A nanoluciferase-based in vitro platform for high-throughput screening of anticoccidial compounds using transgenic Eimeria tenella

Abstract Coccidiosis caused by Eimeria spp. remains a major constraint to poultry production worldwide, and the discovery of new anticoccidial agents is hindered by the lack of scalable in vitro screening systems. Here, we developed a phenotypic high-throughput screening (HTS) platform based on a transgenic E. tenella line coexpressing enhanced yellow fluorescent protein (EYFP) and nanoluciferase (Nluc), enabling sensitive and quantitative assessment of parasite growth in vitro. Using Madin-Darby bovine kidney (MDBK) cells as host cells, parasite replication was reliably monitored within a 45 h infection window via a luminescence readout. The assay exhibited strong linearity with the parasite inoculum and was validated using known anticoccidials, monensin and salinomycin, whose expected dose–response profiles were obtained. The platform was applied to screen 1,770 compounds, identifying 28 primary hits (> 50% inhibition). After the samples were filtered for their cytotoxicity and drug-like properties, three compounds (alendronate, pemetrexed, and torsemide) were selected for further evaluation. These compounds exhibited micromolar in vitro activity (EC 50 ~ 8–10 μM) with favorable selectivity indices, particularly for pemetrexed and torsemide (> 100). In a chicken infection model, compared with the vehicle control, all three compounds significantly reduced oocyst shedding. Using the anticoccidial index (ACI), a composite metric incorporating oocyst shedding, mortality, weight gain, and lesion severity, torsemide was most effective (ACI = 163.3), followed by pemetrexed (ACI = 153.5). Alendronate showed only marginal activity (ACI = 110.8). Overall, this study establishes a robust, scalable in vitro HTS platform for anticoccidial drug discovery and highlights several approved drugs as potential leads for further optimization.