Institute for Parasitology, Centre for Infectious Diseases, University of Veterinary Medicine, Buenteweg 17, 30559 Hannover, Germany.
Vet Parasitol. 2010 May 28;170(1-2):61-70. doi: 10.1016/j.vetpar.2010.01.032. Epub 2010 Feb 1.
Three different in vitro methods, the Larval Development Test (LDT), the Larval Migration Inhibition Test (LMIT) and the Micromotility Meter Test (MMT) have been adapted to detect anthelmintic resistance in cattle nematodes. Nematode eggs and third stage larvae of different Ostertagia ostertagi and Cooperia oncophora isolates were obtained from faecal cultures of experimentally infected calves. Additionally, adult C. oncophora were evaluated in the MMT for the detection of resistance to ivermectin (IVM). For all three in vitro tests standard operating procedures (SOPs) were established and successfully used for the detection of responses of non-parasitic and parasitic stages to different anthelmintic substances and the description of dose-response curves. In the LDT ivermectin (IVM) and thiabendazole (TBZ) were tested, in the LMIT IVM and levamisole (LEV) and in the MMT only IVM was evaluated. Susceptible isolates of C. oncophora and O. ostertagi, an IVM-resistant isolate of C. oncophora and a TBZ-selected isolate of O. ostertagi were used in all (C. oncophora) or only some of these tests (O. ostertagi). For all isolates sigmoidal dose-response curves and EC(50) values for the tested substances were obtained using a four-parameter logistic model. For the LDT, the previously reported problem in development of larvae was successfully overcome with mean development rates between 80% and 87% in negative controls. Following optimization of incubation times, temperatures, mesh sizes (LMIT only), nutritive medium (LDT only) and group size (MMT only) all three test systems reliably detected significant differences in the response to IVM between the susceptible and IVM-resistant isolate of C. oncophora (p<0.0001), resulting in an resistance ratio (RR) value of approximately 5 for IVM and 2.8 for LEV in C. oncophora. The LDT also detected differences in the response to TBZ between the susceptible and BZ-selected O. ostertagi isolates (p<0.001) with an RR of 2 for TBZ. With the standardization of the described tests we report reproducible and reliable in vitro methods for the detection of resistance to IVM (LDT, LMIT and MMT) and TBZ (LDT) for cattle parasitic nematodes.
三种不同的体外方法，幼虫发育试验（LDT）、幼虫迁移抑制试验（LMIT）和微运动计试验（MMT）已被改编用于检测牛线虫的驱虫剂耐药性。从实验感染小牛的粪便培养物中获得了不同的奥斯特拉格氏线虫和库珀氏线虫卵和第三阶段幼虫。此外，在 MMT 中评估了成年 C. oncophora 对伊维菌素（IVM）的耐药性。所有三种体外试验都建立了标准操作程序（SOP），并成功用于检测非寄生和寄生阶段对不同驱虫剂物质的反应，并描述了剂量反应曲线。在 LDT 中测试了伊维菌素（IVM）和噻苯达唑（TBZ），在 LMIT 中测试了 IVM 和左旋咪唑（LEV），而在 MMT 中仅评估了 IVM。在所有（C. oncophora）或仅其中一些试验（O. ostertagi）中使用了对 C. oncophora 敏感的分离株和对 IVM 耐药的 C. oncophora 分离株以及对 TBZ 选择的 O. ostertagi 分离株。使用四参数逻辑模型获得了所有分离株的 S 形剂量反应曲线和测试物质的 EC（50）值。对于 LDT，之前报道的幼虫发育问题已成功克服，阴性对照的平均发育率在 80%至 87%之间。在优化孵育时间、温度、网孔尺寸（仅 LMIT）、营养培养基（仅 LDT）和组大小（仅 MMT）后，所有三种试验系统都可靠地检测到对 C. oncophora 敏感和 IVM 耐药分离株对 IVM 反应的显著差异（p<0.0001），导致 IVM 的耐药比（RR）值约为 5，LEV 的 RR 值约为 2.8。LDT 还检测到对 TBZ 的反应在敏感和 BZ 选择的 O. ostertagi 分离株之间的差异（p<0.001），TBZ 的 RR 值为 2。通过描述的试验的标准化，我们报告了用于检测牛寄生线虫对 IVM（LDT、LMIT 和 MMT）和 TBZ（LDT）耐药性的重现性和可靠的体外方法。
