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

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, beta-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.

RNA interference in parasitic nematodes of animals: a reality check?

“…It is important to demonstrate that observed phenotypes are due to specific transcript reduction and not nonspecific, toxic effects of dsRNA or unsuitable parasite maintenance. Given that the value of RNAi would be in selecting control targets from the wealth of gene information that is becoming available, it is important to examine further the variable efficacy of RNAi in different nematodes, different developmental stages and different target genes [9][10][11].…”

“…In recent studies, we have noted considerable variation in the outcome of RNAi experiments (from total transcript suppression to no effect) between different batches of dsRNA directed at the same target gene [11]. These outcomes could reflect differences in the amount of 'contaminating' siRNA in each batch.…”

“…The ease with which RNAi can be used in C. elegans, together with the fact that the RNAi mechanism itself is conserved in a large number of organisms [4][5][6], has almost automatically led to the assumption that RNAi could be applied to related parasitic nematodes. However, recent evidence indicates that the application of RNAi to parasitic nematodes is not as straightforward as was expected [7,8] and efficacy has been extremely variable [9][10][11]. This variability is particularly intriguing.…”

“…Following the soaking of H. contortus larvae in fluorescently labelled dsRNA, uptake was found to be relatively weak in third-stage larvae (L3) compared with L2 and L4 stages [10], which might reflect the lack of functioning mouthparts in L3s [19]. Electroporation has been used to introduce dsRNA into several trichostrongylid nematodes of sheep and cattle [9][10][11]. This seems to be the most effective means of delivery [9] but it remains unclear at which anatomical site the dsRNA penetrates the worm and to what extent it spreads in the tissues.…”

RNA Interference and Nonspecific Controls in Parasitic Helminths

“…In order to build up information to elucidate helminths biology and host-parasite interaction towards discovery of new drug targets, comes along the feasibility of systems and tools to manipulate gene expression to make sense of so much data generated by genome and transcriptome sequencing. Among very few techniques available for gene characterization in parasitic helminths, RNA interference (RNAi) is the most accessible and employed tool [3][4][5].…”