Development of an in vitro drug sensitivity assay based on newly excysted larvae of Echinostoma caproni

Abstract

BackgroundEchinostomiasis is one of the major food-borne trematodiases and the species Echinostoma caproni serves as a useful model for trematocidal drug discovery. The current in vitro drug sensitivity assay uses adult E. caproni worms that are incubated with candidate drugs and scored microscopically for viability at 72 hrs. The aim of this study was to investigate the use of newly excysted larvae (NEL) of E. caproni for in vitro drug testing, which would be faster, more cost effective and more ethical compared to adult worm assays.MethodsLarvae were obtained by collecting metacercariae from snails and triggering their excystation using the trypsin-bile salt excystation method. Studies concerning various parameters of this chemical transformation process as well as appropriate NEL culturing conditions were carried out and findings evaluated. NEL and adult worms were incubated with praziquantel, tribendimidine, albendazole and quinine and evaluated microscopically 72 hrs post-incubation. In addition, the colorimetric markers resazurin, CellTiter-Glo® and Vybrant® were tested as an alternative assay read-out method.ResultsThe chemical excystation method successfully induced E. caproni metacercariae to excyst at a rate of about 20-60%. NEL remained viable in culture medium for 5–7 days. The results of an in vitro drug assay using NEL mirrored the results of an assay using adult worms incubated with the same drugs. None of the markers could reliably produce signals proportional to NEL viability or cytotoxicity without significant complications.ConclusionNEL are adequate for in vitro drug testing. Challenges remain in further improving the excystation yield and the practicability of the assay setup. Resolving these issues could also improve read-outs using colorimetric markers. Using NEL is in alignment with the 3 R rules of the ethical use of laboratory animals and can greatly increase the rate and affordability with which drugs are screened in vitro against this intestinal trematode.

Paramphistomosis of Ruminants: An Emerging Parasitic Disease in Europe

“…With widespread drug resistance now affecting the control of a large range of parasitic and infectious diseases [75,76], reliance on a single drug compound raises the risk of untreatable isolates emerging, and is never good policy for sustainable parasite control. Future research may focus on the identification of novel compounds with efficacy against both juvenile and mature rumen fluke (or repositioning of existing drugs) and the selection and validation of new drug targets, using in vitro assays to assess anthelmintic efficacy against measurements such as fluke motility [77] or egg viability [78].…”

Use of praziquantel for treatment of flatworm parasites in centrarchid fish

“…In addition, resazurin metabolism was not correlated with the number of metacercariae present in a particular well. These results are similar to those observed for another trematode, Echinostoma caproni (Panic et al, 2013).…”

“…ATP production has been used as a measure of viability for Schistosoma mansoni (Panic et al, 2015), but had been found to be unsuccessful with metacercariae (Panic et al, 2013). In the present study, we utilized a commercial recombinant luciferase that produces light in the presence of ATP.…”

History of echinostomes (Trematoda)

“…These authors have performed a number of in vivo (using mice as definitive host) and in vitro studies with E. caproni as the drug discovery tool. They have assessed the anthelmintic properties of different medicinal plants and compounds which have allowed them to define several drug development candidates (Keiser et al, 2006a, b, c;Saric et al, 2008;Keiser 2010;Ferreira et al, 2011;Kirchhofer et al, 2011;Panic et al, 2013). Moreover, these workers have started the study of metabonomics in helminth infections using E. caproni as an experimental model (Saric et al, 2008).…”