Cryo-archiving of Batrachochytrium dendrobatidis and other chytridiomycetes

Abstract

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...

Batrachochytrium dendrobatidis: requirement for further isolate collection and archiving

“…Many of these challenges have been successfully addressed (e.g. Berger et al 1998, Boyle et al 2003, Hyatt et al 2007, James et al 2009, Voyles et al 2009a), but others are still being investigated. To facilitate projects that will meet these challenges, we recommend that additional Bd isolates be collected and cryo-archived.…”

“…Priority targets for Bd collection and cryo-preservation have been suggested (Voyles et al 2009b), and recording detailed information on any additional isolates will be important for basic disease research. Although protocols for isolating (Longcore et al 1999) and cryo-archiving (Boyle et al 2003) Bd are readily available, some of the required skills are not standard for medical microbiology labs. Obtaining isolates can be difficult, requiring some technical skills and persistence.…”

“…The methods for isolating and purifying Bd were first established in 1999 (Longcore et al 1999). Some Bd isolates have been cryo-archived for future research (Boyle et al 2003), and many are actively passaged under different nutrient and temperature conditions. Here we review available information on known Bd isolates.…”

Experimental infection of self-cured Leiopelma archeyi with the amphibian chytrid Batrachochytrium dendrobatidis

“…As a New Zealand isolate of Bd was not available, a culture of type isolate JEL 197 (Longcore et al 1999) was sourced from a cryo-archived culture (Boyle et al 2003) held at the Department of Biochemistry, University of Otago, by R. Poulter and M. Butler; it had been passaged on 1% tryptone plates at 23°C for about 1 yr prior to this experiment. Zoospores were collected from 4 d old cultures on 1% tryptone agar plates held at 23°C by flooding with 6 ml of dilute salt solution (DSS, imitating pond water; Boyle et al 2004).…”

BASELINE CUTANEOUS BACTERIA OF FREE-LIVING NEW ZEALAND NATIVE FROGS (LEIOPELMA ARCHEYIANDLEIOPELMA HOCHSTETTERI) AND IMPLICATIONS FOR THEIR ROLE IN DEFENSE AGAINST THE AMPHIBIAN CHYTRID (BATRACHOCHYTRIUM DENDROBATIDIS)

“…The bacterial isolates have been cryopreserved at 270 C at the Auckland Zoo and the Bd isolate has been cryopreserved at 270 C at Landcare Research (Boyle et al 2003).…”