Data Sheet 1_Bioluminescence-based in vitro assay for rapid and quantitative anticoccidial screening.docx

Coccidiosis, caused by Eimeria parasites, is a major threat to global poultry production, and increasing restrictions on conventional anticoccidial drugs highlight the need for safer, more sustainable alternatives. Progress has been hindered by the lack of rapid, sensitive, and animal-sparing in vitro assays for quantifying parasite replication and drug efficacy. This study reports the development of a novel bioluminescent platform for anticoccidial screening based on a genetically modified Eimeria tenella line expressing NanoLuc luciferase (EtNluc). Parasite-associated bioluminescence enabled rapid and quantitative monitoring of intracellular development, allowing the tracking of different replication phases through schizont formation and merozoite release. Time course analysis showed minimal changes in relative light units (RLU) between 2 and 24 hours post infection (hpi), followed by a marked increase between 24 and 72 hpi, consistent with parasite replication. Among the tested multiplicities of infection (MOI), 4:1 exhibited the fastest growth, described by a linear model (slope = 2908 RLU/h, R2 = 0.84). A same-well repeated-measure analysis (2 and 72 hpi) confirmed the dose-dependent replication, with mean slopes of 2052.85, 765.07 and 523.63 RLU/h, respectively, supporting the selection of the MOI 4:1 for anticoccidial screening. These experimental conditions were used to evaluate the anticoccidial efficacy of commercial anticoccidial drugs (salinomycin and robenidine) and natural compounds (thyme and oregano essential oils, thymol, and carvacrol) under two experimental designs: short pre-incubation of sporozoites, and continuous exposure throughout intracellular development. Pre-incubation with commercial anticoccidials reduced invasion approximately to 65% for salinomycin and 44% for robenidine, whereas the essential oils and their bioactive constituents inhibited invasion by 30-55%, and reduced the replication slope to 33-60% of control values. Continuous exposure significantly impaired intracellular development for all treatments, reducing replication to 10-30% of controls, providing additional evidence that plant-derived compounds can complement commercial anticoccidials for integrated strategy for coccidiosis control in chickens. Overall, the EtNluc bioluminescent system provided a rapid, sensitive, and scalable method for quantifying E. tenella growth, suitable for in vitro anticoccidial screening, supporting the characterization of novel anticoccidial while reducing reliance on animal experimentation.