University of Georgia College of Veterinary Medicine, Department of Infectious Diseases, Athens, GA, United States.
Savannah River Ecology Laboratory, University of Georgia, Aiken, SC, United States.
Vet Parasitol. 2020 Jul;283:109125. doi: 10.1016/j.vetpar.2020.109125. Epub 2020 May 23.
Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of macrocyclic lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei's distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.
犬心丝虫（Dirofilaria immitis）的感染预防基于大环内酯（ML）类药物的合规使用。犬心丝虫对 ML 类药物的耐药性已有充分的记载；然而，关于耐药分离株的空间分布和流行情况的信息仍然很少。本项目旨在通过种群遗传学方法来提高对 ML 类药物耐药性的认识。我们开发了一个大型微卫星基因座面板，并鉴定了 12 个新的高度多态性标记。这 12 个标记和之前发表的 5 个标记被用于筛选来自 16 个经证实的药物敏感、25 个经证实的药物耐药和 10 个疑似药物耐药的现场分离株的微丝蚴池。在微丝蚴抑制测试显示耐药的分离株中，空间主成分分析（sPCoA）、邻接法树和贝叶斯聚类均显示出治疗前后样本之间存在高度遗传相似性。有点令人惊讶的是，使用成对 Nei 距离生成的邻接法树和 sPCoA 并未显示出耐药分离株的聚类，也未显示出在佐治亚州、路易斯安那州或密西西比州采集的样本的州级地理聚类。相比之下，主成分判别分析能够区分敏感、疑似耐药和耐药样本。然而，未检测到与耐药相关的标记，这种聚类是由多个位点的多个等位基因的综合作用驱动的。此外，我们还测量了来自同一来源感染的不同传代的实验室株之间出人意料的大遗传距离。这一发现强烈表明，实验室分离株的遗传组成可能会随着每次传代而发生很大变化，这可能是由于遗传瓶颈所致。总之，这些数据表明，耐药分离株的遗传变异性大于预期，并且在犬心丝虫总体上也是如此。我们的结果还表明，微卫星基因分型缺乏检测耐药特定遗传特征的敏感性。未来需要使用基因组分析来阐明 ML 类药物耐药分离株的遗传关系。
