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New codon 198 β-tubulin polymorphisms in highly benzimidazole resistant Haemonchus contortus from goats in three different states in Sudan.
Parasites & Vectors ( IF 3.0 ) Pub Date : 2020-03-02 , DOI: 10.1186/s13071-020-3978-6
Khalid M Mohammedsalih 1 , Jürgen Krücken 2 , Amna Khalafalla 3 , Ahmed Bashar 1 , Fathel-Rahman Juma 1 , Adam Abakar 4 , Abdalhakaim A H Abdalmalaik 1 , Gerald Coles 5 , Georg von Samson-Himmelstjerna 2
Affiliation  

BACKGROUND Benzimidazole (BZ) resistance in gastrointestinal nematodes is a worldwide problem for livestock production, particularly in small ruminants. Assignment of the emergence of resistance using sensitive and reliable methods is required to adopt the correct strategies for control. In Sudan, BZ resistant Haemonchus contortus populations were recently reported in goats in South Darfur. This study aimed to provide additional data regarding albendazole efficacy and to describe the prevailing molecular BZ resistance mechanisms. METHODS Faecal egg count reduction and egg hatch tests (EHT) were used to evaluate albendazole efficacy in three different areas of South Darfur using naturally (Rehed Al-Birdi and Tulus) and experimentally infected (Tulus and Um Dafuq) goats. Using samples from Central, East and South Darfur, pyro- and Sanger sequencing were used to detect the polymorphisms F167Y, E198A and F200Y in H. contortus isotype 1 β-tubulin in DNA extracted from pooled third-stage larval (L3) samples (n = 36) on days 0 and 10 during trials, and from pooled adult male H. contortus (treated goats, n = 14; abattoirs, n = 83) including samples from populations previously found to be resistant in South Darfur. RESULTS Albendazole efficacies at 5, 7.5 and 10 mg/kg doses were 73.5-90.2% on day 14 in natural and experimental infections while 12.5 mg/kg showed > 96.6% efficacy. EC50 in the EHT were 0.8 and 0.11 µg/ml thiabendazole in natural and experimental infection trials, respectively. PCRs detected Haemonchus, Trichostrongylus and Cooperia in L3 samples from albendazole-treated goats. Haemonchus contortus allele frequencies in codons 167 and 200 using pyrosequencing assays were ≤ 7.4% while codon 198 assays failed. Sanger sequencing revealed five novel polymorphisms at codon 198. Noteworthy, an E198L substitution was present in 82% of the samples (L3 and adults) including all post-treatment samples. Moreover, E198V, E198K and potentially E198I, and E198Stop were identified in a few samples. CONCLUSIONS To our knowledge, this is the first report of E198L in BZ resistant H. contortus and the second where this is the predominant genotype associated with resistance in any strongyle species. Since this variant cannot be quantified using pyrosequencing, the results highlight important limitations in the general applicability of pyrosequencing to quantify BZ resistance genotypes.

中文翻译:

苏丹三个不同州的山羊对苯并咪唑的高度抗性Haemonchus contortus的新密码子198β-微管蛋白多态性。

背景技术胃肠道线虫中的苯并咪唑(BZ)抗性是畜牧业,特别是在小型反刍动物中的世界性问题。为了采用正确的控制策略,需要使用灵敏可靠的方法来确定电阻的出现。在苏丹,最近在南达尔富尔州的山羊中报告了抗BZ的捻转血矛线虫种群。这项研究旨在提供有关阿苯达唑功效的其他数据,并描述流行的分子BZ耐药机制。方法使用粪便卵数减少和卵孵化试验(EHT)来评估南达尔富尔州三个不同地区使用天然山羊(Rehed Al-Birdi和Tulus)和实验感染的山羊(Tulus和Um Dafuq)的阿苯达唑效力。使用来自达尔富尔中部,东部和南部的样本,在实验期间的第0天和第10天,使用焦磷酸和Sanger测序检测从合并的第三级幼虫(L3)样本(n = 36)提取的DNA中的扭曲的H. contortus同型1β-微管蛋白的多态性F167Y,E198A和F200Y ,以及来自合并的成年雄性弯曲杆菌(经处理的山羊,n = 14;屠宰场,n = 83),包括先前在南达尔富尔州发现有抗药性的种群的样本。结果在第14天,在自然和实验感染中,阿苯达唑在5、7.5和10 mg / kg剂量下的功效为73.5-90.2%,而12.5 mg / kg则显示> 96.6%的功效。自然和实验感染试验中,噻苯达唑的EC50分别为0.8和0.11 µg / ml噻菌灵。PCR检测到从阿苯达唑处理过的山羊的L3样品中检出Haemonchus,Trichostrongylus和Cooperia。使用焦磷酸测序法测定的密码子167和200中的捻转血矛线虫等位基因频率≤7.4%,而198密码子测定失败。Sanger测序揭示了在198位密码子处的五个新的多态性。值得注意的是,包括所有后处理样品在内的82%的样品(L3和成人)中都存在E198L取代。此外,在一些样本中还发现了E198V,E198K以及潜在的E198I和E198Stop。结论据我们所知,这是E198L在BZ耐药的H. contortus中的首次报道,第二次是在任何强链霉菌中与耐药相关的主要基因型。由于不能使用焦磷酸测序对这种变异进行定量,因此结果突出了焦磷酸测序在量化BZ抗性基因型的通用性方面的重要局限性。Sanger测序揭示了在198位密码子处的五个新的多态性。值得注意的是,包括所有后处理样品在内的82%的样品(L3和成人)中都存在E198L取代。此外,在一些样本中还发现了E198V,E198K以及潜在的E198I和E198Stop。结论据我们所知,这是E198L在BZ耐药的H. contortus中的首次报道,第二次是在任何强链霉菌中与耐药相关的主要基因型。由于不能使用焦磷酸测序对这种变异进行定量,因此结果突出了焦磷酸测序在量化BZ抗性基因型的通用性方面的重要局限性。Sanger测序揭示了在198位密码子处的五个新的多态性。值得注意的是,包括所有后处理样品在内的82%的样品(L3和成人)中都存在E198L取代。此外,在一些样本中还发现了E198V,E198K以及潜在的E198I和E198Stop。结论据我们所知,这是E198L在BZ耐药的H. contortus中的首次报道,第二次是在任何强链霉菌中与耐药相关的主要基因型。由于不能使用焦磷酸测序对这种变异进行定量,因此结果突出了焦磷酸测序在量化BZ抗性基因型的通用性方面的重要局限性。此外,在一些样本中还发现了E198V,E198K以及潜在的E198I和E198Stop。结论据我们所知,这是E198L在BZ耐药的H. contortus中的首次报道,第二次是在任何强链霉菌中与耐药相关的主要基因型。由于不能使用焦磷酸测序对这种变异进行定量,因此结果突出了焦磷酸测序在量化BZ抗性基因型的通用性方面的重要局限性。此外,在一些样本中还发现了E198V,E198K以及潜在的E198I和E198Stop。结论据我们所知,这是E198L在BZ抗性弯曲杆菌中的首次报道,而第二个报告是与任何强链菌种抗性相关的主要基因型。由于不能使用焦磷酸测序对这种变异进行定量,因此结果突出了焦磷酸测序在量化BZ抗性基因型的通用性方面的重要局限性。
更新日期:2020-03-03
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