In vitro activity of selected natural products against Eimeria tenella sporozoites using Reproduction Inhibition Assay

Eimeria tenella is the causative agent of cecal coccidiosis characterized by weight loss, hemorrhagic diarrhea, and high mortality rates. Research into herbal candidates to control the disease increased in the last decades by studying plant extracts with possible anticoccidial activity. As 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, 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 μg/ml and139.1 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 by incubating E. tenella sporozoites for 24 h with cinnamon oil at concentrations of 0.3 μg/ml to80.5 μg/ml. Glabridin (0.08 - 41.7 μg/ml) impaired sporozoites to replicate at a rate of14.1% to81.7% of inhibition. The calculated minimum concentrations of glabridin needed to inhibit parasite replication by 75%, 50%, and 30% (MIC 75 , MIC 50 , and MIC 30 ) were 21.43μg/ml, 5.28μg/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 provoker genes (HSP-70, NADPH, and EtPP5) after exposure of E. tenella sporozoites to glabridin at MIC 75 for 0.5 h, 1 h, 2 h, and 4 h (time-dependent experiment). Moreover, a dose-dependent experiment was performed using glabridin at a concentration resembling MIC 75 , MIC 50 , and MIC 30 for 24 h. In the time-dependent experiment, NADPH and EtPP5 were significantly ( p <0.05) overexpressed after 4 h of incubation with glabridin at a concentration of 21.43 μg/ml. The dose-dependent experiment exhibited a gradual overexpression in all studied genes which indicates stress imposed by glabridin on E. tenella sporozoites. In our hands, RIA was suitable toassess the strength of anticoccidial activity exhibited by the tested natural products. Such in vitro assays help to identify novel anticoccidial candidates such as herbal extracts.