing groups and the controls were taken each 2-3 days for structural studies with scanning electron microscope (SEM). Results Control cell culture viability was 2.2 x 106 (100%) after 7 days of incubation. The maximum anthelmintic effect was found with thymol 5 µg/ml, viability was reduced to 9.95 x 105 (45%) after 7 days of incubation (Figure 1). It could also be observed the effect of thymol on the reduction in the number of cells using optical microscopy (Figure 2). We found differences between control and the different treatments but no differences between treatments. The results of viability test coincided with the damage observed at the structural level. Cell culture treated with thymol 10 μg/ml during 7 days showed morphological alterations as loss of turgor, cellular contraction as well as reduction in the number of cells (Figure 3). Conclusions -The treatment with 1, 5 or 10 μg/ml of thymol showed a pronounced anthelmintic effect compared with control. -The employment of SEM allowed us to examine, at a structural level, the effects of thymol on E. granulosus cell culture. - Thymol not only caused cell death but also stopped cellular proliferation. Following this, in a next step, it would be interesting to investigate the in vivo efficacy of thymol on the germinative layer of the cyst.