Excystment of Fasciola Metacercariae

F. hepatica (US Pacific North West wild strain) metacercariae, with outer cyst wall removed, were obtained from Baldwin Aquatics Inc (Monmouth, Oregon, USA), and stored at 4°C until required. F. gigantica metacercariae were obtained from naturally infected wild snails collected in Aligarh, Uttar Pradesh, India, by researchers at Aligarh Muslim University. Outer cyst walls were removed by incubation in a solution of 1% pepsin, 4 mM HCl, for 90 min at 37°C, followed by several washes in distilled water. Metacercariae were then excysted by incubation in 0.6% sodium bicarbonate, 0.45% sodium chloride, 0.4% sodium tauroglycocholate, 0.025 M HCl, 0.4% L-Cysteine, for up to 3 h at 37°C. After approximately 75 min, at 10–15 min intervals, NEJs were transferred to RPMI 1640 without phenol red (Life Technologies), in which they were maintained at 37°C for a maximum of 3 h until transferred to soaking media. F. gigantica cysts were exposed to excystment media for 3 h, after which they were transferred to RPMI for incubation, in which their excystment completed within 18 h. Variations in maintenance/soaking conditions are described below.