Chytrid diagnosis from archived amphibians

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Vol. 84: 163–166, 2009
doi: 10.3354/dao02029

DISEASES OF AQUATIC ORGANISMS Dis Aquat Org

Published April 6

NOTE

Non-invasive sampling methods for the detection of Batrachochytrium dendrobatidis in archived amphibians
C. Soto-Azat1, 2, B. T. Clarke3, M. C. Fisher4, S. F. Walker1, 4, A. A. Cunningham1,*
1 Institute of Zoology, Zoological Society of London, Regent’s Park, London NW14RY, UK Escuela de Medicina Veterinaria, Universidad Andres Bello, Republica 252, Santiago, Chile 3 Department of Zoology, Natural History Museum, Cromwell Rd, London SW7 5BD, UK 4 Department of Infectious Disease Epidemiology, Imperial College, St Mary’s Campus, Norfolk Place, London W2 1PG, UK 2

ABSTRACT: Chytridiomycosis, an emerging infectious disease of amphibians caused by the chytridfungus Batrachochytrium dendrobatidis (Bd), is associated with amphibian population declines worldwide. Investigation of the origin and spread of the pathogen requires examination of archived museum specimens of amphibians. Examination for Bd infection is usually done using histological techniques, which are often too destructive for valuable museum material. Three alternative methods for Bd detection(skin swabbing, brushing and scraping) were evaluated for ability to yield Bd DNA and destructiveness to specimens. Archived amphibians known to be Bd positive and which had been preserved in either formalin or ethanol for many years were used. Samples were analysed using a Bd-specific quantitative real-time Taqman PCR (qPCR) assay. There was no difference in the ability of each of the techniquesto detect Bd infection, with the pathogen being detected in 75 to 81% of the 16 ethanolfixed frogs examined. Visible evidence of sampling was left by scraping, but not by swabbing or brushing. The brush-qPCR technique detected higher counts of genomic equivalents than the other 2 sampling methods, although differences were not statistically significant. The qPCR assay did not detect Bd from anyof the 6 formalin-fixed frogs examined, regardless of the sampling method. Nondestructive sampling techniques enable qPCR analysis of ethanol-preserved museum specimens for Bd. Recently, the incorporation of DNA cleanup steps allowed the detection of Bd in destructively sampled tissues from formalin preserved specimens. Further studies using nondestructive sampling incorporating DNA cleanup stepsfor the detection of Bd in formalin preserved specimens are warranted. KEY WORDS: Batrachochytrium dendrobatidis · Amphibian chytridiomycosis · Amphibian declines · Museum specimens · Emerging infectious disease
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INTRODUCTION Chytridiomycosis is a recently described emerging disease of amphibians caused by thenonhyphal zoosporic chytrid fungus Batrachochytrium dendrobatidis (Bd) (Berger et al. 1998, Longcore et al. 1999). It has been implicated in epizootic amphibian mortality with resultant population declines on a global scale (Skerratt et al. 2007) and amphibian extinctions in Australia and Central and South America (Ron et al. 2003, La Marca et al. 2005, Alan Pounds et al. 2006, Schloegel et al. 2006).This highly pathogenic, virulent
*Corresponding author. Email: a.cunningham@ioz.ac.uk

and readily transmissible emerging disease has no precedent in historical times (Skerratt et al. 2007). It has been described as the worst infectious disease ever recorded among vertebrates in terms of the number of species impacted, and has a propensity to drive species to extinction (Gascon et al. 2007). Arange of diagnostic assays exists for the detection of Bd in live larval, post-metamorphic and adult amphibians (Hyatt et al. 2007). These include histopathology (Berger et al. 1998), immunohistochemistry (Berger et al. 2002, Van Ells et al. 2003, Olsen et al. 2004), electron microscopy (Berger et al. 2002), conventional poly© Inter-Research 2009 · www.int-res.com

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