Larval Motility Assays

A high-throughput larval motility assay was used to test the anthelmintic efficacy of the selected bark extracts. The DP xCELLigence Real-Time Cell Analyzer, which measures electrical impedance-based signals across interdigitated microelectrodes integrated on the bottom of tissue culture e-plates, has previously been developed to diagnose anthelmintic resistance (13). Third-stage (infective larvae) T. circumcincta larvae were recovered from fecal cultures of monospecifically infected donor sheep after a 10-day incubation period at 20°C. Larval suspension [3,000 L3 per 100 μl of phosphate-buffered saline (PBS)] was added to the wells of E-plates, and the impedance was monitored every 15 sec, for 24 h at 37°C. All larvae used in the experiments were freshly produced (within 3 months of development to L3). At the end of this period, bark extracts were added in all wells at the 2 % concentration (1 g of extract in 20 ml of 1% DMSO) except the negative control wells, where 1% DMSO was added; these wells served as negative controls, and the expectation was that these larvae will be maintained alive until the end of the experiment. In addition to the negative controls, positive controls (dead larvae, which were frozen and maintained at −20°C for a month) were also included in three wells. The wells with dead larvae received bark extracts after 24 h, to enable the testing of our hypothesis (see below). Wells with no worms were also included as technical controls. All controls were present in all experiments. Impedance signals were monitored in total for 48 h (24 h prior to and 24 h post extract addition) before stopping the experiments. Each treatment was run in triplicate (technical replicate). Impedance data were analyzed and a motility index was calculated as described by Smout et al. (25). Motility index data were used for the statistical analysis as described below.