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 15ÊC 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 15ÊC 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 15ÊC) 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.