DOI: 10.7589/2013-03-051 Journal of Wildlife Diseases, 49(4), 2013, pp. 879–886 # Wildlife Disease Association 2013 LOW DETECTION OF RANAVIRUS DNA IN WILD POSTMETAMORPHIC GREEN FROGS, RANA (LITHOBATES) CLAMITANS, DESPITE PREVIOUS OR CONCURRENT TADPOLE MORTALITY Marı´a J. Forza´n,1,3 and John Wood2 1 Canadian Cooperative Wildlife Health Centre, Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave., Charlottetown, Prince Edward Island C1A 4P3, Canada 2 Pisces Molecular LLC, 1600 Range St. Suite 201, Boulder, Colorado 80301, USA 3 Corresponding author (email: [email protected]) ABSTRACT: Ranavirus (Iridoviridae) infection is a significant cause of mortality in amphibians. Detection of infected individuals, particularly carriers, is necessary to prevent and control outbreaks. Recently, the use of toe clips to detect ranavirus infection through PCR was proposed as an alternative to the more frequently used lethal liver sampling in green frogs (Rana [Lithobates] clamitans). We attempted reevaluate the use of toe clips, evaluate the potential use of blood onto filter paper and hepatic fine needle aspirates (FNAs) as further alternatives, and explore the adequacy of using green frogs as a target-sampling species when searching for ranavirus infection in the wild. Samples were obtained from 190 postmetamorphic ($1-yr-old) green frogs from five ponds on Prince Edward Island (PEI), Canada. Three of the ponds had contemporary or recent tadpole mortalities due to Frog Virus 3 (FV3) ranavirus. PCR testing for ranavirus DNA was performed on 190 toe clips, 188 blood samples, 72 hepatic FNAs, and 72 liver tissue samples. Only two frogs were ranavirus-positive: liver and toe clip were positive in one, liver only was positive in the other; all blood and FNAs, including those from the two positive frogs, were negative. Results did not yield a definitive answer on the efficacy of testing each type of sample, but resemble what is found in salamanders infected with Ambystoma tigrinum (rana)virus. Findings indicate a low prevalence of FV3 in postmetamorphic green frogs on PEI (#2.78%) and suggest that green frogs are poor reservoirs (carriers) for the virus. Key words: Blood, fine-needle aspirate, FV3, liver, PCR, Rana clamitans, ranavirus, toe clips. INTRODUCTION been enhanced by introduction of foreign amphibian species into naı¨ve populations Amphibian populations worldwide have (Harp and Petranka, 2006). suffered severe recent declines, partly Prevention of the spread of disease, in because of the spread of diseases, includ- the wild or in captivity, requires accurate ing those caused by ranavirus infection diagnosis of the infection status of re- (Daszak et al., 1999). Ranaviruses (Irido- presentative individuals in a population. viridae) can infect a variety of species of Detection of ranavirus infection can be amphibians, fish, and reptiles, but there is achieved by PCR testing on liver tissue some host-dependent susceptibility: Frog (adult and tadpole), whole-body samples Virus 3 (FV3) usually, but not exclusively, (usually tadpoles), or tail clips from infects frogs and toads (Chinchar, 2002) salamanders and tadpoles (Brunner et al., wheras Ambystoma tigrinum virus (ATV) 2004; Gray et al., 2009). Lethal methods of tends to cause infection in salamanders detection are adverse to threatened or (Jancovich et al., 1997). Infection causes endangered populations and preclude acute mortality of tadpoles (98–100%), and long-term studies on individuals. Recently, can result in severe dermal and visceral toe clips were proposed as nonlethal lesions and death of adults (Greer and samples to detect ranavirus infection in Collins, 2007; Cunningham et al., 2008). wild frogs through PCR (St-Amour and Dissemination of ranavirus infection has Lesbarreres, 2007). Although a welcomed 879 880 JOURNAL OF WILDLIFE DISEASES, VOL. 49, NO. 4, OCTOBER 2013 alternative, toe-clipping may cause inflam- mation, necrosis, or decreased survival, so its use is often discouraged (Canadian Council on Animal Care, 2004). Other potentially suitable samples include blood and hepatic fine-needle aspirate (FNA): ranavirus has been detected in circulating lymphocytes (Cunningham et al., 2008) and FNA is an invasive but nonlethal procedure. In the wild, more than one species of amphibian are likely to be present, and thus targeting a particular species may increase or decrease the FIGURE 1. Map of Prince Edward Island, Cana- da, showing ponds were green frogs (Rana [Litho- chances of finding a particular pathogen, bates] clamitans) were sampled and tested for depending on how likely that species is to ranaviruses in 2010 and 2011: MCI (n530 frogs), be infected. We evaluate postmeta- BUE (n557), LVS (n535), DWM (n559), and EW2 morphic green frogs (Rana [Lithobates] (n59). Scale bar525 km. clamitans) as a target species for the detection of ranavirus in a natural envi- Inc., Quebec, Canada) were applied with a syringe onto the first digit of the left caudal limb ronment, and whether hepatic FNA or (Fig. 2). While the lidocaine numbed the area, blood collection onto specialized filter blood was collected by puncture of the facial paper (Smith and Burgoyne, 2004) could (maxillary) vein with a 25-gauge needle and replace toe-clipping or liver sampling in recovered onto a heparinized capillary tube detecting ranavirus infection using PCR. (Microhematocrit Capillary Tubes, Fisher Sci- entific, Pittsburgh, Pennsylvania, USA; Fig. 3; Forza´n et al., 2012). Blood was immediately MATERIALS AND METHODS transferred to an FTATM Minicard (Whatman- GE Healthcare UK Limited, Buckinghamshire, Postmetamorphic ($1-yr-old juveniles and UK; Fig. 3). Once dry, the FTA card was adults) green frogs (n5190) were collected placed in a multi-barrier pouch with a desiccant from five permanent ponds on Prince Edward (Whatman-GE Healthcare), which was instant- Island (PEI), Canada (46u129N, 63u129W), during August 2010 and July–August 2011. ly sealed. The first digit, along with a small Sampling in 2010 was restricted to a toe clip amount of webbing, was then amputated, and blood sample (n5118 and 116, respective- holding it gently with tissue forceps and cutting ly), sampling in 2011 included toe-clip, blood, swiftly with Stevens curved tenotomy scissors liver, and hepatic FNA samples (n572 of each (Fig. 2); the toe clip was placed in a 2-mL sample). The ponds were selected based on screw-capped microcentrifuge tube with 70% their abundant green frog population (all ethanol. The amputation site was sprayed with ponds) and on the diagnosis of tadpole Bactine antiseptic (Bayer Inc., Ontario, Cana- mortalities due to a ranavirus (ponds BUE, da); the frog was placed back in the plastic DWM, and EW2; Fig. 1) the previous or container and, after a few minutes of demon- contemporary summer (Canadian Cooperative strating no adverse effects from the procedures, Wildlife Health Centre, unpubl. data). Frogs released back into the pond. In only two cases were captured using dip nets, placed in were we unable to extract blood from a frog. individual solid plastic containers with breath- Collection of samples in 2011 varied slightly: ing holes and enough water to keep them moist, it included euthanasia and additional collec- and kept in a shady area until capture was tion of liver tissue and hepatic FNA. Frogs completed and sampling began. All equipment were collected and bled as per description was disinfected between captures using a spray above; after bleeding, each frog was eutha- of 5% bleach (sodium hypochlorite) solution nized by applying 20% benzocaine (Orajel and rinsed after at least 30 sec of contact (The PM, Church & Dwight Co., Ontario, Canada) Australian Treatment Abatement Plan, 2006). gel (Altig, 1980) to its ventral abdomen and Each frog was similarly sampled to collect inner thighs using a wooden tongue depressor. blood and a toe clip in 2010. A few drops of Once the frog became nonresponsive, toe- 2% lidocaine (Lurocaine, Ve´toquinol N. A. clipping was performed, and the frog placed in FORZA´ N ET AL.—LOW DETECTION OF RANAVIRUS IN ADULT GREEN FROGS 881 a sealable plastic bag until death. Dead frogs were kept in a Styrofoam cooler with ice packs and transported to the necropsy room (Atlantic Veterinary College, Charlottetown, Canada) where weighing, FNA, and tissue collection were performed. The FNA was obtained using a 5-cc syringe with a 22-gauge needle; collected material was expelled from the syringe onto an FTA card which was dried and subse- quently placed in a sealed pack (Whatman- GE Healthcare). The coelomic cavity was incised with a new scalpel blade, and a small portion of liver was collected, using forceps and that same blade, and placed in a microcentrifuge tube with 70% ethanol. Samples of spleen and kidney were also obtained and preserved in microcentrifuge tubes at 280 C. Nine kidney samples were later thawed to obtain a subsample that was placed in 70% ethanol for PCR testing. The rest of the carcass was placed in 10% buffered formalin. Unlike in 2010, when blood was not obtained from two sampled frogs, in 2011 all samples (blood, toe clip, hepatic FNA, liver and kidney tissues) were collected from each of the 72 frogs collected. The surgical instruments used for toe- clipping in the field (2010 and 2011) were sterilized between each frog with a dip in 70% alcohol immediately followed by exposure to the open flame of a wickless metal alcohol burner (model 97-5320, C&A Scientific, Ma- nassas, Virginia, USA) or a candle, and kept on a clean stainless steel tray while not in use. A new tongue depressor was used to apply the euthanizing benzocaine gel to each frog. The forceps used to collect the liver, kidney, and spleen samples were sterilized with a micro- incinerator (Bacti-cinerator, McCormick Sci- FIGURE 2. Toe-clipping of a green frog (Rana entific LLC, St. Louis, Missouri, USA) and a [Lithobates] clamitans) for sampling and testing for new scalpel blade was used for each frog.