PREVALENCE OF RANAVIRUS IN GREEN FROGS (Lithobates clamitans) ACROSS INDIANA Megan E. Winzeler1,2, Steven J. A. Kimble2 and Rod N. Williams2 1Savannah River Ecology Lab, University of Georgia, Aiken, SC 29802 2Department of Forestry & Natural Resources, Purdue University, West Lafayee, IN 47907 INTRODUCTION ABSTRACT RESULTS & DISCUSSION Ranavirus is considered a primary pathogen currently threatening amphibians, aacking the immune system Worldwide amphibian declines are due to habitat loss & fragmentaon, This study found all green frogs to be Ranavirus negave using of larval amphibians and causing mass die-off events across six connents. This disease can persist in chemical contaminaon of habitat, introducon of exoc species & the populaons through sub-lethal infecon of adults, spreading from one site to the next through dispersal or convenonal PCR methods. spread of infecous disease. anthropogenic disturbance. Indiana has a highly fragmented landscape which can lead to higher degrees of anthropogenic disturbance and possibly a greater likelihood of Ranavirus spreading throughout the state. We We are sll waing on results from quantave PCR. The assay is currently Ranavirus is a viral pathogen infecng fish, reples & amphibians at varying hypothesized that Ranavirus would be present in the state due to this fragmentaon. We sampled 6 sites developed and based on published sequences of the Ranavirus degrees of virulence. This involves complex interacons between reservoirs (Tippecanoe, Randolph, Porter, Jennings, Posey, Whitley Counes), targeng green frog (Lithobates clamitans) FV3 MCP. of the disease, direct & indirect transmission routes, anthropogenic stressors tadpoles for collecon of liver ssue. Tadpole livers (Tippecanoe: 46, Randolph: 24, Porter: 40, Jennings: 40, & immunity of the individual. Due to life history characteriscs like habitat Posey: 3, Whitley: 16) were used to extract DNA, and convenonal PCR and qPCR analysis was performed on We expected there to be a high prevalence of Ranavirus in Indiana due to and a mul-stage life cycle, amphibians have an increased suscepbility to each individual sample. Each sample was extracted and amplified under sterile condions to prevent the the highly fragmented landscape and history of anthropogenic disturbance. Ranavirus. cross-contaminaon of any one individual to another. Posive samples were idenfied in triplicate on agarose The resulng zero prevalence value may be due to the imprecise methods gels run with mulple posive and negave controls. This study found no presence of Ranavirus at the six of convenonal PCR. A more accurate representaon of the prevalence of Indiana’s landscape is highly fragmented with a long history of disturbance. sites using convenonal PCR methods. Further research should include larger sample sizes at mulple Ranavirus in the individuals sampled could be represented using qPCR locaons at more sites should be done to determine actual prevalence of this disease in Indiana. The influence of anthropogenic stressors like fragmentaon, pollutants and methods, which is more sensive and quanfies the load of the disease in other forms of disturbance, aid in the movement of aquac diseases across the animal. the state. METHODS Addionally, an increased effort to cover Indiana’s enre geographic range • Frog virus 3 (FV3) infects amphibian species more completely with larger sample sizes at several wetlands at each site • Signs of infecon include emaciaon & hemorrhaging (Figure 1) Study Areas would result in a beer idea of the distribuon of this disease. • Prevents proper osmoregulaon • All sites were lenc wetlands with established green frog populaons (Figure 3) • Suscepbility varies among species • 4 out of 6 sites near areas of high disturbance Figure 5. A • Ranid species are more suscepble due to natural history • Surrounding habitat is disturbed by agricultural use and developed by roads standard curve characteriscs based on the known Field Sampling • More virulent in larval amphibians but adults act as reservoir Ranavirus Site # Tadpoles standards • 40 minnow traps at each site included on • Ranavirus is more likely to persist in semi-permanent wetlands with a high Tippecanoe Co. 46 • Checked traps twice daily for 2 days qPCR plates. All diversity of amphibian species • Individuals euthanized (Figure 4) in a MS-222 & stored in Randolph Co. 24 unknown samples are fit ethanol Porter Co. 40 to this line to • Tadpoles idenfied by labial tooth rows & placed in individual Figure 1. Clinical signs of Ranavirus Figure 2. Sites sampled in Indiana Jennings Co. 40 determine 50mL conical tubes containing 70% ethanol presence of Posey Co. 3 Ranavirus. Genec Sampling Whitley Co. 16 Table 1. Number of tadpoles caught at each site. • Liver samples were extracted from euthanized individuals • Primers 4 & 5 from Mao et al. (1997) specific for a 510 bp fragment of the Ranavirus major Figure 6. Gel run capsid protein (MCP) were used in convenonal PCR illustrang a negave sample • New primers were developed for qPCR analyses based on Mao et al.’s (1997) primers (Well 2), a • Reacons performed under a BioSafety hood with equipment & tools sterilized using ethyl posive control alcohol & bleach soluons (Well 3) & a • All samples were amplified 4 mes, posive samples 8 mes for convenonal PCR posive sample (Well 4). Ladders are in Wells 1 & Figure 3. Minnow trap array in Whiteley Co. Figure 4. Euthanizing tadpoles at site. 5.

FUTURE

Photos courtesy of Jason Hoverman In the coming weeks, we will be compleng the qPCR analysis in order to compare the results from qPCR and convenonal PCR methods. Future research should focus on GOALS & OBJECTIVES larger sample sizes covering a wider geographic range and using qPCR methods.

The goal of this project is to accurately assess the distribuon and prevalence of ranavirus across the state of Indiana. This will be evaluated by: ACKNOWLEDGEMENTS • The collecon of green frog tadpoles from six sites across Indiana (Figure 2). We would like to thank Purdue University & the Forestry & Natural Resources Department for financial contribuons. We also thank Gliselle Marin and Vanessa • Wuerthner for assistance field sampling. The College of Agriculture provided funds in Tesng for Ranavirus using convenonal and quantave PCR analysis. the College of Agriculture Undergraduate Research Grant.