2013 International Symposium on Ranavirus by Allan Pessier

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. 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. 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. 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. Diagnostic Methods

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. 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. Histopathology
 Formalin-fixed tissues
 Evaluate tissues for
lesions consistent with
ranaviral disease
 Rule out other disease
processes
 Immunohistochemistry
 Pathologists are fun

9. Histopathology
 Insensitive for
subclinical infections
 Multicentric necrosis
(liver, spleen, kidney,
skin, hematopoietic
tissue)
 Intracytoplasmic
basophilic inclusion
bodies

13. Transmission Electron Microscopy
 Demonstration of
characteristic
icosohedral virions
 Cell culture or
tissues
 Definitive only for
an iridovirus
 Good for historical
cases

15. 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

16. PCR Caveats
 Infection vs. Disease
 Viable vs. inactivated
virions
 Encourages tunnel
vision in outbreak
investigation
 Sample selection
(especially naturally
infected animals)
 Contamination

17. Conventional PCR
 Products visualized
on agarose gels
 DNA sequencing to
confirm positives
 Phylogeny of
positive samples*
 Less sensitive than
Real-Time
 Inhibitors
April Johnson

18. “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

19. 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

20. Real-Time/Taqman PCR
 Great for routine
diagnostics
 High analytical
sensitivity
 No need to seq
positives (Taqman)
 Viral Loads
 MCP-based
 Limits on phylogeny

22. 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

23. Mortality Events
 Avoid assumptions as
many conditions look
alike
 Subclininical infections
 Co-infections
 Necropsy and
histopathology
 Ancillary testing
April Johnson

24. 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

25. 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

26. Control of Ranavirus Infections
 Regulatory and
Pathogen Pollution
 Adequate tests
 Strain differentiation
 Vaccination
 Small populations
 Reintroduction programs
 Treatment of
individuals

27. 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

28. Treatment of Ranavirus Infections
 Guanine analogue
antivirals
(acyclovir, valacyclovir)
 Temperature
elevations
 Questions
 Persistent
infections?