Multi-tool diagnosis of an outbreak of ranavirosis in amphibian tadpoles in the Canadian boreal forest

Abstract

Investigation of mortalities in isolated wild amphibian populations presents diagnostic difficulties that can hinder reaching a definitive diagnosis for the cause of death. Disease can only be diagnosed when pathogen presence (e.g. detection by PCR) is linked to tissue lesions (histopathology) in the host. We report a 2-site outbreak of ranavirosis in wild anuran tadpoles in the boreal forest of Wood Buffalo National Park, Canada, diagnosed by histologic and molecular techniques. Mortalities occurred in wood frog Rana sylvatica tadpoles and boreal chorus frog Pseudacris maculata tadpoles. Lack of mortality in sympatric Canadian toad Bufo (Anaxyrus) hemiophrys tadpoles suggested lower disease susceptibility in this species. In the former 2 species, ranavirosis was diagnosed based on consistent histopathology, immunohistochemistry (IHC), in situ hybridization (ISH), and quantitative PCR results. The most common histopathologic lesion present in wood and boreal chorus frog tadpoles was necrosis of the skin, oral mucosa, renal tubular epithelium, renal hematopoietic tissue, and branchial epithelium. Mild hepatic and pancreatic necrosis and rare intracytoplasmic inclusion bodies in hepatocytes were less common. Skeletal and connective tissues in budding limbs often had multifocal to coalescing necrosis and were intensely positive for ranavirus, with IHC staining even in areas where no obvious necrosis could be observed. Abundant IHC and ISH staining in actively growing tissues support a link between disease emergence and amphibian developmental stage. Our findings provide a definitive diagnosis of ranavirosis in free-living amphibians and highlight the effectiveness of multi-tool approaches to mortality investigation and elucidation of pathogenesis of ranavirosis in wild amphibians.

Ranavirus Amplification in Low-Diversity Amphibian Communities

“…Our findings are also consistent with other studies that demonstrated that larval amphibians are more likely to experience lethal RV infections than adults [ (11,12); see also (105) for multi-year data from our study region]. Notably, all positive CT tadpoles were sampled during a single die-off event of the other two species, but did not experience mass-mortality themselves [see (79)]. This could indicate spillover events from WF and/or CF into CT, only occurring when CT are exposed to a large number of infectious particles due to ongoing die-off events in other species.…”

Sublethal effects of wild-type and a vIF-2α-knockout Frog virus 3 on postmetamorphic wood frogs (Rana sylvatica): potential for a stage-specific reservoir

“…two confirmed die-off events in 2017 at wetlands located 7 and 29 km away from the sampled population (Forzán et al 2019) and mortality occurring among wood frog tadpoles within 3.5 km in 2009 (D.M. Schock, unpublished work).…”

“…To investigate ranavirus infection in postmetamorphic amphibians, we exposed terrestrial wood frogs (Rana sylvatica) via bath exposure to environmentally relevant concentrations (Gray et al 2009a) of two isolates of Ranavirus: FV3 wild-type (WT) and the FV3 vIF-2α knockout (KO) as described by Chen et al (2011). We focused our study on wood frogs because FV3-related mortality events in wild anurans in North America are often associated with communities containing this species (e.g., Greer et al 2005;Brunner et al 2011;Forzán et al 2019), suggesting this species to be an important host for FV3. Further, wood frogs have been suggested as a model species for challenge experiments involving North American strains of Ranavirus due to their widespread distribution, sympatry with numerous other amphibian species, and relatively high ranavirus prevalence levels (Lesbarrères et al 2012;Forzán et al 2015).…”

Sublethal Effects of Wild-Type and a vIF-2&alpha;-Knockout <em>Frog virus 3</em> on Post-Metamorphic Wood Frogs (<em>Rana sylvatica</em>)

“…All wood frogs used in our study were raised from four egg clutches collected from a natural population within Wood Buffalo National Park which showed low Ranavirus prevalence and low viral loads in 2017 [32]. However, Ranavirus has been commonly observed in amphibians in the area [32,34], with two confirmed die-off events at wetlands 7 and 29 km away in 2017 [28], and mortality occurring among wood frog tadpoles within 3.5 km in 2009 [49]. Additionally, sequencing led to the identification of two different FV3-like viruses in amphibian populations in the research area [33,50].…”

“…Mortality events in wild anuran populations in North America are often associated with communities containing wood frogs [e.g. 1,27,28], which could indicate their roles as reservoirs for the pathogen in such communities.…”