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

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)

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

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)

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

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

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

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

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

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

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)

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)

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

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

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

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?

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

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

Acknowledgements

• University of Tennessee AgResearch • UT College of Veterinary Medicine• East Tennessee Research & Education Center

– Dr. Bobby Simpson and Roger Long (JARTU)

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).

Questions??

Brand: mbrand1@utk.eduGray: mgray11@utk.eduMiller: dmille42@utk.edu

Photo: M. Niemiller