One of the major stumbling blocks in identifying candidate drugs for the treatment of lymphatic filariasis and river blindness is the lack of a high throughput screening system for these large worms. The filarid nematodes are long and threadlike and cannot be easily assayed in a 96-well format. We therefore developed an automated imaging system in which Brugia malayi could be assayed in 24-well plates using a simple and inexpensive method called the WormAssay.
The WormAssay is a visual imaging system that utilizes a novel software program to capture video recordings to assay the effect of compounds on macroparasites. To test the robustness of the software program, we assayed Brugia malayi
female worms with 3 antihelminthic drugs: albendazole and fenbendazole (benzimidazoles) and ivermectin (macrocyclic lactone). Albendazole is widely used to treat intestinal nematode infections including ascariasisis and hookworm; ivermectin is used in mass drug administration to treat filariasis; and fenbendazole is used in the veterinary field to treat animals infected with intestinal parasites.
data using these compounds with adult filarids are not available. However, the study by Tompkins, Stitt and Ardelli (2010) showed that when adult male and female Brugia malayi
were exposed to ivermectin at
for 3 days, the motility (as measured in movements/minute) decreased from 250 units to 125 units which is approximately equivalent to the
) in our study using ivermectin (molecular weight
875.1 u) for 2 days 
. Townson et al. (1990) showed that adult male Onchocerca gutturosa
of ivermectin in the course of 7 days had greatly reduced motility levels (based on mean motility scores from 0–10) compared to controls 
. Motility scores for male worms exposed to
albendazole for 2 days were similar to those for their control worms. Although Townson et al. used Onchocerca
adults in their study, their results for both albendazole and ivermectin are consistent with our data.
We are currently using the visual imaging system to screen approximately 400 adult Brugia
females per assay. Along with our visual imaging system, we use a Biomek FX (Beckman Coulter) instrument to remove media from each well and dispense compounds. It takes approximately 15 minutes per plate of 24 worms (1 worm/well) to screen compounds at a single concentration and approximately 20 minutes per plate for an
. The system is capable of screening more worms but assay throughput is currently limited by the number of worms produced and delivered. Once we receive the 24-well plates containing individual adult female Brugia
, we estimate that it takes one person approximately 6–7 hours to setup an assay to screen 96 compounds (at single concentrations) using the Biomek FX and run the WormAssay (on Day 0). Plates are assessed every day for 3 days using the visual imaging system which takes approximately 15–20 minutes for 16 plates. Control worms under these conditions remain highly active while worms treated with low micromolar concentrations of ivermectin are killed as evidenced by the lack of motility. We have observed that the lack of motility is correlated with worm death; dead worms appear more opaque (in some cases are slightly tanned) and never regain motility.
Rather than using laborious and subjective methods of analyzing plates (manual examination of individual wells and plates with a dissecting scope and scoring worm movements relative to control worms), the WormAssay quantified each worm's movement simultaneously on the entire plate, with each plate taking approximately 30 seconds to 1 minute to read. Given the short read times, researchers can increase the number of replicates per compound, thus increasing the accuracy of the assay. Currently, the system requires an individual to place the plate into the visual imaging box but this system is amenable for use with a robotic arm, removing and replacing plates to and from a plate hotel. The software application also includes bar code reading capabilities and can easily be exported to spreadsheets for data analysis.
WormAssay is a unique high-throughput screening motility assay that performs a parallel analysis on each well of entire plates simultaneously, but is independent of specific plate geometry and parasite morphology. The application supports 6-, 12-, 24-, 48- and 96-well plates. WormAssay does not track specific organismal characteristics so it can assay the motility of a large range of macroscopic organisms that can be cultured in a microtiter plate, but is capable of tracking very small or refined movements. The assay requires commodity computer equipment and is compatible with a variety of HD 1080p (or greater resolution) cameras and video capture interfaces. This low-cost and simple-to-use system can also be applied to other target organisms as well. Movements of other macroparasites, including adult schistosome worms were also assessed (see ), and studies with other macroorganisms are currently being explored.
In summary, the WormAssay offers several advantages: 1) it is inexpensive with costs of the video camera, LED lights and camera totaling less than $3,000 USD and the software is freely available, 2) it is easy to use, i.e. the plate can be quickly placed into the box housing the video camera and removed, 3) video recordings are saved onto the computer along with the data and can be reanalyzed at a later time, 4) entire plates with 6-, 12-, 24-, 48- and 96-wells can be assayed simultaneously, 5) the phenotype (worm movement) is quantified and stored as CSV files and 6) can be more generally applied to the study of macroparasites or other macroscopic organisms.