Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.
Int J Parasitol Drugs Drug Resist. 2013 Jun 6;3:102-8. doi: 10.1016/j.ijpddr.2013.05.001. eCollection 2013 Dec.
For more than 20 years, anthelmintics of the macrocyclic lactone (ML) drug class have been widely and effectively used as preventives against the canine heartworm, Dirofilaria immitis. However, in recent years an increased number of lack of efficacy (LOE) cases are being reported, in which dogs develop mature heartworm infections despite receiving monthly prophylactic doses of ML drugs. While this situation is raising concerns that heartworms may be developing resistance to MLs, compelling evidence for this is still lacking. Resolution of this dilemma requires validated biological or molecular diagnostic assays, but, unfortunately, no such tests currently exist. To address this need, we developed and optimized a larval migration inhibition assay (LMIA) for use with D. immitis third-stage larvae. The LMIA was used to measure the in vitro dose-response of two ML drugs (ivermectin and eprinomectin) on a known ML-susceptible laboratory strain of D. immitis. A nonlinear regression model was fit to the dose-response data, from which IC50 values were calculated; the mean IC50 and 95% confidence interval for IVM was 4.56 μM (1.26-16.4 μM), greater than that for EPR at 2.02 μM (1.68-2.42 μM), and this difference was significant (p = 0.0428). The R (2) value for EPR assays (0.90) was also greater than that for IVM treatment (0.71). The consistency and reproducibility of the dose-response data obtained with this assay suggests that it may be a useful technique for investigating the relative susceptibilities to ML drugs in other D. immitis populations.
二十多年来，大环内酯类（ML）药物一直被广泛有效地用作预防犬心丝虫病（Dirofilaria immitis）的药物。然而，近年来，越来越多的缺乏疗效（LOE）病例被报道，尽管狗每月接受 ML 药物的预防性剂量，但仍会出现成熟的心丝虫感染。虽然这种情况引起了人们的担忧，即心丝虫可能对 ML 产生了抗药性，但目前仍缺乏确凿的证据。要解决这一困境，需要经过验证的生物学或分子诊断检测，但不幸的是，目前还没有这样的检测方法。为了解决这一需求，我们开发并优化了一种用于第三期幼虫的心丝虫幼虫迁移抑制检测（LMIA）。该 LMIA 用于测量两种 ML 药物（伊维菌素和依普菌素）在已知的 ML 敏感实验室株 D. immitis 上的体外剂量反应。非线性回归模型拟合剂量反应数据，从中计算出 IC50 值；伊维菌素的平均 IC50 和 95%置信区间为 4.56 μM（1.26-16.4 μM），大于依普菌素的 2.02 μM（1.68-2.42 μM），且差异具有统计学意义（p=0.0428）。依普菌素检测的 R（2）值（0.90）也大于伊维菌素治疗的 R（2）值（0.71）。该检测方法获得的剂量反应数据的一致性和可重复性表明，它可能是一种有用的技术，可用于研究其他 D. immitis 群体对 ML 药物的相对敏感性。
