B. malayi in vitro motility studies

Motility assays were carried out on the Worminator system in a 24-well tissue culture plate (1 worm/well) containing 1 mL of RPMI-media with l-glutamine as described (Marcelino et al. 2012). This assay assesses motility through pixel displacement of each worm over time, giving an output of Mean Movement Units (MMUs). An active compound that inhibits motility reduces the MMUs.

To determine the potency of levamisole on adult female B. malayi, worms were treated with various concentrations of levamisole (10 nM, 30 nM, 100 nM, 300 nM, 1 µM, 3 µM, 10 µM, 30 µM and 100 µM). Worm motility was then recorded prior to the addition of levamisole and 30 s post treatment for each drug concentration. From this we were able generate a concentration–response curve at 30 s and determine IC₅₀ values of levamisole. Percent motility was also calculated as a percentage ratio of motility of worms after treatment at the 30 s time point over motility of naïve worms.

Next, to study the long-term effects of various concentrations of levamisole on adult female B. malayi motility, we conducted motility assays over a 4-h period. Worm motility was recorded prior to the addition of levamisole, 16 s following the addition of levamisole and at 10, 20, 30, 40, 50, 60, 90, 120, 150, 180 and 240-min post treatment to generate a concentration- and time-course response analysis.

To quantify the effect of long-term application of levamisole at high concentrations, adult female B. malayi were assigned into control or drug treatment (n = 4/batch) groups (1 worm/well). Worm motility was recorded prior to the addition of levamisole, immediately following the addition of levamisole and at 30, 60, 90, 120, 150, 180, 210 and 240-min post treatment. Control worms were treated with deionized water, while drug treatment worms were exposed to 100 µM levamisole. Motility was recorded for 30 s for all time points. Three independent experiments were carried out for this study.

To investigate the effects of pyrantel, morantel and nicotine individually on adult female B. malayi motility, worms were exposed to each drug individually at a concentration of 10 µM. Control worms were exposed to de-ionized water. Worm motility was recorded as previously described above. Two independent experiments were carried out for this study.

To examine the effects of a second application of levamisole on worms that recovered during the levamisole 4 h treatment, we transferred the worms to fresh RPMI media and added 100 µM after 1 minute or after 120 minutes. As stated previously, worm motility was recorded using the Worminator system prior to the second addition of levamisole, and following addition of the drug. Control worms were treated with de-ionized water.