ranavirus: an emerging pathogen in amphibian, fish and reptile populations in tennessee and beyond
TRANSCRIPT
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Ranavirus: An Emerging Pathogen in Amphibian, Fish and Reptile
Populations in Tennessee and Beyond
University of TennesseeCenter for Wildlife Health
Department of Forestry, Wildlife and Fisheries
Matthew J. Gray
M. Niemiller
6 March 2014, 10:30 AM, TN-TWS Fall Creek Falls State Park
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Outline
I. Ranavirus-Host Characteristics
II.II. Learning about Ranavirus EcologyLearning about Ranavirus Ecology
III. Can Ranaviruses Contribute to Declines?
IV. Mechanisms of Emergence
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Ranavirus Characteristics•dsDNA, 150-280K bp
•120-300 nm in diameter (3x smaller than bacteria)
•Icosahedral Shape (20)
Family: Iridoviridae
Virion
Chinchar et al. (2011)
Iridovirus, Chloriridovirus, Ranavirus, Megalocytivirus, and Lymphocystivirus Genera:
Invertebrates Ectothermic Vertebrates
Paracrystalline Array
Species (6)Ambystoma tigrinum virus (ATV)
Bohle iridovirus (BIV)
Frog virus 3 (FV3)
ICTV (2012)
BalseiroUne
Epizootic haematopoietic necrosis virusEuropean catfish virus
Santee-Cooper Ranavirus
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How Does Ranavirus Infect A Host?
Brunner et al. (2004), Harp & Petranka (2006), Brunner et al. (2007), Hoverman et al. (2010), Robert et al. (2011)
Routes of Transmission
Indirect Transmission
Skin, Gills, Intestines
(epithelial cells)
(3 hrs viral transcription)
Water or
Sediment
Ingestion
Incidental, Necrophagy, Cannibalism,
Predation
(Mortality 2X Faster)
Direct Contact
One Second Skin Contact
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Gross Signs of Infected AmphibiansEdema, Erythema, Hemorrhages, Ulcerations
N. Haislip, UT
A. Duffus, Gordon
D. Green, USGS
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Organ Destruction3 Primary Organs: Liver, Spleen, and Kidney
Spleen Necrosis Kidney Degeneration
Miller et al. (2007, 2008)
D. MillerD. Miller
Target Organ Failure
Heart FailureToxicosis, Anemia
Pathogenesis
Bollinger et al. (1999)
Liver Necrosis
D. Miller
Mortality Can Be Rapid!
Quickly as 3 days!Hoverman et al. (2011a)
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Maine 2013 Die-off
1000 carcasses/m2
>200,000 deadqPCR Confirmed
6/14/13
Wheelwright et al. (in review)
<24 hrs
6/15/13
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Global Distribution of Ranavirus Cases: Amphibians
All Latitudes, All Elevations15 Families: Alytidae, Ranidae, Hylidae, Bufonidae, Leptodactylidae, Dendrobatidae,
Discoglossidae, Pipidae, Myobatrachidae, Rhacophoridae, Scaphiopodidae, Ambystomatidae, Salamandridae, Hynobiidae, Cryptobranchidae
6 Continents: 1992
Miller et al. (2011)
>70 Species
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Reported Ranavirus Cases in North America: Amphibians
>30 States & 5 Provinces;
46 Species
FamiliesBufonidae
HylidaeRanidae
ScaphiopodidaeAmbystomatidae
CryptobranchidaePlethodontidaeSalamandridae
Norman Wells, NWT
Uncommon
Lithobates sylvaticus
2011
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Case ExampleRe-occuring Die-offs
Jamie Barichivich (USGS) and Megan Todd-Thompson (UT)
A. Cressler, USGS A. Cressler, USGSM. Niemiller, UT
GSMNP, Cades CoveGourley Pond
Spotted & Marbled Salamander, Wood Frog, Spring Peeper, Southeastern Chorus Frog
May 1999, 2000, 2009, 2012D. Green, USGS
Green et al. (2002)
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Cases of FV3-like Ranaviral Disease in Reptiles
Over >95% homology with 1000-bp region of FV3 MCP
Gopherus polyphemus, Testudo hermanni, Terrapene carolina carolina, Trionyx sinensis, Uroplatus fimbriatus, and
Chondropython viridis
(Westhouse et al. 1996; Marschang et al. 1999, 2005; Hyatt et al. 2002; DeVoe et al. 2004; Huang et al. 2009; Allender et al. 2006, 2011; Johnson et al. 2007, 2008, 2011)
At least 20 reptile species Marschang (2011)
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Cases of Ranaviral Disease in Fishes
Ictalurus melas, I. nebuosa, Silurus glanis, Psetta maxima, Sander lucioperca, Perca fluviatilis, P. flavescens, Oncorhynchus mykiss, Pomoxis nigromaculatus, Gambusia affinis, Epinephelus tauvina
Journal of Fish Diseases 33:95-122
At least 30 fish speciesEHNV, ECV LMBV, SGIV
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UT CWH ResearchFV3-like Ranaviruses
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0.360.4
0.3
0.15
0
0.1
0.2
0.3
0.4
0.5
Bullfrog Green Frog
FV
3 P
reva
len
ce
Access
Non-access
Cattle Land Use
A
A
A
B
n =104 tadpoles n =80 tadpoles
P =0.78 P =0.02
3.9X More
Likely!!!
Ranavirus in TNCattle Land Use
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Life History and PhylogenyAmphibians
P = 0.354
• Fast development hatching time*• Low aquatic index • Breeding habitat (temporal)
Hoverman et al. (2011); Brenes (2013)
All Three Isolates
No Phylogenetic Signal
35 spp
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Single-species FV3-like ChallengesChelonians
Terrapene carolina, T. ornata, Elseya latisternum, Emydura krefftii , Trachemys scripta
Water bath exposure sufficient for transmission with some species.
Greatest infection and morbidity with IP injection or oral inoculation.
Ariel (1997), Johnson et al. (2007), Allender et al. (2013), Brenes et al. (2014)
Mississippi Map Turtle
Control Turtle Fish Amph
Soft-shelled Turtle
Brenes (2013)
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Single-species FV3-like & ATV Challenges
Fishes
Amelurus melas, Esox luscious, Sander lucioperca, Micropterus salmoides
Cyprinus carpio, Carassius auratus, Lepomis cyanellus
Scaphirhynchus albus
No Transmission:
Low Transmission:
High Mortality:
Jancovich et al. (2001), Bang Jensen et al. (2011a)
Gobbo et al. (2010), Bang Jensen et al. (2009, 2011b), Picco et al. (2010)
Waltzek et al. (in review; DAO)
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Reservoirs or Amplification Hosts?FV3-like Ranaviruses
Low Mortality(Subclinical)
Low Mortality(Subclinical)
Low – High Mortality(Subclinical & Clinical)
Reservoir Reservoir or Amplification
Reservoir
Suitable Hosts
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Community Level TransmissionBrenes, Gray, & Miller (unpubl. data)
Does Exposure Order or Composition Matter?
Inoculated in Lab103 PFU/mL FV3Exposure Order
Appalachian: Wood frog, chorus frog, spotted salamander
Coastal Plains: Gopher frog, chorus, southern toad
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Exposure Order MattersBrenes (2013)
Only Wood FrogsOnly Chorus Frogs
Only Spotted SalamandersControl
n = 5 pools/trt10 larvae/spp
60 days
Exposure Treatments
Design
Wood Frogs 100%43%
12%
Chorus Frogs
Spotted Salam
72%
3%
Wood Frogs
Spotted Salam
24%
18%
Chorus Frogs
Wood Frogs
Chorus Frogs 44%
Spotted Salam 6%
52%
16%
40%
Appalachian Community
(high)
(mod)
(low)
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Community Composition MattersBrenes (2013)
Only Gopher FrogsOnly Chorus Frogs
Only Southern ToadControl
n = 5 pools/trt10 larvae/spp
60 days
Exposure Treatments
Design
Gopher Frogs 100%52%
34%
Chorus Frogs
Southern Toad
70%
58%
Gopher Frog
Southern Toad
32%
80%
Chorus Frogs
Gopher Frog
Chorus Frogs 78%
Southern Toad 76%
62%
62%
68%
Gulf Coastal Plain, USA
(high)
(high)
(high)
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Can Interclass Transmission Occur?
Bandin & Dopazo (2011)
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Experiment
• Direct exposure– Exposed to 103 PFU/mL
– 3 days
• 12-L containers divided in half by a 2000 µm plastic mesh
• Different species in each side of the container
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Turtle and Fish Results
• All classes tested can transmit the virus
• Turtles infected tadpoles– 50% mortality
• Fish infected tadpoles– 10% mortality
50%
10%
Brenes et al. (PLoS ONE, accepted)
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Amphibian Results
• Amphibians transmitted to turtles but not fish
• No mortality of turtles or fish exposed to infected tadpoles
• Suggests that turtles and fish may be reservoirs of ranavirus
• Amphibians may be amplifying species
Brenes et al. (PLoS ONE, accepted)
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Are Ranaviruses Capable of Causing
Local Extirpations and Species Declines?
0
50
100
150
200
250
1960
1963
1966
1969
1972
1975
1978
1981
1984
1987
1990
1993
1996
Nu
mb
er
of
Po
pu
lati
on
s
Collins & Crump (2009)
Muths et al. (2006)
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Evidence of Local ExtinctionDr. Amber TeacherSoutheastern England
Animal Conservation
13:514-522
1996/97 and 2008
Ranavirus (+) populations
81% Median Reduction
Larger PopulationsGreatest
Proportional Declines
A. Teacher
A. Teacher
Teacher et al. 2010
81%
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Evidence of Local ExtinctionDr. Jim Petranka
Tulula Wetland Complex, NC
Rescue Effect
Biological Conservation 138:371-380
Wetlands 23:278-2901998-2006
Recruitment at most wetlands failed due
to ranavirus
Persistence Possible from Source Populations
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Any Concern for Common Species?Wood Frog Example
Most Widely Distributed Species in North America
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p<0.001
Stage Susceptibility of Wood Frogs
Egg = 60% Survival
Hatchling = 20% Survival
Larvae = 0% Survival
Metamorph = 0% Survival
Haislip et al. (2011)
103 PFU/mL
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Wood Frog Population DataDrs. Keith Berven and Elizabeth Harper
Berven (1990), Harper et al. (2008)
Pre-meta 1 yr. old 2 yr. old 3+ yr. old
• Stage-structured discrete-time matrix model (K = 1500 females)• Simulated Exposure for each Stage (egg, hatchling, larva, meta)• Exposure Interval: 50, 25, 10, 5, and 2 years (every year)• Demographic and ranavirus survival probabilities multiplied • 1000 Simulations (one-year steps) • Built in stochasticity in the model• Closed Population
7-year: Beltsville, Maryland
J. Earl
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Extinction Probability in 1000 yearsEarl and Gray (in review; EcoHealth)
Closed Population
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Time to ExtinctionEarl and Gray (in review; EcoHealth)Closed Population
25 years
Every Year = 5 years
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Female Population SizeEarl and Gray (in review; EcoHealth)
Death of Pre-metamorphic Stages
Matters!
Closed Population
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Evidence of Rare Species EffectsSutton et al. (in review)
Endangered Dusky Gopher FrogDiseases of Aquatic
Organisms
n = 18 /trt
ADULTS
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Evidence of Rare Species EffectsChaney, Gray, Miller & Kouba
Threatened Boreal Toad
Tadpoles Metamorphs
2 – 5 d5 – 7 d
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Factors Contributing to Emergence
Other Possible Stressors: Pesticide Mixtures, Nitrogenous Waste, Endocrine Disruptors, Acidification, Climate Change, Heavy Metals
Pathogen Pollution:
Anthropogenic introduction of novel strains to naïve populations
(Cunningham et al. 2003)
•Fishing Bait •Ranaculture Facilities
•Biological Supply Companies•International Food & Pet Trade
•Contaminated FomitesPicco et al. (2007) Schloegel et al. (2009)
Anthropogenic Stressors:
1) Herbicide (Atrazine)
Forson & Storfer (2006); Gray et al. (2007); Greer et al. (2008); Kerby et al. (2011)
ATV SusceptibilityA. tigrinum
2) Cattle Land Use: Prevalence Green Frogs and Tiger Salamanders
Insecticide (Carbaryl)
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Global Trade of Ranavirus Hosts
From 2000-2006, the U.S. imported >1.5 billion individual animals (fish & wildlife; Smith et al. 2009)
90% fish, 2% amphibians, 1% reptiles25 million live amphibians imported to U.S./year
Ranavirus Positive
•Hong Kong = 89%•Dominican Republic = 70%•Madagascar = 57%
Kristine Smith, DVM
Smith et al. (unpubl. data)
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Pe
rce
nt
mo
rta
lity
0
10
20
30
40
50
60
70
80
90
100
FV3
RI
Ranaculture isolate 2X more lethal than FV3
Risk of Pathogen PollutionMajji et al. (2006), Storfer et al. (2007), Mazzoni et al. (2009), Hoverman et al. (2011a), Brenes (2013)
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Should we be concerned?
•Ranavirus are Multi-species Pathogens •Amphibians with fast-developing larvae most susceptible
•Isolated populations (rare species) greatest threat•Interclass Transmission can occur •Community Composition matters
•Transmission is efficient – Multiple Routes •Environmental Persistence is long (1 – 3 mo)
•Anthropogenic Stressors and Pathogen Pollution contribute to Ranavirus Emergence
Ranaviruses represent a significant threat to the global biodiveristy of ectothermic vertebrates
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What can we do?
•Establish surveillance programs (broad then focus on hotspots, >40% infection)
•Identify mechanisms of emergence(natural, stressors, novel strains)
•Identify and implement intervention strategies(break host-pathogen cycle, reduce stressors,
biosecurity precautions)
Gray and Miller (2013)
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Global Ranavirus Consortiumhttp://fwf.ag.utk.edu/mgray/ranavirus/ranavirus.htm
The goal of the GRC is to facilitate communication and collaboration among scientists and veterinarians conducting research on ranaviruses and diagnosing
cases of ranaviral disease
SymposiaDiscussion Groups
WebsiteReporting System
Outreach ResourcesSpringer eBook