**Background:** Chicken coccidiosis, caused by Eimeria parasites, is a major economic burden on the poultry industry (~US$3 billion/year globally). Control relies heavily on anticoccidial drugs, but resistance has emerged to nearly all available compounds. Halofuginone, a halogenated derivative of febrifugine from Dichroa febrifuga, is used as a coccidiostat, yet resistant strains have been reported without known molecular mechanisms. In Plasmodium falciparum, prolyl-tRNA synthetase (PRS) was identified as halofuginone's molecular target. Given high sequence conservation of PRS among apicomplexans, the authors hypothesized that PRS mutations could confer halofuginone resistance in Eimeria.
**Methods:** The authors designed an experimental evolution strategy using Arbor Acres broilers. In the first experiment, 300 one-day-old chicks were divided into two groups: Group 1 (infection control, inoculated with 500 oocysts of wild-type E. tenella XJ strain) and Group 2 (fed 3 mg/kg halofuginone continuously). Litter containing oocysts from Group 1 was dispersed to Group 2 every 7 days from Day 7 to Day 49. Oocyst output was measured daily. Peak oocyst output in Group 2 occurred at ~35 days post-inoculation (dpi). In a second trial, 150 chicks were fed 30 mg/kg halofuginone (10× normal dose) and infected with litter from Group 2. After 6 additional generations of propagation under 30 mg/kg halofuginone, 12 resistant strains were obtained. Whole-genome sequencing (100× average nucleotide coverage) was performed on all 12 resistant strains and wild-type strains. Selective sweep analysis used population differentiation (FST) and heterozygosity (Hp) in 50-kb sliding windows (20-kb step). Candidate mutations were validated by PCR and Sanger sequencing. Overexpression constructs of EtcPRS Mut (T618A) were generated for E. tenella (under EtActin promoter, with C-terminal Flag tag) and T. gondii (under tubulin promoter at UPRT locus, with 3HA tag). Transgenic parasites were assessed by oocyst output, fluorescence microscopy, western blot, IFA, plaque assays, and replication assays.
**Key Results:** The experimental evolution strategy yielded halofuginone-resistant E. tenella strains in less than 2 months (55 days). Under 30 mg/kg halofuginone, resistant strains showed no significant difference in oocyst output compared to wild-type without drug. H&E staining revealed first-generation schizonts in resistant parasites under drug pressure, while wild-type parasites showed no first-generation schizonts when treated. Whole-genome sequencing identified a strong selective sweep on chromosome 10. Two outlier loci met the threshold of FST > 0.95 and Hp < 0.05. After excluding intergenic regions, synonymous mutations, and low-frequency variants, the primary candidate was ETH2_1020900 (EtcPRS, prolyl-tRNA synthetase), with mutations A1852G and A1854G corresponding to a single amino acid substitution T618A at 100% allele frequency in resistant strains. Molecular docking showed the mutation localizes to the prolyl-anticodon-binding domain, altering amino acid polarity and hydrophilicity. Overexpression of EtcPRS Mut in wild-type E. tenella conferred halofuginone resistance: transgenic parasites completed whole endogenous development under 30 mg/kg halofuginone, with fluorescence rates reaching 97% by F5 generation. In T. gondii, the wild-type strain was completely blocked by 5 nM halofuginone, while TgEtcPRS Mut-overexpressing parasites showed normal growth at concentrations up to 10 nM. When used as a selectable marker, EtcPRS Mut achieved positive rates >92% across multiple target loci (H2AX, SDHB) at halofuginone concentrations of 5–10 nM.
**Clinical Implications:** This study provides the first molecular marker (EtcPRS T618A) for halofuginone resistance in Eimeria, enabling rapid field detection of resistant parasites. The experimental evolution strategy (55 days) is substantially faster than traditional dose-escalation methods (which require 18–35 generations). The demonstration that EtcPRS Mut confers resistance in both E. tenella and T. gondii suggests broad-spectrum relevance within Apicomplexa. Additionally, EtcPRS Mut can serve as a stable selectable marker for genetic manipulation in T. gondii, with >92% screening efficiency. Limitations include that resistant strains were laboratory-induced (field strains may harbor additional mutations), lack of conditional gene knockout approaches in E. tenella, and absence of a continuous in vitro culture system for coccidia.