AN OVERVIEW OF RANAVIRUS DIAGNOSTICS, TREATMENT AND MANAGEMENT
Second International RanavirusSymposiumJuly 27-29, 2013
Allan PessierAmphibian Disease LaboratorySan Diego Zoo [email protected]
Introduction Diagnostics
What’s available Pitfalls Interpretation Selecting the right tests
Treatment and Management Wild Populations Captive Populations Endangered Species and
Reintroduction Programs
Brad Wilson
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
Pitfalls in Diagnostic SamplingWhat affects your diagnostic sensitivity and specificity?
Collection of biologically relevant samples Subclinical infections
Variation in how samples are collected
Sample storage PCR Inhibitors Contamination
Diagnostic Methods
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
Serology
Indirect ELISA Marine Toads Gopher Tortoises 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
Histopathology Formalin-fixed
tissues Evaluate tissues for
lesions consistent with ranaviral disease
Rule out other disease processes
Immunohistochemistry
Pathologists are fun
Histopathology Insensitive for
subclinical infections Multicentric necrosis
(liver, spleen, kidney, skin, hematopoietic tissue)
Intracytoplasmic basophilic inclusion bodies
Transmission Electron Microscopy Demonstration of
characteristic icosohedral virions
Cell culture or tissues
Definitive only for an iridovirus
Good for historical cases
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
PCR Caveats
Infection vs. Disease
Viable vs. inactivated virions
Encourages tunnel vision in outbreak investigation
Sample selection (especially naturally infected animals)
Contamination
Conventional PCR Products visualized
on agarose gels DNA sequencing to
confirm positives Phylogeny of
positive samples* Less sensitive than
Real-Time InhibitorsApril Johnson
“The” Ranavirus andThe Problem with Frog Virus 3 • MCP gene is
highly conserved• Different
viruses will have similar or identical sequences• FV3-like viruses
Implications for Research and Animal Management Detection of
unexpected viruses Need to be able to
easily differentiate viruses for Prevalence/
epidemiologic studies
Reintroduction Programs
Real-Time/Taqman PCR
Great for routine diagnostics High analytical
sensitivity No need to seq
positives (Taqman)
Viral Loads MCP-based Limits on
phylogeny
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
Mortality Events
Avoid assumptions as many conditions look alike
Subclininical infections
Co-infections Necropsy and
histopathology Ancillary testing April Johnson
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
Disease Surveillance
Limitations of PCR for detection of subclinical infections PCR Well-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
Control of Ranavirus Infections
Regulatory and Pathogen Pollution Adequate tests Strain
differentiation
Vaccination Small populations Reintroduction programs
Treatment of individuals
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
Treatment of Ranavirus Infections
Guanine analogue antivirals (acyclovir, valacyclovir)
Temperature elevations
Questions Persistent
infections?
