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Water Temperature Affects Susceptibility to Ranavirus
Mabre Brand1,2, Matthew Gray1, Becky Wilkes2, Roberto Brenes1 and Debra Miller1,2
1Center for Wildlife Health2College of Veterinary Medicine
University of Tennessee-Knoxville
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Die-offs in Summer
Ken Dodd (USGS), Jamie Barichivich (USGS), and Megan Todd-Thompson (UT)
A. Cressler, USGS A. Cressler, USGSM. Niemiller, Yale Univ.
Spotted & Marbled Salamander, Wood Frog, Spring Peeper, Southeastern Chorus Frog
May 1999, 2000, 2009, 2012, 2013:
GSMNP: Cades Cove
Green et al. (2002), Todd-Thompson (2010)
Virus Replication increases with Temperature12 – 32 C (in vitro) Chinchar (2002)
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Ranavirus Landscape PrevalenceTennessee Ponds
Green Frog, Bullfrog, Pickerel Frog, Eastern
Newt, Tiger and Spotted Salamanders
Ranavirus Distribution: 83% of Ponds Sampled
Hoverman et al. (2011)
Greatest Prevalence and a Die-off in Autumn
2011
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Seasonal Trends in Prevalence 0.57
0.15 0.15
0.24
0.45
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Bullfrog Green Frog
FV
3 P
reva
len
ce
Winter
Summer
Fall
Season
A
AB
B
n =104 tadpoles n =80 tadpoles
P< 0.02 P =0.006
B
No Winter Captures
DAO 77:97-103
• Increase in pathogenicity of the ranavirus ATV at colder temperatures
• Decrease in immune function of ectothermic vertebrates at colder temperatures
Rojas et al. (2005)
Raffel et al. (2006)
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Does Temperature Play a Role in the Emergence of Ranavirus?
• Seasonal Trends:– Density dependent factors– Changes in natural (predator density, development)
or anthropogenic stressors (nitrogen concentration)
• Water Temperature’s Role: – Viral Replication vs. Immune Function – Function as a Natural Stressor
Chinchar (2002), Raffel et al. (2006)
Long et al. (2012)
– Fish: regulation of transcription, nucleosome assembly, chromatin organization and protein folding
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Competing Hypotheses
• Virus Replication Hypothesis– Ranavirus replication increases with temperature up
to 32 C– Caveat: Immune function in ectotherms also
increases with temperature• Temperature Induced Stress Hypothesis
– Early Spring Breeding Species: • Stressed by Warm Temp
– Summer Breeding Species:• Stressed by Cold Temp
High Pathogenicity at Higher Temperatures
Pathogenicity is Species-specific and Related to Typical
Water Temperature Experienced During
Tadpole Development
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Objective
Test for Differences in Pathogenicity of Ranavirus at Two Temperatures (10 and 25 C) among Four
Amphibian Species (two spring breeding and two summer breeding species)
Indices of Pathogenicity: • Percent Mortality• Infection Prevalence
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Species Tested
• Early spring breeders – Spotted Salamander – Wood Frog
• Summer breeders – Cope’s Gray Tree Frog– Green Frog
Larvae Metamorphose Prior to June
Larvae Metamorphose Prior to Sept
Larvae Overwinter & Metamorphose following Summer
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Egg Collection and Husbandry• Egg masses: Knox, Blount, and Sullivan Counties
• 250-L wading pools with 70 % shade cloth
• Standardized development: – Anurans: Gosner 30– Caudate: 1 month of age
Haislip et al. (2011): Pathogenicity of Ranavirus Differs among Amphibian Developmental Stages
TWRA Scientific Collection Permit
#1990
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Experimental Design
• Two environmental chambers– Low temperature (10°C = 50°F) – High temperature (25°C = 77°F)
• Two treatments in a RBD (n = 20/trt)– Exposed: 103 PFU/mL of FV3-like isolate– Control: Virus culture media (MEM)
• 2-L containers • 3-d Exposure• 28 days
Acclimated 1 wk
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Animal Monitoring• Condition Checked: 2X/day• Signs of Ranaviral Disease: >24 hrs euthanized
•Diet: 3 days• Tadpoles: TetraMin® fish flakes (12 % body mass)• Larval Salamanders: 1 mL brine shrimp
D. Green, USGS
•Water change: 100% every 3 days• New container 3 d following inoculation• Water was de-chlorinated, aged, and maintained at same
temperature as chambers
IACUC Protocol #2074
Methods follow:Hoverman et al.
(2011)
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Necropsy and qPCR• Euthanized in benzocaine hydrochloride (250 mg/L)• Necropsy
– Liver (1/4) and kidney (1/2 of one)– Tissue Homogenate – Stored at -80 C
•gDNA Extraction and Quantification• Qiagen® Dneasy Blood and Tissue Kit• QubitTM flourometer and Quant-iTTM dsDNA BR Assay Kit
•Quantitative PCR• Applied Biosystems® 7900HT Real-time PCR System• Declared infection if CT < 30• 4 controls:
water, negative animal, positive animal, virus
Methods follow:Hoverman et al.
(2011)
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Wood Frog
1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627280
10
20
30
40
50
60
70
80
90
100
Surv
ival
(%)
Warm Cold
Survival and Infection Prevalence
No Control Mortality
100% Mortality in 7 d
= 84
Subclinical
= 152484
Clinical
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Spotted Salamander
Warm Cold
1 3 5 7 9 11 13 15 17 19 21 23 25 270
102030405060708090
100
Days of exposure
Surv
ival
(%)
Survival and Infection Prevalence
No Control Mortality
45% Mortality
45%
15%
10%
= 6837
Clinical
= 1700
Subclinical
= 10
Subclinical
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Green Frog
Warm Cold
1 3 5 7 9 11 13 15 17 19 21 23 25 270
102030405060708090
100
Days of exposure
Surv
ival
(%)
Survival and Infection Prevalence
15% Control
Mortality in Warm Chamber
40% Mortality
40%30%
5%
= 1871
Clinical= 9
Subclinical
= 103
Clinical
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Cope’s Gray Treefrog
Warm Cold
1 3 5 7 9 11 13 15 17 19 21 23 25 270
102030405060708090
100
Days of exposure
Surv
ival
(%)
Survival and Infection Prevalence
65% Control
Mortality in Cold
Chamber
50% Mortality100% Mortality
in 8 d
50%
15%
85%
= 512
Clinical
= 5
Clinical?
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Reilly et al. (unpubl. data)
25oC Chamber 15oC Chamber
1 3 5 7 9 11 13 15 17 19 2102468101214161820
Tennessee
Minnesota
Days
Num
ber
of
Indiv
iduals
1 3 5 7 9 11 13 15 17 19 2102468101214161820
Tennessee
Minnesota
DaysN
um
ber
of
Indiv
iduals
Median days to mortality:
-Minnesota = 5.5 d-Tennessee = 6 d
Median days to mortality:
-Minnesota =15.5 d-Tennessee =18 d
TN and MN Wood Frogs
10 – 12 dFaster
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Hypothesis Support and Future Directions
• Virus Replication Hypothesis– Mortality Greater in Warm:
• Wood frogs, spotted salamanders, and green frogs
– Infection Greater in Warm:• All species (wood frog: 100% infection in both)
• Morbidity-Infection Threshold (Wood Frogs) – 10 C = 100% infection, no mortality– 15 C = 80 – 90% mortality (Reilly et al.)
•Future Directions: •Retest Cope’s Gray Treefrog•Trend Hold with Other Species
In vitro12 – 32oC
Chinchar (2002)
775X greater in warm
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Acknowledgements
• University of Tennessee AgResearch • UT College of Veterinary Medicine• East Tennessee Research & Education Center
– Dr. Bobby Simpson and Roger Long (JARTU)
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References Cited1. Chinchar, VG. 2002. Ranaviruses (family Iridoviridae): emerging cold-blooded killers. Archives of
Virology. 147: 447-4702. Gray, Matthew ; Miller, Debra. 2013. The Rise of Ranavirus. The Wildlife Professional. 7:51-553. Green, DE; Converse, KA; Schrader, AK. 2002. Epizootiology of sixty-four amphibian morbidity and
mortality events in the USA, 1996-2001. Domestic Animal/Wildlife Interface. 969:323-3394. Hoverman, JT; Gray, MJ; Miller, DL; Haislip, NA. 2011.Widespread Occurrence of Ranavirus in
Pond-Breeding Amphibian Populations. Ecohealth. 9:36-485. Hoverman, JT; Gray, MJ; Haislip, NA; Miller, DL. 2012. Phylogeny, Life History, and Ecology
Contribute to Differences in Amphibian Susceptibility to Ranaviruses. Ecohealth. 8:301-3196. Long, Y; Li, LC ; Li, Q ; He, XZ ; Cui, ZB. 2012. Transcriptomic Characterization of Temperature Stress
Responses in Larval Zebrafish. Plos One. 7.7. Raffel, TR; Rohr, JR; Kiesecker, JM; Hudson, PJ. 2006. Negative effects of changing temperature on
amphibian immunity under field conditions.Functional Ecology. 20:819-8288. Rojas, S; Richards, K; Jancovich, JK; Davidson, EW. 2005. Influence of temperature on Ranavirus
infection in larval salamanders Ambystoma tigrinum . Diseases of Aquatic Organisms. 63:95-1009. Schock, DM; Bollinger, TK; Collins, JP. 2009. Mortality Rates Differ Among Amphibian Populations
Exposed to Three Strains of a Lethal Ranavirus. Ecohealth. 6: 438-44810. Todd-Thompson, M. Seasonality, Variation in Species Prevalence, and Localized Disease for Ranavirus in Cades Cove (Great Smoky Mountains National Park) amphibians. Master Thesis, University of Tennessee, Knoxville, TN, USA, 2010. Available online: http://trace.tennessee.edu/utk_gradthes/665 (accessed on 17 November 2011).