Whirling disease in the Crowsnest River: an emerging threat to wild salmonids in Alberta

We provide the first documented case of whirling disease (WD) impacts to wild, self-sustaining rainbow trout (RNTR, Oncorynchus mykiss) populations in Canada. Myxobolus cerebralis (Mc), the causative agent of WD, was first confirmed in Alberta in 2016. However, evidence of disease in local fish populations was unknown. Using a weight-of-evidence approach, we examined multiple parasite life cycle stages in the Crowsnest River, Alberta. Percentage of infected Tubifex tubifex worms actively shedding triactinomyxons (TAMs) exceeded known thresholds of Mc establishment and TAM densities instream exceeded thresholds known to cause ≥90% declines in RNTR populations. Mc was detected at 5 of 6 study sites in water, fish, and worms. Disease severity was highest in the lower watershed where 100% of sentinel fish tested positive for Mc 7 to 14 days post-exposure; up to 85% of wild fingerling RNTR showed clinical signs of disease and yearling trout were largely absent from the river suggesting reduced survival. Our findings indicate conditions necessary for outbreak of WD exist in Alberta, highlighting the need to consider this disease as an emerging threat to wild salmonid populations.

Serine protease inhibitors of the whirling disease parasite Myxobolus cerebralis (Cnidaria, Myxozoa): Expression profiling and functional predictions

Here, we studied the expression pattern and putative function of four, previously identified serine protease inhibitors (serpins) of Myxobolus cerebralis, a pathogenic myxozoan species (Cnidaria: Myxozoa) causing whirling disease of salmonid fishes. The relative expression profiles of serpins were determined at different developmental stages both in fish and in annelid hosts using serpin-specific qPCR assays. The expression of serpin Mc-S1 was similar throughout the life cycle, whereas a significant decrease was detected in the relative expression of Mc-S3 and Mc-S5 during the development in fish, and then in the sporogonic stage in the worm host. A decreasing tendency could also be observed in the expression of Mc-S4 in fish, which was, however, upregulated in the worm host. For the first time, we predicted the function of M. cerebralis serpins by the use of several bioinformatics-based applications. Mc-S1 is putatively a chymotrypsin-like inhibitor that locates extracellularly and is capable of heparin binding. The other three serpins are caspase-like inhibitors, and they are probably involved in protease and cell degradation processes during the early stage of fish invasion.

In vitro and in vivo assays reveal that cations affect nematocyst discharge in Myxobolus cerebralis (Cnidaria: Myxozoa)

Myxozoans are parasitic, microscopic cnidarians that have retained the phylum-characteristic stinging capsules called nematocysts. Free-living cnidarians, like jellyfish and corals, utilize nematocysts for feeding and defence, with discharge powered by osmotic energy. Myxozoans use nematocysts to anchor to their fish hosts in the first step of infection, however, the discharge mechanism is poorly understood. We used Myxobolus cerebralis, a pathogenic myxozoan parasite of salmonid fishes, and developed two assays to explore the nature of its nematocyst discharge. Using parasite actinospores, the infectious stage to fish, we stimulated discharge of the nematocysts with rainbow trout mucus in vitro, in solutions enriched with chloride salts of Na+, K+, Ca2+ and Gd3+, and quantified discharge using microscopy. We then used quantitative polymerase chain reaction to evaluate the in vivo effects of these treatments, plus Mg2+ and the common aquaculture disinfectant KMnO₄, on the ability of M. cerebralis actinospores to infect fish. We found that Mg2+ and Gd3+ reduced infection in vivo, whereas Na+ and K+ over-stimulated nematocyst discharge in vitro and reduced infection in vivo. These findings align with nematocyst discharge behaviour in free-living Cnidaria, and suggest phylum-wide commonalties, which could be exploited to develop novel approaches for controlling myxozoan diseases in aquaculture.

Distinctive site preference of the fish parasite Myxobolus cerebralis (Cnidaria, Myxozoa) during host invasion

Here, we experimentally studied the site preference of Myxobolus cerebralis, one of the most pathogenic myxozoan (Cnidaria, Myxozoa) fish parasites, which causes whirling disease in salmonids. Parasite invasion was examined in three fish species with various susceptibility levels: the type host brown trout, the highly susceptible rainbow trout, and the non-susceptible gibel carp, in which parasite spores do not develop. We investigated the first two hours of fish invasion, and measured the site preference of triactinomyxons (TAMs) during attachment and penetration of fish in three body parts (gills, fins, skin). Infection prevalence and intensity were estimated using a species-specific nested PCR, optimised in the present study. The highest infection prevalence was detected in the most susceptible fish species, rainbow trout. Interestingly, higher prevalence was observed in gibel carp than in the type host, brown trout (95.2% vs. 85.7%). Considering body locations, remarkable differences were detected in infection intensities. The highest intensity was observed in fins, whereas skin was the least infected body part in every fish species examined. Infection prevalence and intensity did not differ significantly among fish species. Thus, we confirmed that M. cerebralis TAMs cannot discern fish species. Furthermore, we proved experimentally that fish fin is significantly more attractive to fish-invading parasite TAMs than gills or skin.