2013 International Symposium on Ranaviruses by Mabre Brand

1. Water Temperature Affects
Susceptibility to Ranavirus
Mabre Brand1,2, Matthew Gray1, Becky Wilkes2,
Roberto Brenes1 and Debra Miller1,2
1Center for Wildlife Health
2College of Veterinary Medicine
University of Tennessee-Knoxville

2. 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 Temperature
12 – 32 C (in vitro) Chinchar (2002)

3. Ranavirus Landscape Prevalence
Tennessee 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

4. 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
FV3Prevalence
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)

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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)

12. 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)

13. Wood Frog
0
10
20
30
40
50
60
70
80
90
100
1 3 5 7 9 11 13 15 17 19 21 23 25 27
Survival(%)
Warm
Cold
Survival and Infection Prevalence
No Control
Mortality
100% Mortality in 7 d
= 84
Subclinical
= 152484
Clinical

14. Spotted Salamander
Warm
Cold
0
10
20
30
40
50
60
70
80
90
100
1 3 5 7 9 11 13 15 17 19 21 23 25 27
Survival(%)
Days of exposure
Survival and Infection Prevalence
No Control
Mortality
45% Mortality
45%
15%
10%
= 6837
Clinical
= 1700
Subclinical
= 10
Subclinical

15. Green Frog
Warm
Cold
0
10
20
30
40
50
60
70
80
90
100
1 3 5 7 9 11 13 15 17 19 21 23 25 27
Survival(%)
Days of exposure
Survival and Infection Prevalence
15%
Control
Mortality
in Warm
Chamber
40% Mortality
40%
30%
5%
= 1871
Clinical
= 9
Subclinical
= 103
Clinical

16. Cope’s Gray Treefrog
Warm
Cold
0
10
20
30
40
50
60
70
80
90
100
1 3 5 7 9 11 13 15 17 19 21 23 25 27
Survival(%)
Days of exposure
Survival and Infection Prevalence
65%
Control
Mortality
in Cold
Chamber
50% Mortality100%
Mortality
in 8 d
50%
15%
85%
= 512
Clinical
= 5
Clinical?

17. Reilly et al. (unpubl. data)
25oC Chamber 15oC Chamber
0
2
4
6
8
10
12
14
16
18
20
1 3 5 7 9 11 13 15 17 19 21
NumberofIndividuals
Days
Tennessee
Minnesota
0
2
4
6
8
10
12
14
16
18
20
1 3 5 7 9 11 13 15 17 19 21
NumberofIndividuals
Days
Tennessee
Minnesota
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 d
Faster

18. 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 vitro
12 – 32oC
Chinchar (2002)
775X greater in warm

19. Acknowledgements
• University of Tennessee AgResearch
• UT College of Veterinary Medicine
• East Tennessee Research & Education Center
– Dr. Bobby Simpson and Roger Long (JARTU)

20. References Cited
1. Chinchar, VG. 2002. Ranaviruses (family Iridoviridae): emerging cold-blooded killers. Archives of
Virology. 147: 447-470
2. Gray, Matthew ; Miller, Debra. 2013. The Rise of Ranavirus. The Wildlife Professional. 7:51-55
3. 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. 969:323-339
4. Hoverman, JT; Gray, MJ; Miller, DL; Haislip, NA. 2011.Widespread Occurrence of Ranavirus in
Pond-Breeding Amphibian Populations. Ecohealth. 9:36-48
5. Hoverman, JT; Gray, MJ; Haislip, NA; Miller, DL. 2012. Phylogeny, Life History, and Ecology
Contribute to Differences in Amphibian Susceptibility to Ranaviruses. Ecohealth. 8:301-319
6. 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. 20:819-828
8. Rojas, S; Richards, K; Jancovich, JK; Davidson, EW. 2005. Influence of temperature on Ranavirus
infection in larval salamanders Ambystoma tigrinum . Diseases of Aquatic Organisms. 63:95-100
9. Schock, DM; Bollinger, TK; Collins, JP. 2009. Mortality Rates Differ Among Amphibian Populations
Exposed to Three Strains of a Lethal Ranavirus. Ecohealth. 6: 438-448
10. 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).

21. Questions??
Brand: mbrand1@utk.edu
Gray: mgray11@utk.edu
Miller: dmille42@utk.edu
Photo: M. Niemiller