Rufener Lucien, Kaminsky Ronald, Mäser Pascal
Novartis Centre de Recherche Santé Animale, 1566 St. Aubin, Switzerland.
Mol Biochem Parasitol. 2009 Nov;168(1):120-2. doi: 10.1016/j.molbiopara.2009.07.002. Epub 2009 Jul 16.
Benzimidazoles were the first broad-spectrum anthelmintics and are still in use today against gastro-intestinal nematodes of ruminants such as Haemonchus contortus. Benzimidazoles block the polymerization of nematode microtubules. However, their efficacy is jeopardized by the spread of drug-resistant parasites that carry point mutations in beta-tubulin. Here we use a novel in vitro selection-in vivo propagation protocol to breed drug-resistant H. contortus. After 8 generations of selection with thiabendazole an in vitro resistance factor of 1000 was reached that was also relevant in vivo in infected sheep. The same procedure carried out with ivermectin produced only a moderate resistance phenotype that was not apparent in sheep. Cloning and sequencing of the beta-tubulin genes from the thiabendazole-resistant H. contortus mutants revealed all of the isotype 1 alleles, and part of the isotype 2 alleles, to carry the mutation glutamate(198) to alanine (E198A). An allele-specific PCR was developed, which may be helpful in monitoring the prevalence of alanine(198) encoding alleles in the beta-tubulin isotype 1 gene pool of H. contortus in the field.
苯并咪唑是最早的广谱驱虫药，至今仍用于防治反刍动物的胃肠道线虫，如捻转血矛线虫。苯并咪唑可阻断线虫微管的聚合。然而，携带β-微管蛋白点突变的耐药寄生虫的传播危及了它们的疗效。在此，我们采用一种新型的体外筛选-体内繁殖方案来培育耐药的捻转血矛线虫。用噻苯达唑进行8代筛选后，达到了1000的体外耐药系数，这在感染绵羊的体内也很显著。用伊维菌素进行相同的操作仅产生了一种中度耐药表型，在绵羊中并不明显。对噻苯达唑耐药的捻转血矛线虫突变体的β-微管蛋白基因进行克隆和测序，发现所有1型等位基因以及部分2型等位基因都携带谷氨酸（198）突变为丙氨酸（E198A）的突变。开发了一种等位基因特异性PCR，这可能有助于监测田间捻转血矛线虫β-微管蛋白1型基因库中编码丙氨酸（198）的等位基因的流行情况。
