Anticoccidial efficacy testing: In vitro Eimeria tenella assays as replacement for animal experiments

Availability of an accurate in vitro assay is a crucial demand to determine sensitivity of Eimeria spp. field strains toward anticoccidials routinely. In this study we tested in vitro models of Eimeria tenella using various polyether ionophores (monensin, salinomycin, maduramicin, and lasalocid) and toltrazuril. Minimum inhibitory concentrations (MIC95, MIC50/95) for the tested anticoccidials were defined based on a susceptible reference (Houghton strain), Ref-1. In vitro sporozoite invasion inhibition assay (SIA) and reproduction inhibition assay (RIA) were applied on sensitive laboratory (Ref-1 and Ref-2) and field (FS-1, FS-2, and FS-3) strains to calculate percent of inhibition under exposure of these strains to the various anticoccidials (%ISIA and%IRIA, respectively). The in vitro data were related to oocyst excretion, lesion scores, performance, and global resistance indices (GI) assessed in experimentally infected chickens.Polyether ionophores applied in the RIA were highly effective at MIC95 against Ref-1 and Ref-2 (%IRIA≥95%). In contrast, all tested field strains displayed reduced to low efficacy (%IRIA<95%).%IRIA values significantly correlated with oocyst excretion determined in the animal model (p<0.01) for polyether ionophores. However, this relationship could not be demonstrated for toltrazuril due to unexpected lack of in vitro sensitivity in Ref-2 (%IRIA=56.1%). In infected chickens, toltrazuril was generally effective (GI>89%) against all strains used in this study. However, adjusted GI (GIadj) for toltrazuril-treated groups exhibited differences between reference and field strains which might indicate varying sensitivity.RIA is a suitable in vitro tool to detect sensitivity of E. tenella towards polyether ionophores, and may thus help to reduce, replace, or refine use of animal experimentation for in vivo sensitivity assays.