a pathogen lurking in the darkness
TRANSCRIPT
University of Tennessee 1Center for Wildlife Health2CVM Department of BDS
Debra L. Miller1,2 and Matthew J. Gray1
A Pathogen Lurking in the Darkness: Global Emergence of Ranavirus
in Ectothermic Vertebrates
M. Niemiller
26 September 2014, 12:00 PM University of Georgia
Outline
I. Ranavirus Characteristics and Die-offs
II.II. Pathology of RanavirusesPathology of Ranaviruses
III.III. Learning about Ranavirus (FV3) EcologyLearning about Ranavirus (FV3) Ecology
IV. Can Ranaviruses Contribute to Declines?
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
History of Ranavirus Die-offsFirst Isolated: •Dr. Allan Granoff
•Rana pipiens (1962)
First Large-scale Die-offs:•Dr. Andrew Cunningham
•Rana temporaria (1992)
•St. Jude Hospital
•Institute of Zoology, ZSL
First North American Die-offs:•Dr. Jim Collins and students
•Arizona State University
•Ambystoma tigrinum stebbinsi (1985, 1997)
A. Duffus
Acta Herpetologica
6 Continents: 1965, 1992Duffus et al. (2015)
Global Distribution of Ranavirus Cases: Amphibians
All Latitudes, All Elevations18 Families: Alytidae, Ranidae, Hylidae, Bufonidae, Centrolenidae, Craugastoridae, Dendrobatidae,
Discoglossidae, Leptodactylidae, Pipidae, Myobatrachidae, Rhacophoridae, Scaphiopodidae, Ambystomatidae, Salamandridae, Hynobiidae, Cryptobranchidae>100 Species
Global Distribution of Ranavirus Cases: Reptiles
12 Families: Agamidae, Anguidae, Boidae, Dactyloidae, Emyididae, Gekkonidae, Iguanidae, Lacertidae, Pythonidae, Testudinidae, Trionychidae, Varanidae
>30 Species
4 Continents: 1982, 1990s
Most FV3-like Ranaviruses: Captivity
Duffus et al. (2015)
Global Distribution of Ranavirus Cases: Fishes
22 Families:>50 Species
4 Continents: 1991
Most non-FV3-like Ranaviruses
Duffus et al. (2015)
Acipenseridae, Anguillidae, Centrarchidae, Channidae, Cobitoidae, Cyprinidae, Eleotridae, Esocidae, Gadidae, Gasterosteidae, Ictaluridae, Labridae, Latidae, Lutjanidae, Moronidae, Percidae, Poeciliidae, Salmonidae, Sciaenidae, Scophthalmidae, Serranidae, Siluridae
>90% of Reports Since
2010
Maine 2013 Die-off
1000 carcasses/m2
>200,000 deadqPCR Confirmed
6/14/13
Wheelwright et al. (2014)
<24 hrs
6/15/13
Ranaviral Disease in Eastern Box Turtles
13 February 2012
26 of 31 Box Turtles Die
from Ranaviral
Disease
Larval anurans and salamanders
dead too
Farnsworth and Seigel, Towson U.
2008 – 2011
North Branch Stream Valley
State Park
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
Ranaviral DiseaseRanaviral Disease
Gross changesGross changes
Lesions can look similar across classes (amphibian, reptile, fish)Lesions can look similar across classes (amphibian, reptile, fish)– Hemorrhage, swelling and necrosis (tissue death) are common gross Hemorrhage, swelling and necrosis (tissue death) are common gross
changeschanges
W. Sutton N. Haislip T. Waltzak
Amphibians: larvae
Photo: J. ChaneyBoreal Toad
Photo: Nathan HaislipBullfrog
affected
unaffectedPhoto: N. HaislipBullfrog
Photo: N. HaislipWood frog
Amphibians: metamorphs
Photo: Jordan ChaneyBoreal Toad
Amphibian: adultsAmphibian: adults
Midwife toad (Alytes obstetricans)Photos: Amanda Duffus
ulceration
Eastern spotted newt (Notophthalmus viridescens)Photo: Betsie B. Rothermel
Hemorrhage
Friable spleen
Hemorrhage
Gopher frogPhotos: B Sutton and R Hardman
Amphibian: adultsAmphibian: adultsEurope: 2 presentationsEurope: 2 presentations
Common frog (Rana temporaria)Photos: Amanda Duffus
ulceration
hemorrhage
Brazil : 2 Brazil : 2 presentations: presentations:
tadpoles vs tadpoles vs adultsadults
(Mazzoni, in prep)(Mazzoni, in prep)
Edema and Hemorrhages
Neurologic: vestibular syndrome
Hemorrhage and necrosis
Photos: R. Mazzoni
Is this Is this ranaviral ranaviral disease in disease in hellbendershellbenders?? Ozark hellbender (K. Irwin)
Ozark hellbender (K. Irwin)
It is in Chinese Giant Salamanders! (Y. Geng)
How about this?How about this?
Cryptobranchus alleganiensis alleganiensisPhoto: Dale McGinnity and Sherri Reinsch
And what role do And what role do ectoparasites (leeches) play?ectoparasites (leeches) play?
Photos: B Sutton and R Hardman
Bullfrog (~10%; 0% FV3)
Cope’s Gray tree frog (~70% RI; ~40% FV3)
Wood frog (~ 100% for both)
Varies by host-susceptibil i ty & virus isolateVaries by host-susceptibil i ty & virus isolate(mortality: RI [ranaculture isolate] vs FV3)(mortality: RI [ranaculture isolate] vs FV3)
Are isolates from captive (culture) facil i t ies more virulent?
Necrotic (white ) areas inside the mouth (circles and arrows). Upper photo with mouth opened. Lower photo with mandible removed
Photo: Mark RuderPhoto: Mark Ruder
Photo: Mark RuderPhoto: Mark Ruder
Photo: Mark RuderPhoto: Mark Ruder
Reptiles(most often chelonian reports)Similar reports in snakes
and l izards(including being off feed, weight
loss, dermatitis)
Eastern Box Turtle (West Virginia)(Terrapene carolina carolina) Eastern Box Turtle (Kentucky)
(Terrapene carolina carolina)
Blue arrows = lef t lungYellow arrows = necrosis
LiverIntest ine
Stomach
Ocular discharge
Ranavirus-NegativeHerpesevirus-NegativeMycoplasma-POSITIVE (M. agassizzi)
Ranavirus-POSITIVEHerpesvirus-NegativeMycoplasma-Negative
Determining etiologyDetermining etiology
Oral mucosal necrosisPhoto: Mark RuderPhoto: Mark Ruder
Lung necrosis
Photo: Mark RuderPhoto: Mark Ruder
Eastern Box Turtle (Virginia)(Terrapene carolina carolina)
Eastern Box Turtle (Kentucky)(Terrapene carolina carolina)
Fish
Photo: Emilie TravisPhotos: Tom Waltzek
Photo: Ted Henry
HistologyHistology
HistologyHistology Lesions can look similar across classes (amphibian, reptile, fish)Lesions can look similar across classes (amphibian, reptile, fish)
– Cellular necrosis of the hematopoietic tissue, vascular endothelium and Cellular necrosis of the hematopoietic tissue, vascular endothelium and epithelial cells and intracytoplasmic inclusion bodies are common epithelial cells and intracytoplasmic inclusion bodies are common microscopic lesionsmicroscopic lesions
Endothel ium
Endothel ial necrosis
Spleen Necrosis
AmphibiansAmphibians
Vestibular hemorrhage and necrosis(Mazzoni, in prep)
FV3 (originally isolated from frog in the wild)
Box turt le isolate(Die-off in wild)
Pall id sturgeon isolate(From culture facil i ty)
Ranaculture isolate(From culture faci l i ty)
Wood Frog Spleen
Repti lesRepti les
Pallid isolate; bath exposure
Pallid isolate; bath exposure
Skeletal muscle (neck): degeneration
Liver: endothelial necrosis, vacuolar degeneration
Trachea: serosal fibrinous necrosisThymus: fibrinous necrosisLung: occasional endothelial necrosis
Lung
Thymus
Trachea
Intestine: endothelial necrosis and hemorrhage
IntestineLu
men
Mucosa: severe epithelial necrosis
unaffected mucosa
vasculitis
Myofibers disrupted
Ulcerated crust on oral mucosa
Disrupted muscle fibers
Photos: Mark RuderPhotos: Mark Ruder
FishFish
Photo: Emilie Travis
Hematopoietic necrosis; tubular epithelial necrosis
Endothelium
Endothelial necrosis
Photo: Tom Waltzek
Visualizing the virusVisualizing the virus
Concurrent Concurrent
InfectionsInfections
Concurrent Concurrent infectionsinfections
URTIURTI
Gopher Tortoise (Gopherus polyphemus)Photo: Jess Gonynor McGuire
Eastern Box Turtle(Terrapene carolina carolina)
Blue arrows = lef t lungYellow arrows = necrosis
LiverIntest ine
Stomach
Ocular discharge
Ranavirus-NegativeHerpesevirus-NegativeMycoplasma-POSITIVE (M. agassizzi)
Ranavirus-POSITIVEHerpesvirus-NegativeMycoplasma-Negative
Determining etiologyDetermining etiology
Oral mucosal necrosisPhoto: Mark RuderPhoto: Mark Ruder
Lung necrosis
Photo: Mark RuderPhoto: Mark Ruder
Eastern Box Turtle (Virginia)(Terrapene carolina carolina)
Eastern Box Turtle (Kentucky)(Terrapene carolina carolina)
46(Y. Geng)
- Antivirals?
- Temperature?
- Vaccine development (Asia)- Much focus on fish
ranaviruses (Japan)- DNA and Live vaccines- Oral delivery
- In China, an inactivated vaccine is being studied for use in Chinese Giant Salamanders.
What about Treatment?What about Treatment?
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 larval development time*• Low aquatic index • Breeding habitat (ephemoral)
Hoverman et al. (2011); Brenes (2013)
All Three Isolates
No Phylogenetic Signal
35 spp
Physiological Trade-off Hypothesis
Susceptibility: 0 – 100%
1/3 of Species: >75%
Mortality
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)
Control Turtle Fish Amph
Graptemys pseudogeographica kohni
Brenes et al (2014a)
Eastern River Cooter – no infection
Apalone ferox
Single-species FV3-like ChallengesFishes
Control Turtle Fish Amph Control Turtle Fish Amph
No Transmission: Nile tilapia, bluegill and fathead minnow
Brenes et al. (2014a)
Journal of Aquatic Animal Health 26:118-126
Gambusia affinis
Ictalurus punctatus
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
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
Amphibian: Hyla chrysocelis ; Turtle: Trachemys scripta elegans; Fish: Gambusia affinis
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. (2014b)
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 FV3-like ranavirus
• Amphibians may be amplifying species
Brenes et al. (2014b)
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
45% of initially exposed tadpoles.
Nearly 0 transmission of ranavirus & no mortality.
Cope’s gray treefrog 100% of initially exposed
tadpoles became infected.
Transmission to and mortality of >80% of unexposed tadpoles.
Wood Frogs
Superspreading and Amplification Superspreading and Amplification of Ranavirusof Ranavirus
Reilly, Gray, Miller (unpubl. data)
Temperature Dependency
FV3-like ranaviruses appears to become pathogenic at 12 C (54 F) but infection in vivo can occur at lower temperatures.
Survival: Brand, Reilly, Chaney et al. (unpubl. data)
FV3 In Vitro Replication Stops at 12 C (Chinchar 2002)
10oC RC Isolate
L. sylvaticus
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%
Rana temporaria
Female Population SizeEarl and Gray (2014)
Closed Population
Time to ExtinctionEarl and Gray (2014)
Closed Population
25 yearsExposed Every Year = 5 years
300 years
Evidence of Rare Species EffectsSutton et al. (accepted)
Endangered Dusky Gopher FrogDiseases of Aquatic
Organisms
n = 18 /trt
ADULTS
Water bath (103 PFU/mL)
Lithobates sevosus
Evidence of Rare Species EffectsChaney et al. (in progress)
Endangered Dusky Gopher Frog
Ranavirus and Small/Isolated Populations
Pearman and Garner (2005)
Isolated Populations had Faster Mortality and Less Genetic Diversity
Isolated Populations
Rana latastei
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)
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)
World Organization for Animal Health
Chytridiomycosis Ranaviral disease
2008
OIE Aquatic Code International Transport of
Animals
•Bleach >4%•EtOH >70%•Virkon >1%
•Nolvasan >0.75%
$75/ bottle
Notifiable Diseases
Schloegel et al. (2010)
Certification for Shipment
Disinfection: Johnson et al. (2003), Bryan et al. (2009), Gold et al. (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
SymposiaDiscussion Groups
WebsiteReporting System
Outreach ResourcesSpringer eBook
Third International Symposium on Ranaviruses
2015 Florida Marine Health Conference
30 May – 1 June 2015 (3 days): Gainesville, FL
Dr. Tom Waltzek
Invited Talks and Poster SessionDirected Topic Discussions
WorkshopsField Trips
Presentation Contributors:
A. Balseiro, SERIDAM. Brand, University of TennesseeR. Brenes, Carroll University J. Chaney, University of TennesseeA. Duffus, Gordon CollegeR. Goodman, Hampden-Sydney CollegeR. Hardman, Laboklin GmbH & Co KGR. Hill, University of TennesseeJ. Hoverman, Purdue UniversityA. Kouba, Memphis ZooJ. Lankton, USGS NWHCR. Mazzoni, Universidade Federal de GoiásD. McGinnity, Nashville ZooP. Reilly, University of TennesseeM. Ruder, USDAS. Schlosshan, UT Histology (CVM)B. Sutton, Tennessee State UniversityT. Waltzek, University of FloridaB. Wilkes, UT Virology (CVM)
Unpublished Data or Gratis Service
