*2.3. Preparation of Excretory/Secretory (E/S) Products and Somatic Extracts from Adult F. hepatica*

Adult liver flukes were collected from the livers of sheep during post-mortem abattoir surveillance in Roscommon, Ireland, as previously described [24]. The parasites were washed with sterile 1× PBS before culturing in RPMI medium (containing 0.1% glucose, 100 U penicillin and 100 mg/mL streptomycin) at a ratio of 1 worm/2 mL at 37 ◦C and 5% CO2. After 2 h, culture media (containing the E/S) was collected and centrifuged at 300× *g* for 10 min and then 700× *g* for 30 min to eliminate large debris. The supernatant was subsequently concentrated using a spin column protein concentrator with a molecular weight cut-off of 3 kDa (Amicon ultra, Merck Millipore, Burlington, MA, USA), aliquoted and frozen at −80 ◦C prior to use.

Adult somatic proteins were extracted from a frozen adult fluke via homogenisation in 100 μL DPBS followed by centrifugation at 300× *g* for 15 min and then 800× *g* for a further 15 min. The protein concentration in the resultant supernatant of the somatic extract and E/S was measured using the Bradford Protein Assay (Bio-Rad).
