Article

Non-invasive sampling methods for the detection of Batrachochytrium dendrobatidis in archived amphibians.

Institute of Zoology, Zoological Society of London, Regent's Park, London NW1 4RY, UK.
Diseases of Aquatic Organisms (Impact Factor: 1.59). 05/2009; 84(2):163-6. DOI: 10.3354/dao02029
Source: PubMed

ABSTRACT Chytridiomycosis, an emerging infectious disease of amphibians caused by the chytrid fungus 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 techniques to detect Bd infection, with the pathogen being detected in 75 to 81% of the 16 ethanol-fixed 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 any of 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 steps for the detection of Bd in formalin preserved specimens are warranted.

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    ABSTRACT: Aim  Amphibian chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), is associated with global amphibian population declines and species extinctions. Current evidence indicates that the pathogen has recently spread globally from an enzootic focus, with Xenopus spp. (family Pipidae) in South Africa having been identified as a likely source. The aim of this study was to investigate further the likelihood of African Xenopus spp. as the original source of Bd.Location  We examined 665 museum specimens of 20 species of African and South American pipid frogs collected between 1844 and 1994 and held in the collection of the Natural History Museum, London.Methods  Skin brushings taken from adult amphibians and brushings from the mouthparts, lips and developing hind limbs of larval pipid frogs were examined for the presence of Bd using real-time PCR.Results  We found six cases of Bd infection in three Xenopus spp. (from Africa), but none of the South American pipids was positive, although only 45 South American frogs were available for examination. The earliest case of Bd infection was in a specimen of Xenopus fraseri collected from Cameroon in 1933. A consistently low prevalence of infection over time indicates that a historical equilibrium existed between Xenopus spp. and Bd infection in Africa.Main conclusions  Our results suggest that Bd infection was present in Xenopus spp. across sub-Saharan Africa by the 1930s, providing additional support for the ‘out of Africa’ hypothesis. If this hypothesis is correct, it strengthens the argument for stringent control of human-assisted movements of amphibians and other wildlife world-wide to minimize the likelihood of pathogen introduction and disease emergence that can threaten species globally. Our findings help inform species selection for conservation in the face of the current Bd pandemic and also guide future research directions for selecting Bd isolates for sequencing and virulence testing.
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