Anuran susceptibilities to ranaviruses: role of species identity, exposure route, and a novel virus isolate

Abstract

Ranaviruses are responsible for widespread amphibian die-offs, particularly with larval anurans. To understand the factors that may be contributing to the emergence of ranaviruses, we conducted 3 experiments that exposed 3 species of larval anurans to either endemic frog virus 3 (FV3) or an FV3-like isolate from a ranaculture facility. Our goals were to (1) determine the susceptibility of each species to each virus, (2) determine whether direct ingestion of virions or exposure to virions in a water bath were similarly lethal routes of transmission, and (3) quantify the effects of exposure duration on disease outcomes. We conducted our research in a controlled aquatic laboratory using a factorial combination of virus isolates, transmission routes, and exposure durations. While ranaviruses can affect many species, we found that larval anurans differ greatly in susceptibility to ranaviruses. Average mortality rates of Cope's gray tree frogs (66%) and pickerel frogs (68%) were similar but 3-fold higher than for eastern narrow-mouthed toads. Direct ingestion of the viruses increased mean infection and mortality rates by 30% and caused death about 2 times faster compared to water bath exposure. However, exposure duration did not impact mean infection or mortality rates. We also found that the ranaculture isolate increased mortality by > 34% compared to FV3. Our results suggest that ranaviruses can rapidly infect and cause disease in multiple amphibian species. Given the risk associated with introducing novel ranaviruses from ranaculture facilities, we recommend that all nations adopt the protocol set forth by the World Organization for Animal Health for testing and certifying that amphibians that are commercially shipped are negative for ranavirus infection.

Clinical signs, pathology and dose-dependent survival of adult wood frogs, Rana sylvatica, inoculated orally with frog virus 3 Ranavirus sp., Iridoviridae

“…Regarding ST 50 , mortality of Cope's grey tree frog tadpoles [Hyla chrysoscelis, family Hylidae, found in the south-eastern United States (Dodd, 2013)] exposed to water containing FV3 began 12 days p.i. and reached 66 % (Hoverman et al, 2010), whereas mortality of tiger salamander tadpoles [Ambystoma tigrinum, family Ambysomatidae, found in southern Canada, the continental USA and north-eastern Mexico (Duellman & Trueb, 1994)] also exposed to water containing FV3 began just under 20 days p.i. and reached 91 % (Brunner et al, 2005).…”

“…ml 21 : a dose very close to the concentration of FV3 that caused 66 % mortalities in Cope's grey tree frogs. Hoverman et al (2010), by using a consistent methodology, demonstrated that tadpoles of three different species [pickerel frog, Rana (Lithobates) palustris, Cope's grey tree frog, and eastern narrow-mouthed toad, Gastrophryne carolinensis] are not equally susceptible to mortality when exposed to FV3. The authors also concluded that oral inoculation of a known dose of FV3 (10 6 p.f.u.)…”

“…both increased mean mortality rate and sped time to death when compared to water-bath exposure. However, as the same viral concentration used in the direct oral infection was diluted in a 1 l water bath, the different outcomes may actually reflect the effect of dose, not of route of infection (Hoverman et al, 2010). …”

“…Therefore, oral administration may be just as relevant in replicating natural exposure to viral particles in water bodies as is immersion in a water bath, and possibly more relevant for terrestrial species like the wood frog. Oral dosing allows for administration of relatively precise doses, and can be effected easily in most post-metamorphic frogs and even tadpoles of some species (Wolf et al, 1968;Hoverman et al, 2010).…”

“…Ranaviruses have been responsible for mass mortalities in wild and captive frogs and salamanders in north America, Asia, Australia and Europe (Gray et al, 2009), and are currently the focus of intense research (Chinchar et al, 2009). Experimental infections with various species and isolates of ranaviruses have been achieved through intraperitoneal injection (Tweedell & Granoff, 1968;Wolf et al, 1968), immersion in viral suspension via water bath (Brunner et al, 2005;Harp & Petranka, 2006;Cullen & Owens, 2002), exposure of cutaneous wounds to virus (Cunningham et al, 2007) and oral administration (Wolf et al, 1968;Hoverman et al, 2010). Although the work of dozens of researchers, past and present, frequently focuses on experimental challenges with the original FV3 isolated in the 1960s (Granoff et al, 1966), reported dosages vary, as do the species and developmental stage of the infected host.…”