Five amphibian mortality events associated with ranavirus infection in south central Ontario, Canada

Abstract

Using field, molecular and histological methods, an epizootic, systemic disease causing death within wood frog Rana sylvatica tadpoles and leopard frog Rana pipiens metamorphs at 3 different locations within Southern Ontario, Canada, has been investigated. Our results demonstrated that the probable cause of this disease was a ranavirus. Affected amphibians were found to exhibit necrosis within the hematopoietic cells. Liver tissue samples were found positive for the virus by PCR amplification of the ranavirus (Family: Iridoviridae) major capsid protein (MCP). Positive samples were confirmed by sequence analysis. Clinically normal, laboratory-raised wood frog egg broods were also found to test weakly positive for ranavirus. The population effects of disease on these amphibian communities have not yet been conclusively associated with population declines, but warrant more focused consideration.KEY WORDS: Frog · Rana sylvatica · Rana pipiens · Ranavirus · Amphibian · Polymerase chain reaction · Canada Resale or republication not permitted without written consent of the publisherDis Aquat Org 67: [9][10][11][12][13][14] 2005 apparent that the infection and death of diverse groups of amphibians testing positive for ranavirus is a serious and potentially devastating occurrence.We report here on the recurring presence of a ranavirus in 3 amphibian populations in Southern Ontario, Canada. The objectives for this work were to determine whether a ranavirus was associated with 5 amphibian mortality events investigated in south central Ontario, Canada. We also examined potential transmission routes for the virus within these populations. MATERIALS AND METHODSField sites. Tadpoles and recent metamorphs were collected from 5 different sites within Southern Ontario. Field sites can be classified into 2 categories, namely (1) sites where disease was observed and subsequently investigated, and (2) sites where no disease was observed. Disease sites included the James Oliver Ecological Research Station ('Oliver Pond'; 44°31' N, 78°32' W), The Kortright Centre for Conservation ('Kortright Pond'; 43°57' N, 79°42' W) and Gannon's Narrows (44°38' N, 78°41' W). Sites that showed no signs of disease and were used as control sites included the Poplar Pond (44°31' N, 78°32' W) and the Trent University Archeology Pond (44°21' N, 78°17' W). To be considered a diseased site, ≥10 ind. had to meet the case definition. This meant that sick and/or dead individuals had to exhibit 4 of 7 clinical signs. Clinical signs included redness in the vent, abdomen, or legs, swollen vent, edema of the body cavity, cutaneous lesions, anorexia and loss of escape behaviour. The Oliver Pond location is unique because there have been 3 consecutive years of similar mass mortality events here. In May 2002, Rana sylvatica egg masses were collected from the Oliver Pond within 12 h of being laid and reared in the laboratory in 5 gallon (19 l) aquaria containing aged river water.Collection of animals. Live animals were transported back to the laboratory. ...

Xenopus Laevis: A Possible Vector of Ranavirus Infection?

“…Ranaviruses (Iridoviridae) are becoming increasingly associated with diseases in wild and cultured fishes, frogs, salamanders, and reptiles (Cunningham et al, 1996;Zupanovic et al, 1998;Hyatt et al, 2000;Zhang et al, 2001;Chinchar, 2002;De Voe et al, 2004;Pearman et al, 2004;Greer et al, 2005). Frog virus 3 (FV3), the best-characterized member and type species of the Ranavirus genus, is a large (165-169 nm) double-stranded DNA icosahedral virus that was originally isolated from the North American leopard frog, Rana pipiens (Chinchar, 2002).…”

Frog Virus 3-Like Infections in Aquatic Amphibian Communities

“…Wood frog tadpoles in both Oliver and Parker ponds previously have been shown to be infected by FV3 (Greer et al, 2005;Charbonneau, 2006). Wood frog tadpoles were sampled at five different dates in Oliver Pond, at eight dates in Parker Pond, and at five dates in Crowe's Pond.…”

“…Tissue samples were incubated at 37 C overnight, and then 15 U of proteinase K (Roche Diagnostics, Manheim, Germany) was added, and samples were incubated for 2 hr at 65 C. An additional aliquot of 15 U of proteinase K was added before a final incubation at 37 C overnight. DNA digests were extracted following the QIAamp DNeasy extraction kit protocol (QIA-GEN, Mississauga, Ontario, Canada) following Greer et al (2005). DNA obtained from the hepatic tissue was assessed by PCR for the presence of FV3 using primers that amplify a 500-base pair (bp) region of the major capsid protein of FV3 (Greer et al, 2005).…”

“…The first reported iridovirus outbreak and mortality event in Canada was caused by the Regina ranavirus, which infected larval and adult tiger salamanders (Ambystoma tigrinum diaboli) in Saskatchewan (Bollinger et al, 1999). More recently, frog virus 3 (FV3) or an FV3-like virus, was identified in populations of wood frogs (Rana sylvatica), leopard frogs (Rana pipiens), and green frogs (Rana clamitans) in southern Ontario where it was associated with wood frog tadpole die-offs (Greer et al, 2005;Charbonneau, 2006). Although the mechanism of FV3/FV3-like virus transmission remains unclear, both vertical (reproduction-dependent) and horizontal (nonreproduction-dependent) transmission may be possible.…”

“…From 1999 through 2001, virtually all of the wood frog tadpoles in one of these ponds (Oliver Pond) died shortly before metamorphosis (Greer et al, 2005). Field observations suggested that only wood frog tadpoles died during these events and that they were positive for FV3-like virus infections; clinical signs were not observed in other amphibian species in the pond, and they were not examined for the presence of FV3-like infections (Greer et al, 2005). Although these outbreaks provided no evidence of interspecies transmission of FV3, interspecies transmission of FV3 has not been documented between the threespined stickleback (Gasterostelus aculeatus) and tadpoles of the red-legged frog (Rana aurora) .…”