AN OVERVIEW OF RANAVIRUS DIAGNOSTICS, TREATMENT AND MANAGEMENT

Second International RanavirusSymposiumJuly 27-29, 2013

Allan PessierAmphibian Disease LaboratorySan Diego Zoo Globalapessier@sandiegozoo.org

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?