Lloyd-Smith et al. (2005) Matthew J. Gray 2 , Patrick N. Reilly 1,2 , - PDF document

3/22/16 ¡ Role of Wood Frogs & Community Composition in Ranavirus Outbreaks Lloyd-Smith et al. (2005) Matthew J. Gray 2 , Patrick N. Reilly 1,2 , Roberto Brenes 2 , Jordan C. Chaney 2 , Rebecca P. Wilkes 1 , Rachel D. Hill 2 , and Debra L. Miller 1,2 Global Ranavirus Consortium Course UT CVM 1 23 March 2016 UT CWH 2 Wood Frog Hoverman et al. (2011) Of the 38 amphibian species tested, among the top 3 most susceptible species. Widespread Cases: Wood Frog Duffus et al. (2015) D’Aoust-Messier et al. (2015) S. Smith (unpubl. data) B. Rothermel (unpubl. data) M. Gahl (unpubl. data) 12 U.S. States Seven Canadian Provinces More Ranavirus Die-offs in the Wild than any other Species 1 ¡

3/22/16 ¡ Classical Cases: Wood Frogs Wheelwright et al. (2014): ME Green et al. (2002), Todd-Thompson (2010): TN >200,000 Tadpoles Community-level Hoverman et al. In 24 hours Effects? (2011) Grim Reaper: Amplifying Species Ranavirus Persistence Dispersal Host Community Contact Rate Disease Hotspots • Gahl and • Green et al. (2002) Calhoun (2010) • Petranka et al. • Uyeharaet al. (2003) 95-100% (2010) • Harp and • Brunner et al. Petranka (2006) (2011) Superspreading Individuals Amplification Species Shedding Rate Contact Susceptibility Contact Rate Lloyd-Smith et al. (2005) Frontiers in Ecology and the Environment 10:75-82 2012 OBJECTIVES u Test whether superspreading occurs for two common amphibian host species (wood frog and Cope’s gray treefrog) u Test for differences in viral shedding and contact rate between host species: which contributes more to initiate outbreaks? u Test whether infected wood frog tadpoles co-housed with uninfected Cope’s gray treefrog tadpoles results in amplified infection & mortality 2 ¡

3/22/16 ¡ Methods: Highly Susceptible Species • Wood frog ( Lithobates sylvaticus ) 95-100% Mortality Moderately Susceptible Species • Cope’s gray treefrog ( Hyla chrysoscelis) 35-70% Mortality Hoverman et al. (2011) Wood frog tadpole FV3-like Ranavirus • American bullfrog (GA) Miller et al. (2007) Standardized: Gosner 30 Haislip et al. (2011) Cope’s gray treefrog tadpole EXPERIMENTAL DESIGN Inoculation 6-Hour Co-habitation Individual Monitoring • 20 individuals • One exposed individual introduced • 20 exposed were euthanized • 1 liter of water to 10 unexposed individuals (LOW) tested for infection using qPCR • 3 day exposure • N = 20 1224-cm 2 (12 qt) tubs with • Others separated into 2-L tubs • 10 3 PFU/ml 5.5 liters of water Robert et al. (2011) with 1-L of water. Hoverman et al. (2010) • Monitored for 14 days 20 exposed, 200 unexposed individuals Individuals from the same tub, RCB design Results: Wood frog Percent Mortality: Day 5 Day 10 20-80 Rule: <20% of Individuals % Infection Day 14 Result in >80% of Transmission Transmission in All Tubs 17/20 = 85% Superspreading Occurred 3 ¡

3/22/16 ¡ Results: Cope’s gray treefrog % Infection • Transmission in 15% of tubs but no superspreading • No mortality due to ranavirus infection Differences in Host Susceptibility (Initially Exposed: 72 + 6 hrs) Mean Viral Load was 43% greater in Wood Frog Tissue Contact Shedding ? Contact Rates Mean = 0.8 – 0.9 Low Density = contacts/min 44 tadpoles/m 2 15 Minute Observations A a a B Infected wood frog tadpoles moved more Uniform Contact = but number of contacts did not differ with 9 Minutes: 30X Cope’s gray treefrog tadpoles. (co-housed 6 hours) 4 ¡

3/22/16 ¡ Shedding Rates Exposed 72 hours: Water Changed Second Experiment eDNA Viral Load 1.6 A 10^X ug/ul (per .25ug DNA) 1.2 10 1.3 PFU/mL Wood Frogs 10 0.85 PFU/mL 10 0.45 PFU/mL 0.8 per 24 hours B 0.4 10 0.45 PFU/mL 10 0.4 PFU/mL 0 24 48 72 Post-Exposure Time (hrs) Water Collected thereafter: No Virus Detected for HYCH RASY 1, 3, 6, 12, 24, 48, 72 hrs 48 hours (Day 5 PE) Wood frog tadpoles shed more virus than Cope’s gray treefrog tadpoles. How Long to Reach Lethal Dose? LD 50 = 10 2.4 PFU/mL 10 0.45 PFU/mL per 24 hours Reach LD 50 in 8.3 days and LD 100 in 10.5 days (assumes minimal virion degradation & no shedding from dead tadpoles) Contact Most Important Initially, with Shedding Playing a Role Later Warne et al. (2011) Cross-Species Transmission Experiments Transmission to Gray Treefrogs from Wood Frog Tadpoles 70% of Wood Frogs that were initially exposed were infected after 3 days when co-habitation occurred More than gray treefrog tadpole transmission 100% Died+Not Inf amongst each 90% Sur+ Not Inf other (15%). 80% Sur+Inf 70% Died+Inf Transmission in 60% 35% of tubs. 50% 40% 30% 1/20 = 5% Superspreading 20% Occurred. 10% 0% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 No Amplification of Mortality. Co-housed for only 6 hours; no necrophagy 5 ¡

3/22/16 ¡ Cross-Species Transmission Experiments Transmission to Wood Frogs from Gray Treefrog Tadpoles 25% of Gray Treefrogs that were initially exposed were infected after 3 days when co-habitation occurred Thus, ranavirus outbreaks probably 100% Died+Not Inf originate from within-species transmission 90% Sur+ Not Inf 80% events associated with Sur+Inf 70% Died+Inf highly susceptible species. 60% 50% Superspreading Did 40% Not Occur 30% 20% 10% 0% One Transmission 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Event Documented SUMMARY u Probability of ranavirus transmission differs among species. u Wood frog tadpoles are superspreaders of ranavirus but amplification may depend on other host species (highly susceptible) present. u Limitations: Co-housed for 6 hours, Necrophagy not included. u Contact of individuals probably initiates an outbreak, but shedding may be more important later and result in high environmental concentrations that result in rapid transmission and mortality of less susceptible species. Community Level Transmission Objective #2: Mesocosm Brenes, Gray, Hoverman & Miller (unpubl. data) Experiment Appalachian: Wood frog, chorus Inoculated in Lab frog, spotted salamander 10 3 PFU/mL FV3 Coastal Plains : Gopher frog, chorus, Exposure Order southern toad Does Exposure Order or Composition Matter? 6 ¡

3/22/16 ¡ Exposure Order Matters Objective #2: Mesocosm Brenes (2013) Experiment Design Exposure Treatments n = 5 pools/trt Only Wood Frogs 10 larvae/spp Only Chorus Frogs 60 days co-habitation Only Spotted Salamanders Control All transmission Appalachian Community routes possible 43% Chorus Frogs Wood Frogs 100% 52% 12% Spotted Salam (high) 72% Wood Frogs 40% Chorus Frogs 44% 3% Spotted Salam (mod) 24% Chorus Frogs 16% Spotted Salam 6% 18% Wood Frogs (low) Community Composition Matters Objective #2: Mesocosm Brenes (2013) Experiment Design Exposure Treatments Only Gopher Frogs n = 5 pools/trt Only Chorus Frogs 10 larvae/spp Only Southern Toad 60 days Control Gulf Coastal Plain, USA 52% Chorus Frogs Gopher Frogs 100% 62% 34% Southern Toad (high) 70% Gopher Frog 68% Chorus Frogs 78% 58% Southern Toad (high) 32% Chorus Frogs 62% Southern Toad 76% 80% Gopher Frog (high) SUMMARY u Our results suggest that the probability of ranavirus transmission differs among species. u Species exposure order affects ranavirus outbreak outcomes, perhaps due to differences in virus shedding. u Probability of an outbreak increases with the number of highly susceptible species in a community. 7 ¡

3/22/16 ¡ What Causes an Outbreak? A. Peace S. O’Regan • Contact Rates • Shedding • Necrophagy • Environmental Persistence • Density (100/m 2 ) • Community Composition Reservoirs or Amplification Hosts? FV3-like Ranaviruses Gray et al. (2009) Brunner et al. Suitable ¡Hosts ¡ (2015) Low Mortality Low Mortality Low – High Mortality (Subclinical) (Subclinical) (Subclinical & Clinical) Reservoir or Reservoir Reservoir Amplification Questions?? mgray11@utk.edu preilly2@utk.edu rbrenes@carrollu.edu dmille42@utk.edu 8 ¡