背景:犬心丝虫对大环内酯抗性的报道增加,Dirofilariaimmitis令人震惊。虽然基于DNA的测试已经得到了很好的验证,它们可能很贵。在之前的研究中,我们表明,适用于96孔板格式和分光光度计读数的两个生化测试可以检测实验室验证的D.immitis分离株之间的差异。两项测试-Resazurin减少和Hoechst33342外排-分别检测了从感染的狗血液中分离出的微丝菌的代谢和P-糖蛋白活性。
方法:我们的目的是通过测试先前未测试的D.immitis分离物中的各种测定参数来进一步优化两种测定。我们测试了微丝接种密度,孵育时间和伊维菌素和多西环素在其他五个D.immitis分离株JYD-34,大头,伯克利,格鲁吉亚III和LOL。所有测定均以3个技术重复和2-4个生物学重复进行。为了了解检测的分子基础,我们还对ABC转运体和细胞色素P450基因家族的选定药物代谢和消除相关基因进行了qPCR。
结果:通过这些测定检测的代谢和ABC转运蛋白活性在菌株之间变化。驱虫状态(耐药或易感)与代谢或P-gp流出无关。在ABC转运蛋白和细胞色素P450基因的菌株之间发现了基本转录变异。
结论:这些测定提供了对D.immitis分离株之间的生化变异的更多了解,将来可以利用它来开发能够区分易感和抗性分离株的体外诊断测试。
BACKGROUND: The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux-detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood.
METHODS: Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates-JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2-4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families.
RESULTS: Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes.
CONCLUSIONS: These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates.