Genetic characterisation of the Theileria annulata cytochrome b locus and their impact on the buparvaquone resistance in ruminants
Control of tropical theileriosis depends on the use of buparvaquone drugs, the efficacy of which has been compromised by the emergence of resistance. The present study aims to understand the molecular genetics of buparvaquone resistance in T. annulata, concerning the role of mutations and the effect of selection pressures on their emergence and spread. First, we investigate the genetic characteristics of the cytochrome b locus in 10 susceptible and 7 resistant T. annulata isolates. 129G (GGC) mutation was found in the Q01 binding pocket and 253S (TCT) and 262S (TCA) mutations were identified within the Q02 binding pocket. Similarly, we have identified cytochrome b mutations 129G (GGC), 253S (TCT) and 262S (TCA) in 21/75 buffalo-derived and 19/119 T. annulata isolates show the evidence of positive selection pressure in ruminants. Both hard and soft selective sweeps have occurred with striking differences between isolates. For example, 19 buffalo-derived and 7 cattle-derived isolates contained 129G (GGC) and 253S (TCT) resistance haplotypes at a high-frequency demonstrating evidence of the emergence of resistance by a single mutation. Two buffalo-derived and 11 cattle-derived isolates contained 129G (GGC), 253S (TCT), 129G (GGC)/253S (TCT) and 262S (TCA) resistance haplotypes had equally high frequencies demonstrating evidence of the emergence of resistance by pre-existing and or recurrent mutations. Phylogenetic analysis further revealed that 9 and 21 unique haplotypes in buffalo and cattle-derived isolates were present in a single lineage, suggesting that there is a single origin of this mutation in T. annulata. The present study also proposed that animal migration between farms is an important factor in the spread of buparvaquone resistance in the endemic regions of Pakistan.