Storey Bob, Marcellino Chris, Miller Melissa, Maclean Mary, Mostafa Eman, Howell Sue, Sakanari Judy, Wolstenholme Adrian, Kaplan Ray
Department of Infectious Diseases, University of Georgia, Athens, GA, USA.
Center for Discovery and Innovation in Parasitic Diseases, University of California San Francisco, San Francisco, CA, USA ; Case Western Reserve University School of Medicine, Cleveland, OH, USA.
Int J Parasitol Drugs Drug Resist. 2014 Aug 28;4(3):233-43. doi: 10.1016/j.ijpddr.2014.08.003. eCollection 2014 Dec.
A major hindrance to evaluating nematode populations for anthelmintic resistance, as well as for screening existing drugs, new compounds, or bioactive plant extracts for anthelmintic properties, is the lack of an efficient, objective, and reproducible in vitro assay that is adaptable to multiple life stages and parasite genera. To address this need we have developed the "Worminator" system, which objectively and quantitatively measures the motility of microscopic stages of parasitic nematodes. The system is built around the computer application "WormAssay", developed at the Center for Discovery and Innovation in Parasitic Diseases at the University of California, San Francisco. WormAssay was designed to assess motility of macroscopic parasites for the purpose of high throughput screening of potential anthelmintic compounds, utilizing high definition video as an input to assess motion of adult stage (macroscopic) parasites (e.g. Brugia malayi). We adapted this assay for use with microscopic parasites by modifying the software to support a full frame analysis mode that applies the motion algorithm to the entire video frame. Thus, the motility of all parasites in a given well are recorded and measured simultaneously. Assays performed on third-stage larvae (L3) of the bovine intestinal nematode Cooperia spp., as well as microfilariae (mf) of the filarioid nematodes B. malayi and Dirofilaria immitis, yielded reproducible dose responses using the macrocyclic lactones ivermectin, doramectin, and moxidectin, as well as the nicotinic agonists, pyrantel, oxantel, morantel, and tribendimidine. This new computer based-assay is simple to use, requires minimal new investment in equipment, is robust across nematode genera and developmental stage, and does not require subjective scoring of motility by an observer. Thus, the "Worminator" provides a relatively low-cost platform for developing genera- and stage-specific assays with high efficiency and reproducibility, low labor input, and yields objective motility data that is not subject to scorer bias.
评估线虫群体的抗驱虫药能力，以及筛选现有药物、新化合物或具有驱虫特性的生物活性植物提取物时，一个主要障碍是缺乏一种高效、客观且可重复的体外检测方法，该方法要能适用于多个生命阶段和寄生虫属。为满足这一需求，我们开发了 “蠕虫分析仪” 系统，该系统能客观且定量地测量寄生线虫微观阶段的活力。该系统围绕计算机应用程序 “蠕虫检测” 构建，此程序由加州大学旧金山分校寄生虫病发现与创新中心开发。“蠕虫检测” 的设计目的是评估宏观寄生虫的活力，以便对潜在驱虫化合物进行高通量筛选，它利用高清视频作为输入来评估成虫阶段（宏观）寄生虫（如马来布鲁线虫）的运动。我们通过修改软件以支持全帧分析模式，将此检测方法应用于微观寄生虫，该模式将运动算法应用于整个视频帧。这样，给定孔中所有寄生虫的活力会被同时记录和测量。对牛肠道线虫古柏属的三期幼虫（L3）以及丝状线虫马来布鲁线虫和犬恶丝虫的微丝蚴（mf）进行的检测，使用大环内酯类药物伊维菌素、多拉菌素和莫西菌素，以及烟碱类激动剂噻嘧啶、奥克太尔、莫仑太尔和三苯双脒时，产生了可重复的剂量反应。这种基于计算机的新检测方法使用简单，设备方面所需的新投资极少，对线虫属和发育阶段具有很强的适应性，并且不需要观察者对活力进行主观评分。因此，“蠕虫分析仪” 提供了一个成本相对较低的平台，可高效、可重复地开发属特异性和阶段特异性检测方法，劳动力投入低，并能产生不受评分者偏差影响的客观活力数据。
