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A rapid method for determining salinomycin and monensin sensitivity in Eimeria tenella
M C Jenkins1, C N O'Brien1, L Fuller2
1Animal Parasitic Diseases Laboratory, Building 1040, Agricultural Research Service, USDA, Beltsville, MD 20705, United States.
Abstract:
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.
Insights
This study introduces a rapid in vitro method to test Eimeria tenella sensitivity to salinomycin and monensin. The new assay accurately assesses drug resistance using molecular techniques, offering a cost-effective alternative to traditional methods.
Area of Science:
- Veterinary Parasitology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Standard Eimeria ionophore sensitivity testing involves in vivo chicken infection, which is time-consuming and resource-intensive.
- Eimeria tenella causes significant economic losses in poultry farming, necessitating efficient methods for monitoring drug resistance.
Purpose of the Study:
- To develop and validate a rapid in vitro assay for determining Eimeria tenella sensitivity to the ionophores salinomycin and monensin.
- To compare the efficacy of molecular methods (qPCR, sqPCR) with traditional sporozoite counting for assessing drug sensitivity.
Main Methods:
- Cultured MDBK cells were infected with E. tenella sporozoites and exposed to varying concentrations of salinomycin or monensin.
- Intracellular sporozoites and DNA were analyzed at 24 hours using direct counting, semi-quantitative PCR (sqPCR), and quantitative PCR (qPCR).
- The assay was validated using E. tenella field isolates known to be resistant to ionophores.
Main Results:
- A dose-dependent decrease in E. tenella sporozoites and DNA was observed with increasing drug concentrations, consistent across all tested methods.
- Quantitative PCR (qPCR) demonstrated higher sensitivity in detecting drug effects compared to sporozoite counting and sqPCR.
- The in vitro assay successfully identified ionophore-resistant E. tenella field isolates, showing higher DNA levels than the sensitive laboratory strain.
Conclusions:
- The developed in vitro assay provides a rapid, cost-effective, and reliable method for assessing Eimeria tenella ionophore sensitivity.
- Molecular techniques, particularly qPCR, enhance the sensitivity and accuracy of in vitro drug resistance testing.
- This assay facilitates timely monitoring of anticoccidial drug resistance in Eimeria, crucial for effective poultry health management.
