Fig. 1

Overview of anticoccidial drugs and drug resistance mechanisms. A Anticoccidial drugs introduced into and used in the poultry industry. B The diagram of the three major mechanisms of drug resistance. a. enzymatic action: drugs’ effectiveness is reduced either because an enzyme degrades it or fails to activate it. b. concentration regulation: internal concentration of the drug is regulated, either by restricting its entry (influx) or enhancing its exit (efflux). c. target alteration: drugs’ effectiveness is compromised because the target molecule of the drug in the organism undergoes mutations or modifications, making the drug unable to bind or act effectively. Star, mutation. C Schematic of direct and reverse genetic study of anticoccidial drug resistance in Eimeria. ① Experimental evolution to obtain resistant strains: Starting with a wild-type strain, the organism is exposed to a drug for the selection of drug-resistant strains. The procedure includes periods of drug exposure and relaxation (no drug). ② Multi bioinformatics methods to track candidate loci: The genomic DNA of drug-resistant strains is analyzed to pinpoint areas (loci) in the genome that might be responsible for resistance. These candidate loci are highlighted by Single Nucleotide Polymorphisms (SNPs), which are variations in a single DNA building block. ③ Reverse genetic approaches to verify candidate genes: To validate the identified candidate genes, two methods are depicted: Overexpression: Overexpression involves inserting one or more copies of a specific gene to evaluate if its increased presence produces or enhances drug resistance. This method aims to assess how amplifying gene copies or its expression influences the organism's ability to combat drug stress; Homologous recombination: Homologous recombination via CRISPR/Cas9 involves creating targeted DNA breaks, enabling the replacement of a normal gene with a mutated one. This precise editing method induces drug resistance in parasites carrying the altered gene. DSB: double-strand break. HR, homologous recombination. FACS: fluorescence-activated cell sorting