title: Factors affecting susceptibility to RNA interference in Haemonchus contortus and in vivo silencing of an H11 aminopeptidase gene.
abstract: Gene silencing by RNA interference (RNAi) has been applied very successfully to Caenorhabditis elegans to study gene function but has proven less effective in parasitic nematodes. In the sheep gastrointestinal nematode Haemonchus contortus, previous studies demonstrated reproducible silencing of β-tubulin but not of other genes targeted. Here we aimed to examine whether the level of target transcript or site of gene expression influence susceptibility to RNAi by soaking. Target genes represented by a high number of expressed sequence tags (ESTs) in the H. contortus L3 stage were not reproducibly silenced. In contrast, four out of six genes putatively expressed in the intestine, excretory cell or amphids were consistently silenced by RNAi. This suggests that genes expressed in sites accessible to the environment are more likely to be susceptible to RNAi by soaking. Silenced genes included those encoding the highly protective gut aminopeptidase H11, secretory protein Hc-ASP-1, β-tubulin and homologues of aquaporin and RNA helicase. To determine whether RNAi silencing of H11 could mimic H11 vaccination in reducing worm and egg counts, we examined the in vivo effects of H11 RNAi. This is the first, to our knowledge, in vivo study of RNAi in an animal parasitic nematode. RNAi of the H11 gene in infective larvae prior to infection resulted in a 57% reduction in faecal egg count (FEC), 40% reduction in worm burden and 64% decrease in aminopeptidase activity compared with pre-soaking in control dsRNA. Thus, in this study we have established that RNAi is a valid and feasible approach to identify essential gene function. However, using current methods, this may be limited to genes expressed in accessible sites.
authors.affiliation: Division of Veterinary Infection and Immunity, Institute of Comparative Medicine, University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, UK.
authors.forename: Buddhini
authors.initials: B
authors.lastname: Samarasinghe
authors.forename: David P
authors.initials: DP
authors.lastname: Knox
authors.forename: Collette
authors.initials: C
authors.lastname: Britton
mesh_terms: D000626:Aminopeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics; D000818:Animals; D018826:CD13 Antigens / Q000037:antagonists & inhibitors* / Q000235:genetics; D005243:Feces / Q000469:parasitology; D006188:Haemonchiasis / Q000662:veterinary; D006189:Haemonchus / Q000201:enzymology* / Q000235:genetics*; D015801:Helminth Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D010270:Parasite Egg Count; D034622:RNA Interference; D012756:Sheep; D012757:Sheep Diseases / Q000473:pathology
chemical_list: D015801:Helminth Proteins; D008565:Membrane Proteins; D000626:Aminopeptidases; D018826:CD13 Antigens; C080908:H11 protein, Haemonchus contortus