Ranavirus Is Common in Wood Frog (Lithobates Sylvaticus) Tadpoles Throughout Connecticut, USA

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Ranavirus is responsible for 40–60% of amphibian mass mor- been reported: four within the Yale-Myers (YM) Research Forest, tality events in the United States (Gray et al. 2009; Green et al. and four within surrounding public lands in the town of Leba- 2002). In 2011, a private citizen in Lebanon, Connecticut shipped non, Connecticut. Wetlands on public properties were identified two live and five dead Wood Frog (Lithobates sylvaticus) larvae via frog-call surveys in late March and early April. In 2013, we and one dead Spotted Salamander (Ambystoma maculatum) randomly selected state-owned properties distributed across larva to the USGS National Wildlife Health Center in Madison, Connecticut, with the condition that two properties were a mini- Wisconsin, after finding moribund and dead animals. Histologi- mum of 15 km apart to ensure samples were from presumed sep- cal abnormalities from the two live frog larvae and one dead am- arate populations. Within each state property, we located three bystomatid were typical of Ranavirus infection (Gray et al. 2009). individual wetlands by performing a series of nighttime frog-call Polymerase chain reaction (PCR) assays for the major capsid surveys during the Wood Frog breeding season in late March and protein confirmed that isolated viruses were Ranavirus. Follow- early April. We then revisited wetlands beginning in early to mid- ing this event, we learned of several instances throughout north- June to collect Wood Frog larvae. All sampling occurred within eastern Connecticut where moribund or dead tadpoles were ob- a time period of two weeks, and thus sampled larvae were at a served, but with no further action taken. We therefore undertook similar stage of development (average Gosner stage ~ 35). a statewide survey for the presence of Ranavirus in the summers In both years we conducted dip net sweeps until at least 30 of 2012 and 2013. Our specific objectives were to survey for the frog larvae were captured, a sample size sufficient to have 95% presence of Ranavirus in wetlands across the state of Connecti- confidence that the prevalence of infection is < 10% if all samples cut and quantify the proportion of infected larvae within each tested negative (Gray et al. 2015). If Wood Frogs were absent from wetland. a site, we did not collect larvae of other species. These wetlands We focused on surveying larval Wood Frogs due to their high contained Gray Tree Frogs and other species not commonly susceptibility to Ranavirus infections and widespread distribu- found with Wood Frogs, suggesting habitat conditions were not tion across the northeastern USA (Hoverman et al. 2012), howev- ideal for Wood Frog breeding activity. We recorded any gross signs er, larvae of some other species were opportunistically collected of disease or Ranavirus infection (e.g., lethargy, emaciation, ar- and tested for Ranavirus. In 2012, we sampled eight wetlands eas of hemorrhaging). We rinsed tadpoles with sterile water and in northeastern Connecticut, near where previous die-offs had then euthanized tadpoles individually in a solution of MS-222 (Argent Laboratories, Richmond, Washington, USA). Once indi- KELLY M. O’CONNOR* viduals were unresponsive to tactile stimuli, we placed individu- TRACY A. G. RITTENHOUSE als in separate whirl-pak bags (Nasco, Fort Atkinson, Wisconsin, Wildlife and Fisheries Conservation Center, USA) containing 90% ethanol. Between sites, we disinfected all Department of Natural Resources and the Environment, field equipment with a 10% bleach solution, scrubbing any mud University of Connecticut, Storrs, Connecticut 06269, USA from the treads on our boots, and allowing equipment to dry JESSE L. BRUNNER fully (adapted from Phillott et al. 2010). School of Biological Sciences, Washington State University, Liver samples of collected Wood Frog larvae, as well as larval Pullman, Washington 99164-4236, USA Green Frogs and Spotted Salamanders that were collected oppor- *Corresponding author; e-mail: [email protected] tunistically at two wetlands, were screened for Ranavirus DNA with a quantitative real time polymerase chain reaction (qPCR)

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Table 1. Average Ranavirus titer in plaque-forming unit equivalents (Avg. PFU) and number of Lithobates sylvaticus individuals infected out of number of individuals sampled from 29 wetlands at 16 properties across the state of Connecticut, USA. All individuals were collected at the larval stage (~ Gosner stage 35) during the month of June. Wetlands were given a four-letter code for identification (ID). Results are presented in chronological order.

Wetland ID Property name GPS coordinates Year sampled Avg. PFU No. infected

ALTW Aldo Leopold WMA 41.47816◦N, -73.28854◦W 2013 5.04E+08 2/3 CMMM Cromwell Meadows WMA 41.59010◦N, -72.66185◦W 2013 2.41E+04 8/10 CMMO Cromwell Meadows WMA 41.58778◦N, -72.66057◦W 2013 1.29 1/10 COCS Cockaponset SF 41.40500◦N, -72.52228◦W 2013 32.1 1/10 COGL Cockaponset SF 41.42698◦N, -72.53826◦W 2013 465.35 9/10 DHGG Devil’s Hopyard SP 41.48024◦N, -72.34840◦W 2013 0 0/10 DHRG Devil’s Hopyard SP 41.48039◦N, -72.34548◦W 2013 3.81 5/10 DHSH Devil’s Hopyard SP 41.48172◦N, -72.34744◦W 2013 81.78 8/10 HOSP Housatonic SF 41.89636◦N, -73.39604◦W 2013 0 0/10 HOTW Housatonic SF 41.89072◦N, -73.39882◦W 2013 0.8 1/10 NATB Naugatuck SF 41.44533◦N, -73.08320◦W 2013 69.81 2/6 NHFP Nathan Hale SF 41.76262◦N, -72.35644◦W 2013 9.51E+03 8/10 NHND Nathan Hale SF 41.76258◦N, -72.34759◦W 2013 1.36E+07 10/10 NHTT Nathan Hale SF 41.75644◦N, -72.34168◦W 2013 8.45E+07 3/3 PAMT Paugusset SF 41.40675◦N, -73.20113◦W 2013 1.99 1/10 PEBH People’s SF 41.94928◦N, -72.99620◦W 2013 14.7 7/10 PEKI People’s SF 41.95935◦N, -73.00730◦W 2013 1.39 1/10 PESS People’s SF 41.94405◦N, -72.99606◦W 2013 10.7 7/10 RODT Roraback WMA 41.73442◦N, -73.06288◦W 2013 0 0/4 ROSY Roraback WMA 41.73449◦N, -73.05755◦W 2013 3.15 1/10 SGRC Sleeping Giant SP 41.43345◦N, -72.88143◦W 2013 0 0/10 LEBB* Bartlett Brook WMA 41.59752◦N, -72.26767◦W 2012, 2013 159, 1.66E+05 20/22, 10/10 LEPO* Pomeroy State Park 41.69548◦N, -72.22221◦W 2012, 2013 1.77E+04, 1,465 20/23, 10/10 LEBR* Lebanon Private Property Private residence, NA 2012, 2013 3.84E+07, 15.5 22/22, 10/10 LEGR Lebanon Airline Trail 41.614320◦N, -72.367532◦W 2012 2.95E+06 7/9 YMAT* Yale Myers Forest 41.96678◦N, -72.15372◦W 2012, 2013 183.2, 115.3 8/10, 10/10 YMWP* Yale Myers Forest 41.95498◦N, -72.12379◦W 2012, 2013 1.67E+07, 1.01E+08 18/18, 10/10 YME8* Yale Myers Forest 41.96442◦N, -72.14852◦W 2012, 2013 48, 4.58E+03 6/6, 7/10 YMLP* Yale Myers Forest 41.96683◦N, -72.15061◦W 2012, 2013 770.9, 1.53E+06 9/10, 10/10 in the Amphibian Disease Diagnostic Laboratory at Washington across the state of Connecticut. For logistical and financial rea- State University. DNA was extracted with Qiagen DNEasy Blood sons we screened a subset of these samples for Ranavirus: 256 and Tissue kit following the manufacturer’s instructions (QIA- Wood Frogs from 28 of 35 wetlands sampled in 2013 (Table 1, GEN Inc, Valencia, California, USA) and then ca. 5 µL of DNA Fig. 1). We submitted up to ten individual Wood Frogs per wet- template was included in triplicate 20 µL reactions on 96-well land for qPCR testing. We detected Ranavirus in 24 of 28 wet- plates qPCR reaction with primers and probe that amplify a 70- lands tested, at 13 of 14 properties (Table 1). Of the 256 larval bp region within the major capsid protein of all known amphib- Wood Frogs tested, 142 (53.6%) were positive for Ranavirus. The ian ranaviruses in North America (Picco and Collins 2008). A prevalence of Ranavirus was ≥ 50% in 16 of 28 wetlands and 10-fold serial dilution of DNA extracted from a Frog Virus 3-like 100% in seven wetlands (Table 1). We also detected Ranavirus in Ranavirus grown and titered in Epithilium papilloma cyprinia all five Lithobates clamitans larvae from the one site they were cells was used as a standard against which unknown samples collected (ALTW; see Table 1 for site name acronyms), but in were quantified. Samples were scored positive if ≥ 2 of the three none of the A. maculatum larvae from two sites (N = 4, NATB; N wells had clear amplification and negative of zero of three wells = 6, RODT). We cannot be certain that the pathogen was absent had amplification. If there was amplification in just one of the in the four wetlands in which Ranavirus was not detected, only three wells, the sample was re-run and considered positive if ≥ 1 that the prevalence was probably ≤ 28% (based on Wilson confi- well was positive and negative if not. Ranavirus titers are report- dence interval on a proportion, Newcombe 1998). Average titers ed as log10 (average plaque-forming units; PFU) of all replicates were generally similar among tadpoles within the same wetland, of the sample. but varied by orders of magnitude among wetlands in both years In 2012, most Wood Frog larvae (110 of 120; 91.7%) tested (Table 1), from low of 1.29 PFU in CMMO to a high of 5.04 × 108 from the eight wetlands were positive for Ranavirus (Table 1), PFU in ALTW. and Ranavirus was found in all eight wetlands (Fig. 1). In 2013, The widespread occurrence of Ranavirus among our sam- we collected a total of 727 larval Wood Frogs from 35 wetlands pled wetlands aligns with other recent surveys (Glenney et al.

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Fig. 1. Distribution of 29 wetlands on 16 properties across Connecticut, USA surveyed for Ranavirus in 2012 and 2013. Symbols denote the presence or absence of Ranavirus-infected Wood Frog (Lithobates sylvaticus) tadpoles by year sampled. Connecticut’s location relative to the extent of the USA is indicated with a star on the inset panel.

2010; Brunner et al. 2011; Hoverman et al. 2012; Rothermel et al. Check-Off Fund. The USGS-NWHC, specifically D. Earl Green and H. 2013; Crespi et al. 2015). We observed signs of a mass mortality Ip performed pathology and diagnostic testing on tadpoles submit- event at only one wetland in one year (YMLP in Table 1). Here on ted by the CT private landowner in 2011. We are grateful for access to Yale-Myers Forest and assistance provided by members of D. Skelly 4 June 2013 we observed ≥ 100 frog larvae floating near the sur- lab group, notably M. Atwood. The private landowner allowed access face of water with little ability to right themselves or control their to her property in both years. C. Macklem and J. O’Connor assisted direction of movement. These larvae showed little to no response with field work. Research was conducted under University of Con- to our movements when we walked through the wetland; we also necticut Institutional Animal Care Committee in protocol A12-004 found individuals that already appeared to be deceased. When and CT DEEP Scientific and Educational collection permit number in the hand, these larvae had noticeably swollen hind limbs, as 1214001. well as petechial hemorrhaging across the ventral surface and on the hind limbs. Titers were high in this wetland (1.53 × 106 PFU Literature Cited on average, Table 1), but were not the highest observed in this sampling season. No larval amphibians collected or observed Brunner, J. L., K. Barnett, C. Gosier, S. McNulty, M. Rubbo, and M. B. at any other wetland showed gross signs of infection. However, Kolozsvary. 2011. Ranavirus infection in die-offs of vernal pool am- Ranavirus-related mortality events can progress rapidly within phibians in New York, USA. Herpetol. Rev. 42:76–79. a wetland (e.g., Wheelwright et al. 2014) and the majority of our Crespi, E. J., L. J. Rissler, N. M. Mattheus, K. Engbrecht, S. I. Duncan, T. Seaborn, E. M. Hall, J. D. Peterson, and J. L. Brunner. 2015. Geophys- sites were visited only once during the larval period, so even iology of wood frogs: landscape patterns of prevalence of disease though carcasses of Wood Frog larvae may remain detectable for and circulating hormone concentrations across the eastern range. a week or more during die-off events (E. M. Hall, pers. comm.) Integr. Comp. Biol. 55:602–617. our odds of detecting a die-off in any given wetland likely was Glenney, G. W., J. T. Julian, and W. M. Quartz. 2010. Preliminary am- low. Still, our results demonstrate that reported mortality events phibian health survey in the Delaware Water Gap National Recre- severely underestimate the distribution of Ranavirus across a ation Area. J. Aquat. Anim. Health 22:102–114. landscape, especially when surveys sacrifice temporal replica- Gray, M. J., D. L. Miller, and J. T. Hoverman. 2009. Ecology and pathol- tion (i.e., many repeat visits to a single site) to improve spatial ogy of amphibian ranaviruses. Dis. Aquat. Org. 87:243–266. coverage of a landscape (i.e., few visits to many sites). More in- ———, J. L. Brunner, J. E. Earl, and E. Ariel. 2015. Design and analysis tensive monitoring within a wetland is needed to elucidate the of ranavirus studies: surveillance and assessing risk. In M. J. Gray and V. G. Chinchar (eds.), Ranaviruses: Lethal Pathogens of Ecto- dynamics and outcomes of Ranavirus epidemics. thermic Vertebrates, pp. 209–240. Springer International Publish- ing, Heidelberg. Acknowledgments.—Funding was provided by Connecticut De- Green, D. E., K. A. Converse, and A. K. Schrader. 2002. Epizootiology of partment of Energy and Environmental Protection, Wildlife Division sixty-four amphibian morbidity and mortality events in the USA, via Connecticut Endangered Species/Watchable Wildlife Income Tax 1996–2001. Ann. New York Acad. Sci. 969:323–339.

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Hoverman, J. T., M. J. Gray, and D. L. Miller. 2010. Anuran susceptibili- Picco, A. M., and J. P. Collins. 2008. Amphibian commerce as a likely ties to ranaviruses: Role of species identity, exposure route, and a source of pathogen pollution. Conserv. Biol. 22:1582–1589. novel virus isolate. Dis. Aquat. Organ. 89:97–107. Rothermel, B. B., E. R. Travis, D. L. Miller, R. L. Hill, J. L. McGuire, and ———, ———, ———, and N. A. Haislip. 2012. Widespread occur- M. J. Yabsley. 2013. High occupancy of stream salamanders despite rence of ranavirus in pond-breeding amphibian populations. Eco- high ranavirus prevalence in a southern Appalachians watershed. health 9:36–48. Ecohealth 10:184–189. Newcombe, R. G. 1998. Two-sided confidence intervals for the single Warne, R., E. J. Crespi, and J. L. Brunner. 2011. Escape the pond: stress proportion: comparison of seven methods. Stat. Med. 17:857–872. and developmental responses to ranavirus infections in wood frog Phillott, A. D., R. Speare, H. B. Hines, L. F. Skerratt, E. Meyer, K. R. Mc- tadpoles. Funct. Ecol. 25:139–146. Donald, S. D. Cashins, D. Mendez, and L. Berger. 2010. Minimizing Wheelwright, N. T., M. J. Gray, R. D. Hill, and D. L. Miller. 2014. Sud- exposure of amphibians to pathogens during field studies. Dis. den mass die-off of a large population of wood frog (Lithobates syl- Aquat. Org. 92:175–185. vaticus) tadpoles in Maine, USA, likely due to ranavirus. Herpetol. Rev. 45:240–242.

Herpetological Review, 2016, 47(3), 397–400. © 2016 by Society for the Study of Amphibians and Reptiles First Reported Detection of the Amphibian Pathogens Batrachochytrium dendrobatidis and Ranavirus in the Common Mudpuppy (Necturus m. maculosus)

Assessments of the taxonomic and geographic distribution of United States. In this study, we assessed the distribution and re- amphibian pathogens provide the basic information required for gional prevalence of Bd and Ranavirus for N. maculosus in west- comparisons among species with different life histories. Skerratt ern Pennsylvania, USA streams. et al. (2008) suggested that surveillance efforts focus on ecologi- During July and August, we sampled adult mudpuppies in cal groups that are most likely to be exposed to pathogens. Per- eight streams from six counties distributed among the Western manently aquatic salamanders (i.e., obligate paedomorphs) are Glaciated, Northern Unglaciated, and Southern Unglaciated Al- potentially exposed to Batrachochytrium dendrobatidis (Bd) and legheny Plateau ecoregions of Pennsylvania (Fig. 1). Four streams Ranavirus throughout their lifetime in freshwater environments were visited for Bd sampling during 2009–2011 whereas four dif- where these pathogens or infected individuals are present. How- ferent streams were visited for Bd and Ranavirus sampling in ever, few detailed studies have been conducted on salamanders 2015. We collected mudpuppies by searching under relatively with fully aquatic life histories compared to those with complex large rocks (0.25–0.75 m2) in streams, capturing salamanders us- life cycles or direct development. With the exception of the Chi- ing a dip net or by hand, and placing them in a cloth mesh bag. To nese Giant Salamander (Andrias davidanus) and Eastern Hell- minimize cross-contamination during processing we wore new bender (Cryptobranchus a. alleganiensis), permanently aquatic latex gloves and used a different bag for each individual. We used salamanders have not been examined for Ranavirus infection (Duffus et al. 2015). In North America, Bd has been reported within representatives of the salamander genera Amphiuma, Cryptobranchus, Necturus, Pseudobranchus, and Siren. Among the mudpuppies and waterdogs (Necturus spp.), Bd has been reported in the Gulf Coast Waterdog complex (Necturus cf. beyeri) (Chatfield et al. 2012). Bd was not detected in Common Mudpup- pies (Necturus maculosus) in Canada but was reported from one individual collected in the north-central United States (Ouellet et al. 2005; Global Bd Mapping Project: www.Bd-maps.net). Nec- turus maculosus is a relatively abundant salamander that occu- pies diverse freshwater habitats and is widely distributed from southern central Canada south to most of the middle and eastern

KURT J. REGESTER* HALEY C. EISENMAN JENNA L. STRAGAND Department of Biology and Geosciences, Clarion University, Clarion, Pennsylvania 16214, USA CHAD S. BROOKS Fig. 1. Watershed-level distribution of Batrachochytrium dendroba- Department of Biology, Austin Peay State University, tidis (blue border) and both B. dendrobatidis and Ranavirus (yel- Clarksville, Tennessee 37044, USA low border) in the Common Mudpuppy (Necturus m. maculosus) in western Pennsylvania, USA. Black border only indicates neither *Corresponding author; e-mail: [email protected] pathogen detected (Sugar Creek).

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