Immersion of SPF rainbow trout fry in water inhabited by siRNA-treated <i>T</i>. <i>tubifex</i>.

<p>Immersion of SPF rainbow trout fry in water inhabited by siRNA-treated <i>T</i>. <i>tubifex</i>.</p

Immersion of SPF rainbow trout fry (n = 30; in triplicates of 10 fish per aquarium) in water inhabited by siRNA-treated <i>T</i>. <i>tubifex</i> 3mpi.

<p>Representative image of rainbow trout fry photographed alive <b>(A, C)</b> and post-mortem <b>(B, D)</b> three months post-immersion in water inhabited by negative control siRNA-treated <i>T</i>. <i>tubifex</i> 3mpi <b>(A, B)</b> or <i>MyxSP-1</i> siRNA-treated <i>T</i>. <i>tubifex</i> 3mpi <b>(C, D)</b>. Arrow indicates clinical signs of whirling disease–shortening of the gill operculum <b>(A, B)</b>; scale bar = 2cm. 3 months post-immersion, fish were euthanized and <i>T</i>. <i>tubifex</i> were harvested. <i>MyxSP-1</i> gene expression was evaluated using qPCR. <i>MyxSP-1</i> gene expression was normalized to that of <i>M</i>. <i>cerebralis β-actin</i>. <b>(E)</b> Normalized gene expression of <i>MyxSP-1</i> in rainbow trout fry post-mortem. Data represent mean normalized expression (n = 6–8; +SE; ^****<i>p</i><0.01). <b>(F)</b> Normalized gene expression of <i>MyxSP-1</i> in siRNA-treated <i>T</i>. <i>tubifex</i> (3mpi) harvested at the end of the experiment. Data represent mean normalized expression (n = 6–8; +SE; ^****<i>p</i><0.01). Abbreviations: SPF = specific-pathogen-free; qPCR = real-time quantitative PCR.</p

A RNAi-based therapeutic <i>proof of concept</i> targets salmonid whirling disease <i>in vivo</i>

<div><p><i>Myxobolus cerebralis</i> is a cnidarian-myxozoan parasite that causes salmonid whirling disease. <i>M</i>. <i>cerebralis</i> alternates between two hosts: (1) a vertebrate salmonid and (2) an invertebrate oligochaete, <i>Tubifex tubifex</i>. There is no successful treatment for salmonid whirling disease. MyxSP-1 is a <i>M</i>. <i>cerebralis</i> serine protease implicated in whirling disease pathogenesis. We hypothesized that short-interfering RNA (siRNA)-induced RNA interference (RNAi) can silence <i>MyxSP-1</i> in the invertebrate host and abrogate the <i>M</i>. <i>cerebralis</i> life cycle. This would preclude whirling disease infection in the salmonid host. To test this hypothesis, we first developed a siRNA delivery protocol in <i>T</i>. <i>tubifex</i>. Second, we determined the effective dose for siRNA treatment of <i>M</i>. <i>cerebralis</i>-infected <i>T</i>. <i>tubifex</i>. <i>M</i>. <i>cerebralis</i>-infected <i>T</i>. <i>tubifex</i> were treated with different concentrations of <i>MyxSP-1</i> or negative control siRNAs (1μM, 2μM, 5μM or 7μM) at 15°C for 24h, 48h, 72h and 96h, respectively. We monitored <i>MyxSP-1</i> knockdown using real-time quantitative PCR (qPCR). siRNA treatment with <i>MyxSP-1</i> siRNA at 2μM concentration for 24h at 15°C showed maximum significant <i>MyxSP-1</i> knockdown in <i>T</i>. <i>tubifex</i>. Third, we determined the time points in the <i>M</i>. <i>cerebralis</i> life cycle in <i>T</i>. <i>tubifex</i> at which siRNA treatment was most effective. <i>M</i>. <i>cerebralis-</i>infected <i>T</i>. <i>tubifex</i> were treated with <i>MyxSP-1</i> 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 <i>T</i>. <i>tubifex</i> was most effective at 1mpi, 2mpi and 3mpi. Fourth, we immersed specific-pathogen-free rainbow trout fry in water inhabited by <i>MyxSP-1</i> siRNA-treated <i>T</i>. <i>tubifex</i> (at 1mpi, 2mpi and 3mpi). The salmonids did not develop whirling disease and showed significant <i>MyxSP-1</i> knockdown. We also observed long-term RNAi in <i>T</i>. <i>tubifex</i>. Together these results demonstrate a novel RNAi-based therapeutic <i>proof of concept in vivo</i> against salmonid whirling disease.</p></div

Effective dose for siRNA treatment of <i>M</i>. <i>cerebralis</i>-infected <i>T</i>. <i>tubifex</i> oligochaetes at period of peak release of TAMs.

<p>Effective dose for siRNA treatment of <i>M</i>. <i>cerebralis</i>-infected <i>T</i>. <i>tubifex</i> oligochaetes at period of peak release of TAMs.</p

Immersion of SPF rainbow trout fry (n = 30; in triplicates of 10 fish per aquarium) in water inhabited by siRNA-treated <i>T</i>. <i>tubifex</i> 2mpi.

<p>Representative image of rainbow trout fry photographed alive <b>(A, C)</b> and post-mortem <b>(B, D)</b> three months post-immersion in water inhabited by negative control siRNA-treated <i>T</i>. <i>tubifex</i> 2mpi <b>(A, B)</b> or <i>MyxSP-1</i> siRNA-treated <i>T</i>. <i>tubifex</i> 2mpi <b>(C, D)</b>. Arrows indicate clinical signs of whirling disease–darkening of the caudal region and shortening of the gill operculum <b>(A, B)</b>; scale bar = 2cm. 3 months post-immersion, fish were euthanized and <i>T</i>. <i>tubifex</i> were harvested. <i>MyxSP-1</i> gene expression was evaluated using qPCR. <i>MyxSP-1</i> gene expression was normalized to that of <i>M</i>. <i>cerebralis β-actin</i>. <b>(E)</b> Normalized gene expression of <i>MyxSP-1</i> in rainbow trout fry post-mortem. Data represent mean normalized expression (n = 6–8; +SE; ^****<i>p</i><0.01). <b>(F)</b> Normalized gene expression of <i>MyxSP-1</i> in siRNA-treated <i>T</i>. <i>tubifex</i> (2mpi) harvested at the end of the experiment. Data represent mean normalized expression (n = 6–8; +SE; ^****<i>p</i><0.01). Abbreviations: SPF = specific-pathogen-free; qPCR = real-time quantitative PCR.</p

Thermal cycling parameters for quantitative real-time PCR (qPCR).

<p>Thermal cycling parameters for quantitative real-time PCR (qPCR).</p

siRNA treatment of <i>T</i>. <i>tubifex</i> at different concentrations for 24h, 48h, 72h and 96h.

<p>1600 SPF <i>T</i>. <i>tubifex</i> were collected and then divided into 32 groups with each having 50 SPF <i>T</i>. <i>tubifex</i>. All groups of SPF <i>T</i>. <i>tubifex</i> were infected with <i>M</i>. <i>cerebralis</i> myxospores at the same time. At 3mpi, infected <i>T</i>. <i>tubifex</i> oligochaetes were treated with different concentrations of <i>MyxSP-1</i> siRNA or negative control siRNA (1μM, 2μM, 5μM or 7μM, respectively) at 15°C for 24h <b>(A; n = 6–8; +SE;</b> ^<b>**</b><b><i>p</i><0.001)</b>, 48h <b>(B; n = 6–8; +SE;</b> ^<b>*</b><b><i>p</i><0.0001)</b>, 72h <b>(C; n = 6–8; +SE;</b> ^<b>*</b><b><i>p</i><0.0001)</b> and 96h <b>(D; n = 6–8; +SE;</b> ^<b>*</b><b><i>p</i><0.0001)</b>. Post-soaking, siRNA-treated <i>T</i>. <i>tubifex</i> were harvested and <i>MyxSP-1</i> gene expression was evaluated using qPCR. <i>MyxSP-1</i> gene expression was normalized to that of <i>M</i>. <i>cerebralis β-actin</i>. Data represent mean normalized expression +SE. Abbreviations: SPF = specific-pathogen-free; mpi = months post-infection; qPCR = real-time quantitative PCR.</p

Schematic representation of the experimental workflow.

<p>What happens if SPF rainbow trout fry are immersed in water inhabited by live siRNA-treated <i>T</i>. <i>tubifex</i>?</p

Normalized gene expression of <i>MyxSP-1</i> gene after siRNA treatment of <i>T</i>. <i>tubifex</i> at different time points post-infection.

<p>700 SPF <i>T</i>. <i>tubifex</i> were collected and then divided into 14 groups with each having 50 SPF <i>T</i>. <i>tubifex</i>. All groups of SPF <i>T</i>. <i>tubifex</i> were infected with <i>M</i>. <i>cerebralis</i> myxospores at the same time. Infected <i>T</i>. <i>tubifex</i> were treated with <i>MyxSP-1</i> siRNA or negative control siRNA (2μM concentration for 24h at 15°C) at 24hpi, 48hpi, 72hpi, 96hpi, 1mpi, 2mpi and 3mpi, respectively. 24 hours after the final siRNA treatment at 3mpi, siRNA-treated <i>T</i>. <i>tubifex</i> were harvested and <i>MyxSP-1</i> gene expression was evaluated using qPCR. <i>MyxSP-1</i> gene expression was normalized to that of <i>M</i>. <i>cerebralis β-actin</i>. Data represent mean normalized expression (n = 6–8; +SE; ^*<i>p</i><0.0001; ^**<i>p</i><0.001; ^***<i>p</i><0.005). Abbreviations: SPF = specific-pathogen-free; hpi = hours post-infection; mpi = months post-infection; qPCR = real-time quantitative PCR.</p

siRNA treatment of <i>M</i>. <i>cerebralis</i> infected-<i>T</i>. <i>tubifex</i> at different time points post-infection.

<p>siRNA treatment of <i>M</i>. <i>cerebralis</i> infected-<i>T</i>. <i>tubifex</i> at different time points post-infection.</p