Establishment Of In Vitro Culture Of Transgenic Eimeria Tenella And Application In Anticoccidial Drug Screening

Chicken coccidiosis is an acute epidemic intestinal infection of poultry caused by coccidia,of which Eimeria tenella is the most pathogenic one,causing huge economic losses to the chicken industry.Currently,the main focus is on drug and vaccine prevention,but the emergence of problems such as drug resistance,drug residues and dispersal toxicity has created an urgent need to find new methods and tools to address the current limitations of the disease treatment.Therefore,the development of new anticoccidial drugs is imperative.The development of anticoccidial drugs is hampered by the lack of methods available for high-throughput in vitro drug screening and validation.In vitro culture systems for coccidia may provide an important technical platform for exploring the growth and developmental mechanisms of coccidia and for screening new drugs and vaccine candidate antigens for the prevention and treatment of E.tenella.In this study,we first obtained an eYFP-nLuc stable transgenic strain of E.tenella and,for the first time,used the luciferase(nLuc)transgenic E.tenella strain as a model to establish a method that can be used for high-throughput in vitro screening of small molecule inhibitors,providing a new platform for anticoccidial drug development.The advantage of this model is that it can be directly examined optically and observed microscopically.Through validation,this study applied MDBK cells(Madin-Darby Bovine Kidney Cells)as a cell model for in vitro culture of transgenic E.tenella as well as drug screening,and established and optimized the in vitro culture method of transgenic E.tenalla.We observed the growth and development of transgenic E.tenella in MDBK cells under inverted fluorescence microscope,and photographed the development of coccidia at 48 h,68 h and 72 h after infection of host cells;and then we applied fluorescence signal detection to detect the relationship between the number of coccidia and fluorescence signal and found that they showed a positive correlation;next we used two positive drugs that have been applied in clinical treatment of E.tenella Next,monensin and salinomycin,which have been used in clinical treatment of E.tenella infection,were used to evaluate the relationship between the drugs and fluorescence signal in this model.The results showed that their effects were dose-dependent.In conclusion,an in vitro drug-screening model of with transgenic E.tenella was successfully established.Finally,1263 compounds from the library of FDA-approved drugs were screened by the model;and the cytotoxicity of these drugs were evaluated the compounds with high cytotoxicity were excluded.And made a control with fluorescent signal intensity.The three screened drugs Co-(1686),pemetrexed disodium and nilotinib hydrochloride were examined for their inhibition of intracellular survival activity of coccidia at different concentration gradients to determine their in vitro inhibition kinetic parameters.The results showed that Co-(1686),Pemetrexed disodium,and Nilotinib hydrochloride exhibited significant in vitro anticoccidial effects and low cytotoxicity.This study provided candidate molecules for the development of novel anticoccidial drugs.