An overview of ranavirus diagnostics, treatment and management

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2013 International Symposium on Ranavirus
by Allan Pessier

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An overview of ranavirus diagnostics, treatment and management

  1. 1. AN OVERVIEW OF RANAVIRUS DIAGNOSTICS, TREATMENT AND MANAGEMENT Second International Ranavirus Symposium July 27-29, 2013 Allan Pessier Amphibian Disease Laboratory San Diego Zoo Global apessier@sandiegozoo.org
  2. 2. Introduction  Diagnostics  What’s available  Pitfalls  Interpretation  Selecting the right tests  Treatment and Management  Wild Populations  Captive Populations  Endangered Species and Reintroduction Programs BradWilson
  3. 3. Sensitivity and Specificity of Diagnostic Tests  Gold Standard: The standard accepted diagnostic test for a condition to which all other diagnostic tests are compared  Analytical Sensitivity: Lowest concentration of pathogen DNA that can be detected by the test (PCR).  Diagnostic Sensitivity: Proportion of true positives detected.  Diagnostic Specificity: Proportions of true negatives detected
  4. 4. Pitfalls in Diagnostic Sampling What affectsyour diagnostic sensitivity and specificity?  Collection of biologically relevant samples  Subclinical infections  Variation in how samples are collected  Sample storage  PCR Inhibitors  Contamination
  5. 5. Diagnostic Methods
  6. 6. Virus Isolation  Usually tissue samples  Fish and amphibian cell lines  CPE and ID by ELISA, IPX or PCR  Greater cost, not widely available  Needed for purified virus, transmission experiments, best quality DNA OIE Gold Standard
  7. 7. Serology  Indirect ELISA  MarineToads  GopherTortoises  May be useful for detection of prior exposure in populations (? Adjunct to PCR surveillance)  Does not provide information on current infection status or RV strain type
  8. 8. Histopathology  Formalin-fixed tissues  Evaluate tissues for lesions consistent with ranaviral disease  Rule out other disease processes  Immunohistochemistry  Pathologists are fun
  9. 9. Histopathology  Insensitive for subclinical infections  Multicentric necrosis (liver, spleen, kidney, skin, hematopoietic tissue)  Intracytoplasmic basophilic inclusion bodies
  10. 10. Transmission Electron Microscopy  Demonstration of characteristic icosohedral virions  Cell culture or tissues  Definitive only for an iridovirus  Good for historical cases
  11. 11. Polymerase Chain Reaction (PCR)  Accessible and fast  Conventional and Real-Time  Major capsid protein (MCP) is most common  Samples include tissues, blood, oral and cloacal swabs, and FFPE
  12. 12. PCR Caveats  Infection vs. Disease  Viable vs. inactivated virions  Encourages tunnel vision in outbreak investigation  Sample selection (especially naturally infected animals)  Contamination
  13. 13. Conventional PCR  Products visualized on agarose gels  DNA sequencing to confirm positives  Phylogeny of positive samples*  Less sensitive than Real-Time  Inhibitors April Johnson
  14. 14. “The” Ranavirus and The Problem with Frog Virus 3 • MCP gene is highly conserved • Different viruses will have similar or identical sequences • FV3-like viruses
  15. 15. Implications for Research and Animal Management  Detection of unexpected viruses  Need to be able to easily differentiate viruses for  Prevalence/epidemio logic studies  Reintroduction Programs
  16. 16. Real-Time/Taqman PCR  Great for routine diagnostics  High analytical sensitivity  No need to seq positives (Taqman)  Viral Loads  MCP-based  Limits on phylogeny
  17. 17. Am I doing the right test(s)?  Investigation of mortality events  Did animals die of ranaviral disease  Co-infections  Positive PCR doesn’t equal disease  Population Surveillance and Management  High sensitivity  Adequate samples (type and number)  Ability to differentiate strains  Standardization
  18. 18. Mortality Events  Avoid assumptions as many conditions look alike  Subclininical infections  Co-infections  Necropsy and histopathology  Ancillary testing April Johnson
  19. 19. When there is no pathologist…..  Save tissues for multiple types of diagnostic investigation  Fixed tissues or carcasses for histopathology (not frozen)  Frozen tissues or carcasses for other diagnostics
  20. 20. Disease Surveillance  Limitations of PCR for detection of subclinical infections  PCRWell-validated for sick animals  What is the appropriate sample for subclinical infection  ? Macrophages/leukocytes; kidney  Adequate sample numbers  Consideration of diagnostic sensitivity  Differentiation of strains  Implications for epidemiology and risk assessments  Implications for regulatory requirements  Interpretation of prevalence data
  21. 21. Control of Ranavirus Infections  Regulatory and Pathogen Pollution  Adequate tests  Strain differentiation  Vaccination  Small populations  Reintroduction programs  Treatment of individuals
  22. 22. Reintroduction/Translocation Programs  Positive PCR tests on routine surveillance  Paralysis even when animals held in isolation  Need for easy strain differentiation  ? Role of treatment protocols for valuable animals/populations
  23. 23. Treatment of Ranavirus Infections  Guanine analogue antivirals (acyclovir, valacyclovir)  Temperature elevations  Questions  Persistent infections?

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