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

Outline

I. Ranavirus-Host Characteristics

II.II. Learning about Ranavirus EcologyLearning about Ranavirus Ecology

III. Can Ranaviruses Contribute to Declines?

IV. Mechanisms of Emergence

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

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

Gross Signs of Infected AmphibiansEdema, Erythema, Hemorrhages, Ulcerations

N. Haislip, UT

A. Duffus, Gordon

D. Green, USGS

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)

Maine 2013 Die-off

1000 carcasses/m2

>200,000 deadqPCR Confirmed

6/14/13

Wheelwright et al. (in review)

<24 hrs

6/15/13

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

Reported Ranavirus Cases in North America: Amphibians

>30 States & 5 Provinces;

46 Species

FamiliesBufonidae

HylidaeRanidae

ScaphiopodidaeAmbystomatidae

CryptobranchidaePlethodontidaeSalamandridae

Norman Wells, NWT

Uncommon

Lithobates sylvaticus

2011

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)

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)

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

UT CWH ResearchFV3-like Ranaviruses

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

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

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)

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)

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

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

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)

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)

Can Interclass Transmission Occur?

Bandin & Dopazo (2011)

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

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)

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)

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)

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%

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

Any Concern for Common Species?Wood Frog Example

Most Widely Distributed Species in North America

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

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

Extinction Probability in 1000 yearsEarl and Gray (in review; EcoHealth)

Closed Population

Time to ExtinctionEarl and Gray (in review; EcoHealth)Closed Population

25 years

Every Year = 5 years

Female Population SizeEarl and Gray (in review; EcoHealth)

Death of Pre-metamorphic Stages

Matters!

Closed Population

Evidence of Rare Species EffectsSutton et al. (in review)

Endangered Dusky Gopher FrogDiseases of Aquatic

Organisms

n = 18 /trt

ADULTS

Evidence of Rare Species EffectsChaney, Gray, Miller & Kouba

Threatened Boreal Toad

Tadpoles Metamorphs

2 – 5 d5 – 7 d

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)

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)

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)

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

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)

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

GRC@LISTSERV.UTK.EDU

SymposiaDiscussion Groups

WebsiteReporting System

Outreach ResourcesSpringer eBook

Questions??

mgray11@utk.edu865-974-2740

Photo: N. Wheelwright