In vitro determination of anticryptosporidial activity of phytogenic extracts and compounds

Abstract

Cryptosporidiosis caused by Cryptosporidium spp. is an important diarrhoeal disease observed in farm animals and humans, especially in young or immunocompromised individuals. A novel cell culture assay for testing extracts and pure compounds against Cryptosporidium parvum in 96-well microplate format was established and evaluated. It is based on previously described indirect fluorescent antibody techniques and was optimised for higher sample throughput. Rapid assessment of minimal inhibitory concentrations (MICs) was done by checking each well microscopically for the presence or absence of parasite stages. As a novelty, parasite development was quantified by enumeration of clusters of secondary infection (CSI), which typically appeared upon infection with a distinct parasite inoculum after a defined incubation time. Host cell (HCT-8) viability was measured by an integrated non-destructive water-soluble tetrazolium salt assay (WST-1), which facilitated discrimination of antiparasitic activity from possible cytotoxic effects of a test compound against the host cells. Host cell viability was regarded unimpaired when cultures had 75% or more viability when compared to control cultures without test substance. In this study, a maximum density of distinguishable CSI was obtained when cultures were infected with 2.5 × 10³ oocysts and incubated for 48 h. The applicable inoculum has to be optimised for each batch of oocysts and before each experimental series. Parasite development was inhibited completely by monensin at 134 nM and silymarin at 50 mg/mL. These concentrations were non-toxic to the host cells and comparable to literature data. The percentages of parasite inhibition were determined for monensin and a 50% inhibitory concentration (IC₅₀) of 36.6 nM (27.4–45.5) and a 90% inhibitory concentration of 65.9 nM (54.8–90.2) were calculated. The introduced assay is economic because relatively low parasite numbers may be used. If MICs are determined, evaluation is fast, as each well is viewed only briefly under the fluorescence microscope for presence or absence of CSI. Furthermore it is highly critical because only full parasite inhibition is assessed. Counting of CSI is more laborious and time-consuming, but it allows calculation of parasite inhibition rates and parameters like the half maximal inhibitory concentration (IC₅₀). This assay shall be used to assess anticryptosporidial activities of various plant waste materials and by-products from the food and the pharmaceutical industries in the course of the EU project SAFEWASTES. Comparison with in vivo models should be performed to further corroborate the results. Automated evaluation by flow cytometry might facilitate higher sample throughput and reduce operator bias.