In vitro activity of selected natural products against Eimeria tenella sporozoites using reproduction inhibition assay.

Eimeria tenella is the causative agent of cecal coccidiosis in poultry characterized by weight loss, hemorrhagic diarrhea, and high mortality rates. Research into herbal candidates with possible anticoccidial activity has increased lately. As an alternative to animal experiments, an in vitro reproduction inhibition assay (RIA) was previously designed to determine the sensitivity of E. tenella isolates against ionophores. In this study, the RIA was used to test the anticoccidial activity of nutmeg oil, cinnamon oil, and glabridin. The concentration of nutmeg oil used in this study ranged between 1.1 and 139.1 μg/ml. Nutmeg oil exhibited a moderate in vitro inhibitory activity ranging from 35.5 to 49.5%. In contrast, no inhibitory effect was detected when incubating E. tenella sporozoites for 24 h with cinnamon oil at concentrations of 0.3 to 80.5 μg/ml. Glabridin (0.08-41.7 μg/ml) prevented the replication of sporozoites at a rate of 14.1 to 81.7% of inhibition. The calculated minimum concentrations of glabridin needed to inhibit parasite replication by 75%, 50%, and 30% (MIC75, MIC50, and MIC30) were 21.43 μg/ml, 5.28 μg/ml, and 0.96 μg/ml, respectively. Further studies to assess the in vitro efficacy of glabridin were performed by studying mRNA gene expression of stress-induced protein genes (HSP-70, NADPH, and EtPP5) after exposure of E. tenella sporozoites to glabridin at MIC75 for 0.5 h, 1 h, 2 h, and 4 h (a time-dependent experiment). Moreover, a dose-dependent experiment was performed using glabridin at a concentration matching MIC75, MIC50, and MIC30 for 24 h. In the time-dependent experiment, a significant (p < 0.05) increase of expression in NADPH and EtPP5 were detected after 4 h of incubation with glabridin at a concentration of 21.43 μg/ml. The dose-dependent experiment exhibited a gradual increase of expression in all studied genes, which indicates stress imposed on E. tenella sporozoites by glabridin. In our hands, RIA was suitable to assess the anticoccidial activity exhibited by the tested natural products as a precursor to in vivo studies which will help in the identification of novel anticoccidial candidates.