Water temperatures affects susceptibility to ranavirus
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2013 International Symposium on Ranaviruses

2013 International Symposium on Ranaviruses
by Mabre Brand

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Water temperatures affects susceptibility to ranavirus Presentation Transcript

  • 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