A rapid method for determining salinomycin and monensin sensitivity in Eimeria tenella

Standard methods of determining the ionophore sensitivity of Eimeria rely on infecting chickens with an isolate or a mixture of Eimeria spp. oocysts in the presence of different anti-coccidial drugs. The purpose of this study was to develop a rapid in vitro method for assessing salinomycin and monensin sensitivity in Eimeria tenella. Cultures of MDBK cells were grown to 85% confluency, and then inoculated with excysted E. tenella laboratory strain (APU-1) sporozoites in the presence of different concentrations of salinomycin or monensin. At various timepoints, the monolayers were fixed for counting intraceullar sporozoites, or were subjected to DNA extraction, followed by molecular analysis using quantitative (qPCR) or semi-quantitative PCR (sqPCR). Preliminary experiments showed that 24h was the optimum time for harvesting the E. tenella-infected cell cultures. The average number of E. tenella sporozoites relative to untreated controls displayed a linear decrease between 0.3 and 33.0 μg/ml salinomycin and between 0.3 and 3.3 μg/ml monensin. A similar pattern was observed in the relative amount of E. tenella DNA as measured by sqPCR. A linear decrease in the relative amount of E. tenella DNA was observed over the entire range of salinomycin and monensin concentrations as measured by qPCR possibly reflecting the greater sensitivity of this assay. Comparison of sporozoite counting, sqPCR, and qPCR signals using a criterion of 50% inhibition in sporozoite numbers or level of PCR amplification product showed good agreement between the three assays. E. tenella field isolates (FS-1 and FS-2) displaying resistance to salinomycin and monensin were evaluated in the in vitro assay using qPCR and sqPCR. Compared to E. tenella APU-1, the E. tenella FS-1 and FS-2 isolates showed higher levels of E. tenella DNA at 24h by both qPCR and sqPCR. This in vitro assay represents a significant advance in developing rapid, cost-effective methods for assessing ionophore sensitivity in E. tenella.