A new large-scale technique to prospect for plant extract activity against Encephalitozoon cuniculi

Microsporidiosis is an emerging zoonotic disease caused by several species of microsporidia, among which Encephalitozoon cuniculi is particularly important. To date, therapeutic options are limited to albendazole, which can control the infection transiently but does not eliminate the parasite. Brazil is a country rich in natural diversity and has several plants with therapeutic potential. This work aims to report the development of a fast, low-cost in vitro method for large-scale prospecting plant extracts against Encephalitozoon cuniculi and to select the most effective anti-infective extracts. For this, a cell cytotoxicity assay was performed with MDCK Madin-Darby canine kidney cells using 15 plant extracts. Additionally, a spore viability assay was performed after treatment with 15 plant extracts and albendazole, and the results were qualitatively evaluated based on cell culture viability and spore cloud observations, with assigned scores. Of the 15 extracts used, five were found to be potentially active against the spores: ANN1, GEN1, LAU, OLA, and SOL. At the same time, CLU and GEN2 showed strong sporicidal activity and very low cytotoxicity against MDCK, the host cell. Furthermore, gas chromatography-mass spectrometry (GC-MS) analysis of the extracts revealed the presence of alkaloids, phenolic compounds, terpenes, and fatty acids. This study contributed to the establishment of an innovative qualitative test for large-scale testing of plant extracts against E. cuniculi.