Abstract

Purpose Cultivation of Theileria annulata schizont-transformed cell lines typically relies on fetal bovine serum (FBS), limiting standardization. This study evaluated three serum-free media (SFMs) for the in-vitro cultivation of T. annulata -transformed cell lines derived from different isolates. Methods TaNM1 and TaXJS cell lines were adapted to AIM-V, VP-SFM, and HL-1, and compared against FBS-supplemented RPMI-1640. Proliferation, apoptosis, and parasite burden were assessed via viable cell counting, flow cytometry, and a novel TaSP -targeted absolute quantitative PCR (qPCR). Results AIM-V SFM and VP-SFM supported sustained proliferation of both cell lines after adaptation. AIM-V SFM showed the most stable performance among the tested serum-free conditions, with mean doubling times of 35.45 h for TaNM1 and 52.45 h for TaXJS. In contrast, unsupplemented HL-1 did not support robust growth, whereas supplementation with hypoxanthine-thymidine(HT) and AlbuMax I partially restored growth. Flow cytometry showed no marked increase in apoptosis under serum-free conditions, and qPCR indicated maintenance of T. annulata genome copy numbers during serum-free culture. Conclusions Serum-free culture of T. annulata schizont-transformed cell lines is feasible but strongly medium-dependent. AIM-V SFM and VP-SFM supported serum-free maintenance after adaptation, whereas HL-1 required supplementation. These findings provide a basis for further optimization of serum-free culture systems for standardized research and vaccine-related applications.