Institute of Parasitology of the Slovak Academy of Sciences, Hlinkova 3, 040 01, Košice, Slovakia.
Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Robert-Von-Ostertag-Str. 7-13, 14163, Berlin, Germany.
Sci Rep. 2023 Jan 23;13(1):1296. doi: 10.1038/s41598-023-28168-0.
We evaluated the variation in the frequency of benzimidazole (BZ) resistance-associated alleles at codons 200, 167 and 368 (F200Y, F167Y, V368L) of the β-tubulin isotype 1 gene during the patent period in isolates of Haemonchus contortus susceptible and resistant to BZ using pyrosequencing. Four lambs 5-6 months old were infected with 5000-6000 infective third-stage larvae (L3) of the susceptible MHco1 and the multi-resistant MHco4 isolates, respectively. Faecal samples were collected 28-times during 20-90 days post-infection (dpi). Coprocultures were subsequently prepared to produce L3 for genotyping. The frequency of the resistant allele (TAC) at codon 200 in MHco1 was lowest at 43 and 76 dpi with at each time point 0% and highest at 36 dpi with 10.85%, with a mean of 6.47% ± 2.39 and a coefficient of variation of 37.01%. The frequency of the TAC at codon 200 in MHco4 was lowest at 76 dpi with 25.6% and highest at 90 dpi with 49.25%, with a mean of 35.7% ± 4.42 and a coefficient of variation of 12.39%. No resistance alleles were detected in MHco1 at either codon 167 or 368. For MHco4 isolate, resistance alleles were detected only on codon 167 with a mean of 8.00% ± 4.83 and a mean coefficient of variation of 60.40%. Our results demonstrate the considerable variation in the frequency of resistant alleles in the susceptible and resistant isolates during the patent period. This variation should be considered when testing for the presence of BZ resistance in populations of gastrointestinal parasites, especially those with a low frequency of TAC.
我们使用焦磷酸测序法评估了在易感性和抗苯并咪唑（BZ）的旋毛虫分离株的专利期内，β-微管蛋白同工型 1 基因的 200、167 和 368 密码子（F200Y、F167Y、V368L）处与 BZ 抗性相关的等位基因的频率变化。四只 5-6 月龄的羔羊分别感染了 5000-6000 条易感性 MHco1 和多抗性 MHco4 分离株的感染性 3 期幼虫（L3）。在感染后 20-90 天内，共采集了 28 次粪便样本。随后准备粪培养物以产生 L3 进行基因分型。MHco1 中 200 密码子处抗性等位基因（TAC）的频率在 43 和 76 dpi 时最低，每个时间点均为 0%，在 36 dpi 时最高，为 10.85%，平均值为 6.47%±2.39，变异系数为 37.01%。MHco4 中 200 密码子处 TAC 的频率在 76 dpi 时最低，为 25.6%，在 90 dpi 时最高，为 49.25%，平均值为 35.7%±4.42，变异系数为 12.39%。在 MHco1 中未检测到 167 或 368 密码子处的抗性等位基因。对于 MHco4 分离株，仅在 167 密码子处检测到抗性等位基因，平均值为 8.00%±4.83，平均变异系数为 60.40%。我们的研究结果表明，在专利期内，敏感和抗性分离株中抗性等位基因的频率存在很大差异。在检测胃肠道寄生虫种群中是否存在 BZ 抗性时，特别是在 TAC 频率较低时，应考虑这种差异。
