Journal of Wildlife Diseases, 43(2), 2007, pp. 286–290 # Wildlife Disease Association 2007 Susceptibility of the Endangered California Tiger Salamander, Ambystoma californiense, to Ranavirus Infection Angela M. Picco,1,3 Jesse L. Brunner,1,2 and James P. Collins11School of Life Sciences, Arizona State University, Tempe, Arizona 85287, USA; 2 Current address: Institute of Ecosystem Studies, Millbrook, New York 12545, USA; 3 Corresponding author (email: [email protected]) ABSTRACT: Emerging infectious diseases are food, pets, and research (Jancovich et al., implicated in the declines and extinctions of 2005; Schlaepfer et al., 2005), which can amphibians worldwide. Ranaviruses in the potentially move amphibian pathogens family Iridoviridae are a global concern and have caused amphibian die-offs in wild popula- from their native range into na1¨ve popula- tions in North America, Europe, South Amer- tions (Cunningham et al., 2003). These ica, and in commercial populations in Asia and pathogens include ranaviruses, such as South America. The movement of amphibians Ambystoma tigrinum virus (ATV; Iridovir- for bait, food, pets, and research provides idae), that have caused amphibian die-offs a route for the introduction of ranaviruses into naive and potentially endangered species. In (Chinchar, 2002; Jancovich et al., 2005). this report, we demonstrate that the California The potential for moving these viruses tiger salamander, Ambystoma californiense,is through the commercial trade of amphi- susceptible to Ambystoma tigrinum virus bians, such as fishing bait, has been (ATV). This virus has not been previously reported in California tiger salamander, but demonstrated by the detection of rana- observed mortality in experimentally infected virus-infected tiger salamander larvae in animals suggests that California tiger salaman- 85% of surveyed bait shops in Arizona der populations could be adversely affected by during the 2005 fishing season (Picco, an ATV introduction. unpubl. data). Jancovich et al. (2005) Key words: Ambystoma californiense, Am- bystoma tigrinum virus, ranaviruses, tiger reported that an ATV isolate from tiger salamander. salamanders purchased from a bait shop in Phoenix, Arizona, was most closely related Amphibians are declining and becoming to isolates from Colorado. There is clear extinct on a global scale, at a rate exceed- evidence that ATV is present in the tiger ing that of birds and mammals (Stuart et salamander bait trade, but the impact of al., 2004). Three relatively well under- potential introduction to na1¨ve hosts is less stood causes of declines include the clear. Thus, we tested whether the endan- introduction of exotic species, commercial gered California tiger salamander, Ambys- overexploitation, and land use change. toma californiense, from which ranaviruses However, amphibians also have declined have not been isolated, is vulnerable to in protected parks and reserves where ATV. these factors should not be operating, and Adults of the endangered California in such areas, three additional factors are tiger salamander and three subspecies of suspected: global climate change, con- Ambystoma tigrinum (A. t. nebulosum, taminants, and emerging infectious dis- Arizona tiger salamander; A. t. mavortium, ease (EID) (Collins and Storfer, 2003; barred tiger salamander; and the endan- Stuart et al., 2004). Emerging infectious gered A. t. stebbinsi, Sonora tiger sala- diseases have been implicated in amphib- mander) were experimentally infected ian declines in Central America and with ATV. Three subspecies of tiger Australia (Lips et al., 2006; Pounds et al., salamanders were included in this study 2006), and they are increasingly reported to compare mortality and time course of in amphibian populations around the infection between known susceptible spe- world (Retallick et al., 2004; Ron, 2005). cies/subspecies and the endangered Cali- Amphibians are moved globally for bait, fornia tiger salamander. The A. t. stebbinsi 286 SHORT COMMUNICATIONS 287 and A. t. nebulosum were obtained from were injected with 200 ml of APBS solu- our breeding colony, and the A. califor- tion. The ATV isolate used was collected niense and A. t. mavortium were provided from an A. t. nebulosum on 23 July 2002 by H. B. Shaffer (University of California, near Prescott, Arizona, and it was grown Davis, California) (12 salamanders of each on epithelioma papilloma cyprini cells species/subspecies); all animals were sur- (Fijan et al., 1983) for three passes before plus animals that could not be reintro- this experiment (Jancovich et al., 2005). duced into natural populations. The A. t. Prescott, Arizona, is an area with salaman- mavortium were originally collected from ders known to be involved in tiger Clearlake Oaks, California, and the A. salamander translocations for bait pur- californiense were from White Slough and poses (Collins, 1981). Great Valley Grasslands in California. The All animals were observed daily for A. t. stebbinsi were laboratory-bred ani- signs of infection for 60 days and then mals from stock originally collected from twice a week for an additional 90 days. the San Rafael Valley, Arizona. The A. t. Signs of ranavirus infection were re- nebulosum were offspring of stock origi- corded, including lesions, papules, and nally collected from the White Mountains, exudates from cloaca (Jancovich et al., Arizona. Sample sizes were limited due to 1997). To monitor infections, tail clips the endangered status of A. californiense. were taken from all surviving control and Of the 12 individuals from each species/ treatment animals each month and subspecies, six individuals (three males screened by polymerase chain reaction and three females) were assigned to the (PCR). The experiment was terminated treatment group, and the other six indi- after 150 days, at which point all surviving viduals (three males and three females) animals were euthanized with an overdose were assigned to the control group. of MS-222 (Poole, 1987). Upon death, Animals from similar localities and samples were immediately collected from clutches were evenly distributed between liver, stomach, spleen, intestine, and body treatment and control groups to avoid wall using sterile equipment for each confounding factors associated with local- individual. All samples were homogenized ity and clutch. in 2 ml of APBS in individual sterile bags All animals were individually housed at with a Stomacher 80H (Seward Ltd., 20 C in 5.7-l plastic boxes (35 3 21 3 London, England) and frozen at 280 C 11 cm) placed at an incline filled with for later screening. 700 ml water on one side, and fed five DNA was extracted from 150 ml of each crickets once per week. Animals were of the homogenized samples by using a salt acclimated to laboratory conditions for extraction protocol (Sambrook and Rus- 2 mo before the experiment. Thirty days sell, 2001). Extracted DNA was tested for before the challenge experiment, tail and the presence or absence of ranaviruses toe tissue samples were taken from all using PCR primers amplifying an ,500 animals for ATV screening to verify that base pair (bp) region of the major capsid animals were not infected before the protein (MCP) of ranaviruses using pri- experiment. mers 4 and 5 described in Mao et al. Treatment animals were intraperitone- (1997). Samples were visualized by elec- ally injected with 200 ml of inoculum trophoresis on 1.5% agarose gels. Quanti- containing 1,000 virions (determined by tative PCR also was used to test animals plaque assay) of ATV suspended in for ATV infection. Animals were consid- amphibian phosphate-buffered saline ered infected if either diagnostic or (APBS) solution (6.6 g of NaCl, 1.15 g of quantitative PCR was positive. The quan- anhydrous Na2HPO4, 0.2 g of KH2PO4, titative PCR protocol amplified a 70-bp and sterile water to 1 l). Control animals region of the ranavirus MCP (Brunner, 288 JOURNAL OF WILDLIFE DISEASES, VOL. 43, NO. 2, APRIL 2007 TABLE 1. Mortality rates and detection of Ambystoma tigrinum virus (ATV) in experimentally exposed and control animals of each of four species/subspecies of tiger salamander. Mortalitya PCR resultsb Species Control ATV-exposed Control ATV-exposed Ambystoma californiense 2/6 5/6 0/6 6/6 A. t. stebbinsi 0/6 5/6 0/6 6/6 A. t. mavortium 0/6 4/6 0/6 5/6 A. t. nebulosum 1/6 6/6 0/6 6/6 a Number dead/number injected. b Number positive/number injected. 2004). Each sample was analyzed in pules, hemorrhaging from lesions in the triplicate in 20-ml reactions containing skin, and exudate from the cloaca. Five of 10 mlof23 TaqMan Universal PCR the six salamanders died, three of them on Master mix (Applied Biosystems, Foster day 14, and one each on day 16 and day City, California, USA), 2 ml (100 ng) of 18. ATV was detected by PCR in all six of template DNA, 300 nmol rtMCP-for (59- these animals, confirming their suscepti- ACACCACCGCCCAAAAGTAC-39)and bility to ATV infection (Table 1). Two of 900 nmol rtMCP-rev (59- CCGTTCAT- six control A. californiense died on days 2 GATGCGGATAATG-39), and 250 nmol and 68, but ATV was not detected by PCR fluorescent probe rtMCP-probe (59-FAM- in these or any other control animals CCTCATCGTTCTGGCCATCAACCAC- (Table 1). TAMRA-39). Quantitative PCR analyses Mortality differed between treatment were conducted in 384-well optical PCR and control animals across all species/sub- plates on an ABI Prism 7900 Sequence species (x2525.696, df51, P,0.0001), but Detection system (Applied Biosciences). it did not differ among species/subspecies Negative and positive controls were in- (Table 1; x253.175, df53, P50.36). The cluded in each run for both diagnostic mean time to death did not differ among PCR and quantitative PCR. No attempt species/subspecies (x253.8063, df53, was made to quantify the amount of viral P50.28). DNA in the samples; quantitative PCR California tiger salamanders were in- was only used in this study to detect the fected with ATV; all six ATV-exposed presence or absence of ranaviruses.