Open AccessA RNAi-based therapeutic proof of concept targets salmonid whirling disease in vivo
Author(s) -
Subhodeep Sarker,
Simon Menanteau–Ledouble,
Mohamed H. Kotob,
Mansour El-Matbouli
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0178687
Subject(s) - tubifex tubifex , tubifex , gene knockdown , rna interference , biology , small interfering rna , microbiology and biotechnology , rna , ecology , gene , genetics
Myxobolus cerebralis is a cnidarian-myxozoan parasite that causes salmonid whirling disease. M . cerebralis alternates between two hosts: (1) a vertebrate salmonid and (2) an invertebrate oligochaete, Tubifex tubifex . There is no successful treatment for salmonid whirling disease. MyxSP-1 is a M . cerebralis serine protease implicated in whirling disease pathogenesis. We hypothesized that short-interfering RNA (siRNA)-induced RNA interference (RNAi) can silence MyxSP-1 in the invertebrate host and abrogate the M . cerebralis life cycle. This would preclude whirling disease infection in the salmonid host. To test this hypothesis, we first developed a siRNA delivery protocol in T . tubifex . Second, we determined the effective dose for siRNA treatment of M . cerebralis -infected T . tubifex . M . cerebralis -infected T . tubifex were treated with different concentrations of MyxSP-1 or negative control siRNAs (1μM, 2μM, 5μM or 7μM) at 15°C for 24h, 48h, 72h and 96h, respectively. We monitored MyxSP-1 knockdown using real-time quantitative PCR (qPCR). siRNA treatment with MyxSP-1 siRNA at 2μM concentration for 24h at 15°C showed maximum significant MyxSP-1 knockdown in T . tubifex . Third, we determined the time points in the M . cerebralis life cycle in T . tubifex at which siRNA treatment was most effective. M . cerebralis- infected T . tubifex were treated with MyxSP-1 or negative control siRNAs (2μM concentration for 24h at 15°C) at 24 hours post-infection (24hpi), 48hpi, 72hpi, 96hpi, 1 month post-infection (1mpi), 2mpi and 3mpi, respectively. We observed that siRNA treatment of T . tubifex was most effective at 1mpi, 2mpi and 3mpi. Fourth, we immersed specific-pathogen-free rainbow trout fry in water inhabited by MyxSP-1 siRNA-treated T . tubifex (at 1mpi, 2mpi and 3mpi). The salmonids did not develop whirling disease and showed significant MyxSP-1 knockdown. We also observed long-term RNAi in T . tubifex . Together these results demonstrate a novel RNAi-based therapeutic proof of concept in vivo against salmonid whirling disease.