Abstract
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.
Highlights
Myxobolus cerebralis is a cnidarian-myxozoan parasite and causative agent of whirling disease, an ecologically and economically debilitating disease of salmonids [1,2,3]
At 3mpi, infected T. tubifex oligochaetes were treated with different concentrations of MyxSP-1 short-interfering RNA (siRNA) or negative control siRNA (1μM, 2μM, 5μM or 7μM) at 15 ̊C for 24h, 48h, 72h and 96h, respectively
Specific-pathogen-free T. tubifex oligochaetes were infected with M. cerebralis and treated with MyxSP-1 siRNA or negative control siRNA (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. 24 hours after the final siRNA treatment at 3mpi, we harvested the oligochaetes in all treatment groups and monitored the effect of siRNA treatment on MyxSP-1 gene expression in infected T. tubifex using quantitative PCR (qPCR) (Figs 3 and 4; Table 2). siRNA treatment of M. cerebralis-infected T. tubifex was most effective at 1mpi, 2mpi and 3mpi
Summary
Myxobolus cerebralis is a cnidarian-myxozoan parasite and causative agent of whirling disease, an ecologically and economically debilitating disease of salmonids [1,2,3]. The disease was responsible for the significant decrease in rainbow trout (Oncorhynchus mykiss) populations in North America since the 1950s and was further exacerbated by a major outbreak in the mid-1990s [4] Alarmed by this menace, the United States Congress awarded funding for the period between 1997 and 2006 to two organizations instituted for research associated with salmonid whirling disease viz., the Whirling Disease Foundation and the Whirling Disease Initiative through a cooperative agreement with the U.S Fish and Wildlife Service [5]. In 2016, the Canadian Food Inspection Agency (CFIA; Government of Canada) confirmed the first cases of salmonid whirling disease in Canada in 10 locations including the Banff National Park, Alberta [6]. This prompted the Alberta Environment and Parks to issue a Ministerial Fish Quarantine Order under section 32(2) of the Fisheries (Alberta) Act on September 6, 2016 for all commercial fish culture operations in the region [7]
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