Plant extract
B. aegyptiaca fruits were obtained from the nearby markets in Aswan, Upper Egypt, and validated at the Herbarium of National Research Centre. Their methanolic extract was acquired as prescribed by Shalaby et al. (2010).
Adult D. caninum
Adult D. caninum were collected from the intestines of naturally infected stray dogs that were executed by the Egyptian police. After recovery, the worms were identified based on the macroscopic appearance of proglottids (Edwards and Herbert 1981) and washed in body-warm normal saline.
In vitro treatment
The entire worms, under sterile conditions in a laminar flow cabinet, were transferred to a normal saline solution containing B. aegyptiaca extract at a concentration of 240 μg/ml. This concentration was chosen based on concentrations utilized in vitro with trials involving Toxocara vitulorum (Shalaby et al. 2012) and Paramphistomum microbothrium (Shalaby et al. 2016). A stock solution of methanolic extract at 10 mg/ml was prepared with a mixture of liquid paraffin and Tween 80 (v/v) for immediate use. Then, the entire worms were incubated for 12 and 24 h at 37 °C in an atmosphere of 5% CO2. Solvent control worms were incubated for 12 and 24 h in normal saline solution containing 0.2 % (v/v) mixture of liquid paraffin and Tween 80. Normal control worms were fixed immediately following the initial washing. Five worms were examined for each time period. The activity of worms was checked at 12- and 24-h incubation by visual observation and if necessary by physical excitation of the worms via gently agitating the culture media. The percent inhibition of motility of Balanites-treated cestodes was estimated using the following formula:
$$ \mathrm{Inhibitory}\ \mathrm{activity}=\frac{C-E}{C}\times 100 $$
where
C, mean number of control motile worms
E, mean number of exposed motile worms
Statistical analysis
Analysis of data was performed using the statistical program for the social sciences SPSS version 11. The significance of Balanites extract-induced inhibition in the motility of the cestodes was assessed using one-way analysis of variance (ANOVA) for each period of incubation.
Light microscopy
Following incubation, the gravid segments of Balanites-treated and untreated worms were cut into small, 5-mm pieces before being fixed at 10% buffered formol saline, and processed according to the method of Bancroft et al. (1996). The body wall of the gravid segments was studied and photographed using an Olympus CX41 microscope.
Scanning electron microscopy (SEM)
Following incubation, the anterior end of Balanites-treated and untreated worms was fixed intact for 12 h in a 3:1 mixture of 4% (w/v) glutaraldehyde in 0.12 M Millonig’s buffer, pH 7.4 and 1% aqueous osmium tetroxide. After this, the specimens were processed for SEM following a method previously reported (Shalaby et al. 2012).