Development of an in vitro drug screening assay using Schistosoma haematobium schistosomula

Abstract

Background: The development of novel antischistosomal drugs is crucial, as currently no vaccine and only a single drug is available for the treatment of schistosomiasis. Fast and accurate in vitro assays are urgently needed to identify new drug candidates and research efforts should include Schistosoma haematobium. The aim of the present study was to develop a S. haematobium drug sensitivity assay based on newly transformed schistosomula (NTS).

Methods: We first undertook comparative studies on the cercarial emergence rhythms of the intermediate host snails Biomphalaria glabrata (S. mansoni) and Bulinus truncatus (S. haematobium). Two transformation methods as well as three purification methods were studied on S. haematobium cercariae in order to produce a large number of viable and clean NTS. Known antischistosomal drugs were tested in the established NTS assay in vitro. Drug effects were evaluated either microscopically or fluorometrically, using a resazurin based viability marker. Microscopically obtained IC₅₀ values were compared with results obtained for S. mansoni.

Results: A circadian rhythm existed in both snail species. Infected B. truncatus snails shed less cercariae than B. glabrata during the testing period. The highest transformation rate (69%) of S. haematobium cercariae into NTS was obtained with the vortex transformation (mechanical input) and the highest purification factor was observed using Percoll®. The fluorimetric readout based on resazurin was very precise in detecting dead or/and severely damaged schistosomula.

Conclusions: With the use of viability markers such as resazurin, drug screening assays using S. haematobium NTS can be efficiently performed. However, drugs acting on the morphology and motility of S. haematobium NTS, such as metrifonate are missed. Drug sensitivity assays with NTS of both species, S. haematobium and S. mansoni, showed very similar results using known antischistosomal drugs. The S. mansoni NTS assay might be more suitable as primary screen in drug discovery efforts, which ultimately aim for a broad-spectrum antischistosomal drug as a larger number of S. mansoni NTS can be generated.

Figure 1

Circadian rhythm of cercariae released from S. haematobium infected B. truncatus and S. mansoni infected B. glabrata snails. Mean values of (n = 3) performed experiments are shown, bars indicate standard deviations.

Figure 2

Survival time of S. haematobium NTS (150–200 NTS per well) in different culture media.

Figure 3

Relationship between fluorescence signal and number of schistosomula (25–250 schistosomula per well) in the resazurin based assay. Figure shows an average curve of four replicates, bars indicate standard deviations of the performed experiments. Fluorescence was measured after 24 hours of incubation. The fluorescence decreased with schistosomula concentrations above 200 per well. Linear regression is shown for data of 25-200 NTS.