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Efficacy and specificity of RNA interference in larval life-stages of Ostertagia ostertagi

Published online by Cambridge University Press:  31 July 2006

A. VISSER ,
P. GELDHOF ,
V. DE MAERE ,
D. P. KNOX ,
J. VERCRUYSSE  and
E. CLAEREBOUT
A. VISSER
Affiliation:
Department of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B9820 Merelbeke, Belgium
P. GELDHOF
Affiliation:
Moredun Research Institute, Pentlands Science Park, Penicuik, Bush Loan, Midlothian, UK
V. DE MAERE
Affiliation:
Department of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B9820 Merelbeke, Belgium
D. P. KNOX
Affiliation:
Moredun Research Institute, Pentlands Science Park, Penicuik, Bush Loan, Midlothian, UK
J. VERCRUYSSE
Affiliation:
Department of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B9820 Merelbeke, Belgium
E. CLAEREBOUT
Affiliation:
Department of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B9820 Merelbeke, Belgium
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Abstract

RNA interference (RNAi) on parasitic nematodes has been described as successful and useful for the identification of novel drug and vaccine candidates. In this study we have evaluated this technology on the cattle parasite Ostertagia ostertagi. Eight different genes were targeted in L1 and L3 O. ostertagi larvae, by electroporation and soaking in dsRNA respectively. Down-regulation of target transcript levels was evaluated by semi-quantitative reverse transcriptase (RT) PCR. In L3 larvae, variable decreases in mRNA levels were observed for 5 genes, ranging from a complete knock down (tropomyosin, β-tubulin) to a minor decrease (ATPsynthase, superoxide dismutase, polyprotein allergen). However, repeated experiments indicated that effects were sometimes difficult to reproduce. RNAi for ubiquitin, a transthyretin-like protein and a 17 kDa excretion secretion (ES) protein never resulted in a knock down of the transcript. The mRNA levels of 7 non-target genes showed no difference between larvae soaked in C. elegans control dsRNA versus O. ostertagi tropomyosin dsRNA, supporting that the observed reductions are specific for the target gene. Electroporation of L1 larvae proved to be less effective. Reductions in mRNA levels were only noticed for 2 genes and were not reproducible. In conclusion, the results indicate that the RNAi pathway is probably present in O. ostertagi but that the current RNAi techniques can not be used as a reliable screening method.

Keywords

Ostertagia ostertagiRNA interferencescreening
Type
Research Article
Information
Parasitology , Volume 133 , Issue 6 , December 2006 , pp. 777 - 783
Copyright
2006 Cambridge University Press

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