Paramphistomum spp: improved artificial excystment and in vitro culture of immature and adult stages

Abstract

The aims of the present study were to establish an adequate excystment method for metacercariae of Paramphistomum spp. and to compare the in vitro survival of paramphistomids in three different culture media. In a preliminary study, 31 different assays were performed solely with the purpose of obtaining the best percentage of metacercariae excystment. Once the best excystment method was selected, 21 further assays using 100 metacercariae/assay were carried out to determine the average percentage of excystment. For the in vitro culture of paramphistomids, the Hedon-Fleig, Rohrbacher, and RPMI-1640 culture media were compared by using 24-well immunoplates containing newly excysted flukes or 50-ml capacity tissue-culture flasks for adult paramphistomids. Results indicated an average of 69.0% excystment, the best culture medium to support the survival of immature and adult stages of the trematode being Hedon-Fleig and Rohrbacher, respectively. The best percentage of survival obtained was on day 4 (80%), with some immature and adult trematodes remaining alive until days 10 and 11, respectively.

In Vitro Maintenance of Clonorchis sinensis Adult Worms

“…[19], and Cymatocarpus solearis [20]. M. novaezealandensis juvenile worms were cultured up to 5 days in NCTC-109 medium with or without serum supplements but the media showed no significant differences [17].…”

“…Paramphistomum spp. adults were maintained in Hedon-Fleig, Rohrbacher, and RPMI-1640 media and lasted for 11 days in Rohrbacher medium [19]. Furthermore, ovoculture technique were applied to culture C. solearis from metacercarial stage to adult and the authors recovered only 6 adults with eggs in the uterus after 24 days from about 100 metacercariae [20].…”

Optimized conditions for thein vitroexcystment ofCalicophoron daubneyimetacercariae

“…In vitro excystment of C. daubneyi metacercariae Six methods which have been previously described for the in vitro excystment of various trematode parasite species; F. hepatica (McGonigle et al 2008), Fasciola gigantica (Nagar et al 2010), Zygocotyle lunata (Fried et al 1978), Paramphistomum spp (Huesca-guillén et al 2007), Acanthoparyphium spinulosum (Bass and LeFlore, 1984) and Neascus pyriformis (Schroeder et al 1981) were selected to test with C. daubneyi metacercariae. Whilst other methods were available, the selected methods were chosen to avoid testing highly similar protocols.…”