Liver fluke extract and excretory/secretory (ES) product preparation F. hepatica adults were collected, cultured in vitro for 4 h and the ES products collected and prepared as previously described [40] . Newly excysted juveniles (NEJ) were excysted from metacercariea in vitro and cultured in Fasciola saline for 4 h post excystment as previously described [41] . F. hepatica (adult and NEJ) soluble fractions were obtained by homogenisation of frozen fluke at 4uC in a glass grinder in lysis buffer (20 mM KHPO 4 , pH 7.0, 0.1% Triton-X100 and a cocktail of protease inhibitors [Roche, Complete-Mini, EDTA-free]). Homogenates were centrifuged at 100,0006 g for 1 h at 4uC. Supernatants were considered as the soluble cytosolic fraction. Cytosolic protein extracts were treated and resolved by 2DE as described previously [11] . F. hepatica eggs were isolated, cultured and protein extracted as previously described [42] .