Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide, associated with declines in amphibian populations. Diagnosis of chytridiomycosis to date has largely relied upon histological and immunohistochemical examination of toe clips. This technique is invasive and insensitive particularly at early stages of infection when treatment may be possible. We have developed a real-time PCR Taqman assay that can accurately detect and quantify one zoospore in a diagnostic sample. This assay will assist the early detection of B. dendrobatidis in both captive and wild populations, with a high degree of sensitivity and specificity, thus facilitating treatment and protection of endangered populations, monitoring of pristine environments and preventing further global spread via amphibian trade. KEY WORDS: Chytrids · Batrachochytrium dendrobatidis · Amphibian declines · Real-time PCR Taqman assay · Chytridiomycosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 60: [141][142][143][144][145][146][147][148] 2004 examination via haematoxylin and eosin staining of toe clips or skin scrapings (Daszak et al. 1999, see also www.jcu.edu.au/school/phtm/phtm/frogs/histo/ chhisto.htm). Immunoperoxidase staining with polyclonal antibodies to B. dendrobatidis increases the specificity and sensitivity of detection (Berger et al. 2002). More recently, a staining protocol has been described which enhances diagnosis by the colocalisation of B. dendrobatidis and keratin in the skin of amphibians (V. Olsen, D. Boyle, D. Mendez, A. D. Hyatt unpubl.). However, the drawbacks of histological testing include the degree of expertise needed for identification, the invasiveness of sampling technique -typically performed by toe-clipping of live animals -the length of time required, the low sensitivity of the test, and the variability of infection levels amongst toes. In the field, examination of oral disc abnormalities in tadpoles with a 10 × hand lens has been recommended as a preliminary indication of chytridiomycosis (Fellers et al. 2001), although these abnormalities can also be caused by DDT intoxication. The fungus can be identified by isolation and culture , but this requires a high degree of expertise and time.Early detection of Batrachochytrium dendrobatidis is vital to the control of spread of the disease via global amphibian trade. B. dendrobatidis has been identified in animals imported for zoo collections , in international pet trade (Mutschmann et al. 2000), in the food trade (Mazzoni et al. 2003) and in laboratory animal trade (Parker et al. 2002). Early detection of infection would allow possible curative treatment to be undertaken, at least in captive populations where formalin/malachite green solution has been shown to be effective on adult Xenopus tropicalis (Parker et al. 2002). Itraconazole baths (0.01%) were found to be effective in treating the terrestrial species Dendrobates tinctorius (Nichols et al. 2001), although whether this treatment is effective and safe...

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide. It has been associated with catastrophic declines of frog populations including those in pristine habitats in Queensland, Australia. To facilitate genetic and disease studies of this fungus and related species, it is essential to have a reliable long-term storage method to maintain genetic integrity of isolates. We have adapted well-established techniques used for the long-term storage of tissue-culture cell lines to the preservation of B. dendrobatidis and other chytridiomycetes. This simple method has allowed us to recover these fungi from storage at -80°C and in liquid nitrogen over an extended period. With this technique it is now possible to preserve saprobic and parasitic isolates from a variety of environmental and disease situations for comparative genetic and biological studies. KEY WORDS: Chytrids · Batrachochytrium dendrobatidis · Storage · Cryopreservation · Amphibian declines Resale or republication not permitted without written consent of the publisherDis Aquat Org 56: [59][60][61][62][63][64] 2003 to the origin and spread/transmission of disease and specific cause of death must be addressed. To address these questions it is essential that the many isolates be effectively frozen and archived until required. The development of such a procedure would be invaluable for future research as questions relating to attenuation (via passage), contamination and alterations in the genome (e.g. site mutations) can be minimized. B. dendrobatidis can be isolated from infected frogs using TGhL agar and then maintained on agar or in TGhL broth at 23°C (Longcore et al. 1999). Cultures also grow well at 15°C and survive many months at 4°C. These procedures are, however, labour intensive since cultures require regular examination for contamination, growth failure and passage every 14 to 21 d on nutrient agar plates at room temperature or every 4 to 5 mo when grown in liquid medium and stored at 4 to 6°C.Previous methods for long-term storage of Chytridiomycetes at low temperatures have produced survival rates of only 16% (Smith 1982) while Hohl & Iselin (1986) had limited success with storage in liquid nitrogen. We have developed a simple method of freezing, based on established methods for freezing tissue-culture cell lines that have had 100% success in retrieval of Batrachochytrium dendrobatidis from storage over 12 mo. The method was also successfully applied to other fungi from the phylum Chytridiomycota. MATERIALS AND METHODSCulture and harvest of Batrachochytrium dendrobatidis. TGhL (13 ml) broth (16 g tryptone, 4 g gelatin hydrolysate, 2 g lactose, 1000 ml distilled water) in a 25 cm 2 disposable tissue-culture flask was inoculated with 2 ml of an actively growing 7 d old broth culture of B. dendrobatidis (Isolate A98 1810/3) and incubated at 17 to 23°C for 3 to 21 d (Longcore et al. 1999). All cultures contained active released zoospores and sporangia (empty or containing zoospores), both in the media and attached to the plasti...