Institute of Parasitology, McGill University, Sainte Anne-de-Bellevue, QC, Canada.
Institute of Parasitology, McGill University, Sainte Anne-de-Bellevue, QC, Canada.
Vet Parasitol. 2022 Apr;304:109696. doi: 10.1016/j.vetpar.2022.109696. Epub 2022 Mar 11.
The filarial parasite Dirofilaria immitis causes dirofilariosis, a potentially fatal pulmonary condition in canids and felines. Dirofilariosis can be prevented by treatment with a prophylactic macrocyclic lactone (ML) regimen. Unfortunately, ML-resistant D. immitis isolates, genetically distinct from the wildtype population, have been confirmed via molecular markers. DNA-based tests for ML-resistance are costly and time-consuming. There lacks a simple and reliable in vitro biological test to differentiate D. immitis infections resulting from inadequate adherence to recommended prophylaxis regimens from those caused by truly resistant D. immitis isolates. The goal of the current study was to develop a minimally invasive rapid diagnostic in vitro biological assay to differentiate ML-susceptible from ML-resistant D. immitis isolates. The in vitro assay was developed based on the concept that MLs act on the microfilariae (mf) by paralyzing the excretory pore muscle, inhibiting the release of molecules, including enzymes, that regulate host immunity. The basis of the in vitro diagnostic assay is to assess the effects of ivermectin (IVM) exposure on the secretion of enzymes by the D. immitis mf at a concentration that distinguishes the ML-susceptible from ML-resistant isolates. The metabolic enzyme, triosephosphate isomerase (TPI), was chosen due to high abundance in the D. immitis secretome. In this study, the in vitro TPI enzymatic assay was optimized and tested in eight laboratory-maintained isolates. The ML-susceptible Missouri, Berkeley, and Georgia II isolate; the putative ML-susceptible Georgia III, and Big Head; and the ML-resistant JYD-34, Metairie, and WildCat. We observed mixed results, Missouri and Berkeley had statistically significant decreases in TPI activity following 24-hour in vitro IVM exposure. The three resistant isolates, JYD-34, Metairie, and WildCat showed no change in TPI activity following IVM exposure. The susceptible, or putative susceptible Georgia II, Georgia III, and Big Head isolates had a phenotypic response consistent with ML-resistance based on the in vitro assay. However, increasing genotypic evidence has presented a mixed genotype for the three isolates, indicating they may be partially selected for ML-resistance. The measurement of changes in enzymatic activity and the in vitro TPI enzymatic activity assay consequently does not appear to be a reliable detection method for ML-resistance but may be useful for identifying fully susceptible isolates.
犬恶丝虫（Dirofilaria immitis）寄生虫会引起犬恶丝虫病，这是一种潜在致命的犬猫肺部疾病。通过使用预防性大环内酯类药物（ML）方案进行治疗，可以预防犬恶丝虫病。不幸的是，通过分子标记物已经证实，具有遗传差异的耐 ML 犬恶丝虫分离株对 ML 具有耐药性。用于 ML 耐药性的基于 DNA 的检测既昂贵又费时。目前缺乏一种简单可靠的体外生物学检测方法来区分由于未遵循推荐的预防方案而导致的犬恶丝虫感染与真正的耐 ML 犬恶丝虫分离株感染。本研究的目的是开发一种微创快速体外生物学诊断检测方法，以区分 ML 敏感和 ML 耐药的犬恶丝虫分离株。该体外检测方法基于以下概念：ML 通过麻痹排泄孔肌肉作用于微丝蚴（mf），抑制包括酶在内的分子的释放，这些分子调节宿主免疫。体外诊断检测的基础是评估伊维菌素（IVM）暴露对 mf 分泌酶的影响，其浓度可区分 ML 敏感和 ML 耐药分离株。选择代谢酶三磷酸异丙酮（TPI）是因为其在犬恶丝虫分泌组中含量丰富。在这项研究中，优化了体外 TPI 酶检测方法，并在 8 个实验室维持的分离株中进行了测试。ML 敏感的密苏里州、伯克利和乔治亚 II 分离株；假定的 ML 敏感的乔治亚 III、大头和大头皮；以及 ML 耐药的 JYD-34、梅泰里和 WildCat。我们观察到混合结果，密苏里州和伯克利在 24 小时体外 IVM 暴露后 TPI 活性有统计学意义的降低。三个耐药分离株 JYD-34、梅泰里和 WildCat 在 IVM 暴露后 TPI 活性没有变化。敏感或假定敏感的乔治亚 II、乔治亚 III 和大头分离株的表型反应与体外检测方法一致，表明它们可能对 ML 耐药具有部分选择。然而，越来越多的基因型证据表明，这三个分离株具有混合基因型，表明它们可能部分选择了 ML 耐药性。因此，酶活性变化的测量和体外 TPI 酶活性检测似乎不是 ML 耐药性的可靠检测方法，但可能有助于识别完全敏感的分离株。
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