Techniques for detecting chytridiomycosis in wild frogs: comparing histology with real-time Taqman PCR

Abstract

Chytridiomycosis is a lethal disease of amphibians associated with mass mortalities and population declines worldwide. An accurate, non-invasive technique for detecting chytridiomycosis is urgently needed to determine the current geographical distribution of the disease, and its prevalence in wild amphibian populations. Herein we evaluate a recently devised, rapid, non-invasive, swab-PCR assay. We sampled 101 wild juvenile Mixophyes iteratus by both a skin swab for use in PCR analysis, and a toe-clip for examination by histological methods. The swab-PCR assay detected chytridiomycosis infection in a minimum of 14.9% of frogs, whereas histology detected infection in no more than 6.9% of frogs. We conclude that the swab-PCR technique is the more reliable means of detecting chytridiomycosis in wild amphibians, and that it precludes the need for toe-clipping as a means of sampling for the presence of the disease in future surveys. Further, we document a significant negative relationship between a juvenile frog's snout-vent length and its likelihood of being infected with the disease.KEY WORDS: Batrachochytrium dendrobatidis · Amphibian declines · Chytridiomycosis · Diagnosis · Real-time Taqman PCR assay · Chytrid Resale or republication not permitted without written consent of the publisherDis Aquat Org 71: [141][142][143][144][145][146][147][148] 2006 gers et al. 1993), and the determination of differential effects the pathogen may have on various amphibian populations. However, disease surveys to date have been severely limited by the shortcomings of available diagnostic techniques, which until very recently have been: (1) insensitive, yielding many false-negatives (Berger et al. 2002; (2) non-specific, leaving open the possibility for false-positives and observer bias; (3) invasive, requiring a skin sample such as webbing or toes (Berger et al. 2002, Lips et al. 2003; and (4) timeconsuming. While it has been suggested that chytridiomycosis in severely infected postmetamorphic individuals can be easily diagnosed by the presence of abnormal epidermal sloughing, reddening of the ventral surfaces, and behavioral changes such as lethargy and loss of righting reflex (Berger et al. 1999a), animals at these later stages of infection are rarely found in the wild, due both to the short time span over which they are likely to survive, and the high rate at which they are scavenged (Green et al. 2002). Testing of wild amphibians is therefore dependent upon laboratory analysis of samples taken from apparently healthy individuals that do not exhibit clinical signs of disease.Diagnosis of chytridiomycosis to date has relied largely on histological examination of skin tissue stained with haemotoxylin & eosin (H&E) (Berger et al. 1998, 1999b, Aplin & Kirkpatrick 2000, Waldman 2001, Bonaccorso et al. 2003, Hopkins & Channing 2003, McDonald et al. 2005. Sensitivity and specificity of this technique has been improved slightly by the use of polyclonal antibodies and immunoperoxidase (IPX) staining (Berger et al. 2002), b...

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