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Title:   Author  item
                                            Jenkins, Markitem
                                            Obrien, Celiaitem
                                            FULLER, L - University Of Georgiaitem
                                            MATHIS, G - Georgia Poultry Laboratory Networkitem
                                            Fetterer, Raymond
  Submitted to: Veterinary Parasitology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 8/22/2014
                                    Publication  Date: 12/15/2014
                                    Citation: Jenkins, M.C., Obrien, C.N., Fuller, L., Mathis, G.F., Fetterer, R.H. 2014. A rapid method for determining salinomycin and monensin sensitivity in Eimeria tenella. Veterinary Parasitology. 206:153-158.Interpretive Summary: Avian coccidiosis is an intestinal parasitic disease of poultry caused by protozoa in the genus Eimeria.  Although vaccination is gaining acceptance in the poultry industry, outbreaks of coccidiosis are primarily controlled by the medication of feed with ionophore drugs or synthetic chemicals.  Drug treatment is being compromised by the ability of Eimeria parasites to develop resistance to ionophores and synthetic chemicals.  Poultry producers have no way of knowing beforehand the level of drug sensitivity on a particular poultry farm, and thus risk economic losses if drug resistant Eimeria are present in the litter.  Standard methods of assessing drug resistance in coccidia require isolating and expanding the Eimeria oocysts from litter, and then testing in susceptible chickens.  These assays are time-consuming and labor intensive.  The present paper describes an in vitro assay for drug sensitivity in Eimeria based on counting intracellular parasites in cell culture and analyzing DNA using sensitive molecular methods.  The data from parasite counting and molecular analyses provided similar findings suggesting that the in vitro method may be useful in rapidly assessing drug sensitivity in Eimeria.  Moreover, the assay may provide poultry producers a more rapid way of determining Eimeria drug sensitivity, preventing outbreaks of coccidiosis due to drug resistant parasites.
                                Technical 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 E. tenella.  Cultures of MDBK cells were grown to 85% confluency, and then inoculated with excysted E. tenella 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 or semi-quantitative PCR.  Preliminary experiments showed that 24 hr 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 ug/ml salinomycin and between 0.3 and 3.3 ug/ml monensin.  A similar pattern was observed in the relative amount of E. tenella DNA as measured by semi-quantitative PCR.  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 quantitative PCR possibly reflecting the greater sensitivity of this assay.  Comparison of sporozoite numbers, sqPCR, and qPCR signals using a criterion of 50% inhibition in sporozoite numbers or PCR amplicons showed good agreement between the 3 assays.  The described method represents a significant advance in developing rapid, cost-effective methods for assessing ionophore resistance in E. tenella.


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