An overview of ranavirus diagnostics, treatment and management
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
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
13. Transmission Electron Microscopy
Demonstration of
characteristic
icosohedral virions
Cell culture or
tissues
Definitive only for
an iridovirus
Good for historical
cases
14.
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
21.
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?