Evans Christopher C, Moorhead Andrew R, Storey Bobby E, Blagburn Byron L, Wolstenholme Adrian J, Kaplan Ray M
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.
Vet Parasitol. 2017 Nov 15;246:76-81. doi: 10.1016/j.vetpar.2017.09.003. Epub 2017 Sep 8.
Anthelmintics of the macrocyclic lactone (ML) drug class are widely used as preventives against the canine heartworm (Dirofilaria immitis). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC values were calculated. The D. immitis LMIA yielded consistent and reproducible dose-response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L were much higher than those third- and fourth-stage larvae would experience in vivo. IC values ranged from 1.57 to 5.56μM (p≥0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage.
大环内酯类（ML）药物类驱虫剂被广泛用作犬心丝虫（犬恶丝虫）的预防药物。然而，在过去几年中，出现了ML缺乏疗效（LOE）的报道，即尽管每月进行预防性给药，但犬仍感染了成熟的心丝虫。最近，从LOE病例中分离出的虫株已被用于感染实验犬，并且在以正常预防剂量进行ML治疗的情况下，通过成虫的建立证实了耐药表型。检测和监测犬恶丝虫的耐药性需要经过验证的生物学或分子诊断方法。在本研究中，我们评估了一种幼虫迁移抑制试验（LMIA），该试验是我们之前为犬恶丝虫第三期幼虫（L3）优化的。我们使用该试验来测量已知易感的犬恶丝虫实验室株和来自三个不同LOE病例的三个高度疑似ML耐药分离株的体外ML敏感性；其中两个分离株的后代已通过在受控环境中对感染犬的治疗被确认为ML耐药株。将非线性回归模型拟合到剂量反应数据，从中计算出IC值。犬恶丝虫LMIA产生了一致且可重复的剂量反应数据；然而，在对照和LOE寄生虫之间未观察到药物敏感性的统计学显著差异。此外，使L3麻痹所需的药物浓度远高于第三期和第四期幼虫在体内所经历的浓度。IC值范围为1.57至5.56μM（p≥0.19）。这些数据可能表明，该寄生虫中的ML耐药性不是通过L3对ML药物麻痹作用的敏感性降低来介导的，因此基于运动性的试验可能不适用于测量ML对该目标阶段犬恶丝虫的作用。
