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.热带泰勒虫病（tropical theileriosis）的防控依赖巴普喹酮（buparvaquone）药物的使用，但耐药性的出现已削弱了其临床药效。本研究旨在解析环形泰勒虫（T. annulata）中巴普喹酮耐药性的分子遗传学机制，探究突变的作用及选择压力对耐药性产生与传播的影响。首先，我们对10株敏感株与7株耐药株的环形泰勒虫细胞色素b（cytochrome b）基因座开展遗传特征分析：在Q01结合口袋中检出129G（GGC）突变，在Q02结合口袋中鉴定出253S（TCT）与262S（TCA）突变。同样地，我们在21/75份水牛来源的环形泰勒虫分离株、19/119株环形泰勒虫分离株中鉴定出上述细胞色素b突变，这些分离株均展现出反刍动物体内正向选择压力的特征。不同分离株间存在显著差异，且均发生了强硬选择清扫（hard selective sweeps）与软选择清扫（soft selective sweeps）。例如，19株水牛来源株与7株牛来源株均高频携带129G（GGC）与253S（TCT）耐药单倍型（haplotype），证实耐药性可通过单突变产生；另有2株水牛来源株与11株牛来源株，其携带的129G（GGC）、253S（TCT）、129G（GGC）/253S（TCT）及262S（TCA）耐药单倍型均处于高频状态，表明耐药性可通过预先存在的突变或反复突变产生。系统发育分析进一步显示，水牛来源株与牛来源株中分别存在9种与21种独特的耐药单倍型，且均聚类于同一进化枝，提示环形泰勒虫中的此类突变起源单一。本研究同时提出，农场间的牲畜迁徙是巴基斯坦流行区中巴普喹酮耐药性传播的重要影响因素。