Embryonated chicken eggs: An experimental model for Pythium insidiosum infection

Abstract

Pythiosis is a severe disease caused by Pythium insidiosum. Currently, the research on the treatment of pythiosis uses rabbits as an experimental infection model. To reduce the use of animals in scientific experimentation, alternative models are increasingly necessary options. The objective of this study was to establish a new experimental infection model for pythiosis using embryonated chicken eggs. First, we tested the inoculation of four zoospore concentrations into the egg allantoic cavity at three embryonic days. We observed that increased zoospore concentration causes a decrease in survival time, and at a later embryonic day (the 14^th) of infection, embryos showed delayed mortality. To confirm the reproducibility of the model, we chose the 14^th embryonic day for the inoculation of 50 zoospores/egg, and the experiment was repeated twice. Mortality began with 30% embryos 48 h after inoculation, and 95% embryos died within 72 h. There was no mortality in the uninfected control group. The infection was confirmed by culture, PCR and histopathology. Immunohistochemistry confirmed the presence of hyphae in blood vessels in the umbilical cords in 95% of embryos and only one liver (5%). Our results suggest that embryonated eggs can be a very useful alternative infection model to study pythiosis.

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