University of Georgia College of Veterinary Medicine Department of Infectious Diseases, 501 D.W. Brooks Dr., Athens, GA, 30602, Greece.
University of Georgia College of Veterinary Medicine Department of Infectious Diseases, 501 D.W. Brooks Dr., Athens, GA, 30602, Greece.
Vet Parasitol. 2020 Aug;284:109197. doi: 10.1016/j.vetpar.2020.109197. Epub 2020 Jul 30.
The fecal egg count reduction test (FECRT) is the only method commonly used for diagnosing anthelmintic resistance in gastrointestinal nematodes of cattle, but this method has several drawbacks that have limited its widescale implementation. Consequently, there exists a need to develop better methods for diagnosing resistance. Assays based on larval motility are used commonly for screening potential drug candidates, and for detecting drug resistance, but previous work in our lab demonstrated that the L3 stage failed to discriminate between avermectin-resistant and susceptible isolates of Cooperia spp. We hypothesized that the L4 may be a better stage for this purpose because it is a parasitic and actively feeding life stage without a double cuticle. L3 larvae of Cooperia spp. were exsheathed and cultured to L4 by maintaining them in media at 37 °C and 20 % CO, with media changes and observation every 48 h for nine days. Three avermectin-resistant and two avermectin-susceptible GIN isolates (diagnosed by FECRT) containing >88 % Cooperia spp., were used. Three biological replicates were performed for each parasite isolate using both eprinomectin and ivermectin. Eleven drug concentrations from 0.01um to 40um and negative controls were evaluated. Motility readings were taken using the Worminator system before addition of the drug and at 24- and 48 -hs post drug exposure. Resistance ratios for ivermectin and eprinomectin ranged from 0.35 to 2.75 and 0.54-1.03, respectively. Though significant differences (p < 0.05) in percent inhibition were found at some drug concentrations in some assays, there were no consistent significant differences in the dose-response between susceptible and resistant isolates. Inhibition was greater in about half of the assays for the susceptible isolates, and in half the assays for the resistant isolates. The lack of consistency in these data indicate that motility of L4 is not a reliable diagnostic phenotype for measuring resistance to avermectin drugs in Cooperia spp.
粪便卵囊减少试验（FECRT）是目前用于诊断牛胃肠道线虫抗药性的唯一方法，但该方法存在一些缺陷，限制了其广泛应用。因此，需要开发更好的方法来诊断抗药性。基于幼虫活力的测定方法常用于筛选潜在的药物候选物，并用于检测抗药性，但我们实验室之前的工作表明，L3 期无法区分阿维菌素耐药和敏感的 Coopertia 分离株。我们假设 L4 期可能是一个更好的阶段，因为它是一个寄生的、主动摄食的生活阶段，没有双层角质层。Coopertia spp.的 L3 幼虫在 37°C 和 20%CO 的培养基中蜕皮并培养到 L4 期，每 48 小时更换一次培养基并观察 9 天。使用 FECRT 诊断，有 3 个阿维菌素耐药和 2 个阿维菌素敏感的 GIN 分离株（含 >88%Coopertia spp.）。每个寄生虫分离株使用依维菌素和伊维菌素进行了 3 个生物学重复。评估了从 0.01µm 到 40µm 的 11 个药物浓度和阴性对照。在添加药物之前和药物暴露后 24 小时和 48 小时使用 Worminator 系统进行活力读数。伊维菌素和依维菌素的耐药比值范围分别为 0.35-2.75 和 0.54-1.03。虽然在一些试验中，一些药物浓度下的抑制百分比有显著差异（p<0.05），但在敏感和耐药分离株之间的剂量反应中没有一致的显著差异。在大约一半的敏感分离株试验中，抑制作用更大，在一半的耐药分离株试验中，抑制作用更大。这些数据的不一致表明，L4 的活力不是衡量 Coopertia spp.对阿维菌素类药物抗药性的可靠诊断表型。
