Title:
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The development of RNA interference tools for the validation of new control targets in the parasite, Fasciola hepatica
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The liver fluke Fasciola hepatica seriously undermines food production and is now recognised as
a neglected tropical disease with up to 17 million humans infected. This thesis reports the
development of in vitro maintenance methods for both juvenile and adult life stages of F. hepatica
so that functional assays can be developed using RNA interference (RNAi). Juvenile fluke were
maintained for 201 days (more than two fold longer than reported previously) in chicken serum in
RPMI 1640. These worms grew considerably (- 240x their size at excystment) and exhibited
significant development of reproductive and digestive tissues. The best balance of growth and
survival in NEJs was seen with 50% chicken serum in RPM I and is therefore recommended as in
vitro maintenance medium for juvenile F. hepatica. Two novel Fasciola specific tegumental genes
(designated Teg1 and Teg5) were characterized and their potential as new control targets
examined using RNAi. In juveniles, Teg1 and Teg5 exhibit temporal expression and
immunostaining revealed the expression of both proteins on the tegumental surface where they
are likely to be involved in the host-parasite interaction. Teg5 was also localised to the nervous
system of juvenile flu~e. Teg1 and Teg5 genes were knocked down in juvenile F. hepatica in both
RPMI 1640 and in 20% chicken serum in RPMI (80-90% knockdown was achieved) over a variety
of timescales. However, no significant RNAi phenotypes were recorded. The first successful RNAi
of adult genes (Teg1, Teg5, and cathepsin L) is also reported following the development of an
injection-based RNAi-trigger system. The work in this thesis has significantly advanced the
development of new tools to support functional genomics efforts in liver fluke.
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