Optimized conditions for thein vitroexcystment ofCalicophoron daubneyimetacercariae

Paramphistomosis, caused byCalicophoron daubneyi, is an emerging infection of ruminants throughout Western Europe. Despite its prevalence, many questions remain regarding the basic biology of this parasite and how it interacts with its host. Consequently, there is a need to develop methods to studyC. daubneyi in vitroto improve our understanding of rumen fluke biology. Towards this, we aimed to identify a suitable protocol forin vitroexcystment ofC. daubneyimetacercariae. Six methods that have been used to excyst metacercariae from a number of trematode species were tested withC. daubneyimetacercariae. Three of these achieved an average of >50% excystment whilst one method, which included an acid-pepsin treatment, incubation in reducing conditions and an alkaline/bile salt solution to activate the larvae, consistently gave >80% excystment. The latter protocol also showed no detrimental effect on the motility of newly excysted juvenile (NEJ) parasites when observed for up to 24 h in RPMI 1640 medium post-excystment. The successful production ofC. daubneyiNEJsin vitrois a significant step forward, and will enable the discovery of infective stage-specific parasite antigens and facilitate drug screening trials, to aid the development of much needed diagnostic and therapeutic options for paramphistomosis.