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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:678822 |
| Date | January 2015 |
| Creators | McCusker, Paul |
| Publisher | Queen's University Belfast |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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