Distinct non-synonymous mutations in cytochrome b highly correlate with decoquinate resistance in apicomplexan parasite Eimeria tenella

Abstract

Abstract Background Protozoan parasites of the genus Eimeria are the causative agents of chicken coccidiosis. Parasite resistance to most anticoccidial drugs is one of the major challenges in controlling this disease. There is an urgent need for a molecular marker to monitor the emergence of resistance against anticoccidial drugs, such as decoquinate. Methods In this study, we developed decoquinate-resistant strains by successively exposing the Houghton (H) and Xinjiang (XJ) strains of E. tenella to incremental concentrations of the drug in chickens. Additionally, we isolated a decoquinate-resistant strain from the field. The resistance of these three strains was tested using the criteria of weight gain (WG), relative oocyst production (ROP), and reduction of lesion scores (RLS). Whole-genome sequencing was used to identify the non-synonymous mutations in coding genes that were highly associated with the decoquinate-resistant phenotype in the two laboratory-induced strains. Subsequently, we further scrutinized the missense mutation in a field-resistant strain for verification. We employed AlphaFold and PyMOL to model the alterations in the binding affinity of the mutants towards the drug molecule. Results We obtained two decoquinate-resistant strains, DecR_H and XJ, originating from the H and XJ strains, respectively, as well as a field-resistant E. tenella strain, DecR_SC. These three strains displayed resistant to 120 mg/kg decoquinate administered through feed. Through whole-genome sequencing analysis, we identified the cytochrome b gene (ETH2_MIT00100) as the sole mutated gene shared between the DecR_H and XJ strains and was also detected in the DecR_SC strain. Distinct non-synonymous mutations, namely Gln131Lys in the DecR_H, Phe263Leu in the DecR_XJ, and Phe283Leu in the DecR_SC were observed in the three resistant strains. Notably, these mutations were located in the extracellular segment of cytochrome b, in close proximity to the ubiquinol oxidation site Qo. Drug molecular docking studies revealed that these mutants exhibited varying degrees of reduced binding ability to decoquinate. Conclusions Our findings emphasize the critical role of cytochrome b mutations in the development of decoquinate resistance in E. tenella. The strong correlation observed between cytochrome b mutant alleles and resistance indicates their potential as valuable molecular markers for the rapid detection of decoquinate resistance.