Fitness and Apoptotic Capacity of Buparvaquone-Resistant Theileria annulata Cell Lines with Mutations in Cytochrome B

Tropical theileriosis, caused by Theileria annulata and transmitted by Hyalomma ticks, is one of the fundamental diseases affecting cattle and resulting in substantial economic losses. In recent years, there has been an increase in the rate of buparvaquone (BPQ) treatment failure cases, particularly in infections caused by parasite populations with certain mutations on the parasite's cytochrome b (Cyto b) gene. Biological fitness of the mutated, drug-resistant parasite is an important parameter to limit the spread and delay the emergence of drug resistance in new areas. Programmed cell death (apoptosis) is a defense mechanism against intracellular infection agents such as T. annulata that have the unique ability to induce uncontrolled but fully reversible proliferation of the host cells following invasion. The cells treated with BPQ in vitro survive for a few more days and undergo apoptosis. Besides, the capacity of drug-resistant T. annulata populations to inhibit apoptosis is still questionable. Here, we have evaluated the fitness costs in both mutated drug-resistant and wild-type clonal cell lines under long-term in vitro conditions in the absence of BPQ pressure. This study also addressed the question of what occurs when the parasite population develops drug resistance by evaluating the effects of the P253S mutation, which confers BPQ resistance, on the apoptotic activity of T. annulata populations in vitro. The fluctuations observed in both the intensity of the bands and the half-maximal inhibitory concentration values indicated the presence of a dynamic change. However, this did not impose a fitness cost on either population during the routine in vitro maintenance for up to 50 passages. The drug-resistant parasite population harboring the P253S mutation successfully adapted to in vitro culture alongside the drug-sensitive population in the absence of drug pressure. The percentage of apoptotic cells observed in the BPQ-resistant clonal cell line between 0 and 72 hr after BPQ treatment remained almost the same as that of the drug-sensitive one. Thus, the mutated parasite populations exhibit resistance to apoptosis, and this resistance persists even after the withdrawal of BPQ. This study represents a foundational step in understanding the fitness costs associated with BPQ resistance in T. annulata under in vitro conditions, with a particular focus on the effects of the P253S mutation in the Cyto b gene on the fitness of BPQ-resistant parasite populations. Although these results do not directly quantify fitness costs, they indicate that drug-resistant mutations such as P253S may persist and spread in natural populations, even in the absence of drug pressure.