Edward John Marr - The University of Edinburgh.
Sheep scab is a highly contagious ectoparasitic disease caused by the mite Psoroptes ovis. It has a significant economic impact through its effects on animal performance and welfare, lamb growth rates, wool damage and reduced quality of sheep skin. It also presents an important welfare issue due to the intense pruritis and severe exudative dermatitis associated with the lesion. Since deregulation in 1992, sheep scab has become endemic throughout all areas of the UK. Current disease control strategies are heavily reliant upon chemotherapy; however concerns over residues, ecotoxicity and the development of parasite resistance have led to questions being raised regarding the sustainability of our current strategies and an interest in the development of alternatives. There is a renewed interest in the development of alternative methods of controlling sheep scab and the presence of a protective immune response provides justification for a vaccine-based approach to control. Vaccination of sheep with fractions of mite extracts has demonstrated protective immunity, however the identification of the individual proteins involved has proven difficult. The approach of reverse vaccinology based on P. ovis transcriptome sequences has enabled the identification of potential candidates for a recombinant vaccine approach, however new tools are required to further screen these candidate factors prior to their validation during in vivo vaccination trials.
We aim to develop an RNAi knockdown system in P. ovis mites, similar to current systems demonstrated in ticks and Varroa mites. The system will be optimised to control the persistence of the knockdown and minimise mortality. A mu-class GST gene will be used to initially optimise gene knockdown and the extent will be assessed by SYBR green-based RT-PCR and compared to control mites exposed to dsRNA coding for an irrelevant gene.
Following optimisation of the RNAi gene knockdown system, the candidate gene approach will be adopted. dsRNA will be produced from P. ovis cDNA and the extent and persistence of target gene knockdown assessed by RT-PCR. Mortality will be monitored daily for 5 days after RNAi treatment and 2 control treatments will be run simultaneously, mites treated with irrelevant gene dsRNA and untreated mites. Promising targets will be studied for additive and synergistic effects by comparing mites immersed in dsRNA for 3 genes to those immersed in dsRNA coding for one gene. Chimeric dsRNA of 2 or 3 individual targets will be investigated in order to develop RNAi as a relatively high throughput screen for vaccine development. If successful, this could triple the speed of RNAi screens.
siRNA will be investigated as a potential vaccine screen by designing a 21bp synthetic siRNA based on a sequence encoding an enzymatically-active P. ovis protein - allowing a fluorescence-based enzyme assay to monitor gene knockdown. Following optimisation of this system, promising candidates from the dsRNAi trials will be assessed. If successful, the number of candidates to be tested will be reassessed. This would facilitate cheaper and quicker gene knockdown screening of higher specificity.
The overall aim of this proposal is therefore to develop an RNA interference (RNAi)-based screen to silence the expression of candidate genes in a knockdown system in P. ovis mites to assess their importance in mite survival and biology and therefore inform their use as vaccine candidate molecules. This system will facilitate a novel approach to the screening of potential vaccine candidates for sheep scab with high specificity, speed and at a relatively low cost.