Lee Berger
CSIRO Australian Animal Health Laboratory
Geelong, Victoria, 3220
(lee.berger@dah.csiro.au)

Rick Speare
School of Public Health and Tropical Medicine
James Cook University
Townsville, Queensland, 4811
(richard.speare@jcu.edu.a u)

Chytrid fungi and amphibian declines

Many amphibian population declines are obviously due to habitat degradation, but in the last twenty years there have been mysterious population crashes in protected high altitude areas where no habitat problems have been detected. This pattern is particularly evident in the montane rainforests of Queensland and Central and South America (Laurance et al., 1996, 1997; Lips, 1998). In some cases, these crashes have resulted in extinction of stream dwelling rainforest species. A new species of chytrid fungus has been found infecting the skin of frogs dying during mass mortality events in forests in Queensland and Panama, and may be the cause of these precipitous population declines (Berger et al., 1998). Chytrids are small spherical fungi that produce motile infective stages called zoospores. Some species are commonly found free living in soil and water where they degrade organic matter such as chitin or keratin, and others are parasites of algae, plants, nematodes or insects (Barr, 1990). Before the discovery of the amphibian chytrid, none had been found to cause disease in vertebrates. The epidemiological data supports the hypothesis that this fungus has been introduced to these rainforest areas and is the cause of the population crashes.

In Queensland, seven rainforest frog species disappeared during the past twenty years (Richards et al., 1993; Mahony, 1996). The first extinctions occurred in the D'Aguilar and Conondale mountains near Brisbane in the late seventies/early eighties. The amazing southern gastric brooding frog was last seen in 1979. This was an incredible species whose tadpoles developed in the stomach of the female and the newly metamorphosed froglets emerged through the mouth. In the mid eighties, frog populations in central eastern Queensland declined, and the northern gastric breeding frog (the only other gastric brooding frog in the world) has not been seen since. By then it was clear that our frogs were in trouble, and so remaining high altitude populations were intensively monitored. In the early nineties, populations in north Queensland suffered similar sudden declines, but this time zoologists were present to witness ill, dying and dead frogs as mass mortalities occurred (Laurance et al., 1996). Interestingly, in many of these episodes of declines, tadpoles were seen for months after adult frogs had disappeared.

A range of causes have been proposed to explain the declines, but introduction of a waterborne infectious disease fatal to adult frogs appears the most reasonable explanation. Abnormal levels of water pollutants were not detected, water pH was stable and population changes were not associated with habitat disturbance or unusual weather (Richards et al.,1993; Laurance et al., 1996). Increased UV radiation can be discounted as ground level solar UV radiation has not increased significantly at tropical latitudes, and most of these frogs are nocturnal and live in dense rainforest. The asynchronous timing of the declines and apparent spread of the declines from south to north is consistent with a new epidemic agent progressing through a naïve population. All species of frogs that suffered significant declines are stream-breeding and stream-dwelling, suggesting the problem is waterborne (Williams and Hero, 1998).

During the mass mortality in north Queensland in 1993, about twenty dying frogs were collected for diagnostic investigations (Berger et al., 1998). The species found dying included the sharp-snouted day frog, waterfall frog and common mist frog. Pathology revealed the presence of chytrid fungi in the keratinised layer of the skin, and acute, non specific degenerate lesions in some internal organs. Bacteriological and virological studies did not identify any causative agent. The chytrid fungi appeared to be associated with minor local changes in the skin, and the reasons why frogs died was not apparent. This fungus had never been seen before, and as there was no background information available on diseases in healthy populations of these frogs, it was difficult to determine the significance of its occurrence.

Between 1995 and 1998, a network was set up around Australia to collect any sick wild or captive frogs found by zoologists, and to investigate their diseases pathologically. A range of new diseases were detected, but most importantly, the chytrid fungus was found to be widespread and associated with mass mortality in a range of species where no alternative causes of the deaths were detected (Berger et al., 1998). Infections were found in frogs from a wide range of habitats in both unpopulated and populated regions, including cities such as Brisbane and Adelaide.

In two instances where tadpoles were being raised in captivity, almost all frogs died with chytridiomycosis in the weeks after metamorphosis. When healthy tadpoles were euthanased and examined, the fungus was only found in the mouthparts, which is the only keratinised area on tadpoles. As the fungus appears to be keratinophilic, this could explain why tadpoles survive while adults die. We suspect that the chytrid spreads from the mouthparts to the skin on the body when it becomes keratinised after metamorphosis.

As the chytrid fungus is difficult to culture, a preliminary experiment was conducted using skin scrapings collected from a dead great barred frog and added to the water of six captive bred frogs (Berger et al., 1998). Four controls were given filtered skin scrapings with the fungus removed, and four controls were untreated. Between 10 and 18 days later, all six frogs given skin scrapings become lethargic and were shown to be infected with the fungus. One died and the rest were euthanased with MS 222. The eight controls remained uninfected and healthy.

In 1997, a mass mortality event in rainforest frogs and toads in Panama was detected (Lips, unpub) and an apparently identical chytrid fungus was present in the skin (Berger et al., 1998). As the pattern of declines in Central America and Queensland was remarkably similar, it seems the chytrid may be one of the most significant causes of global amphibian population declines, particularly in isolated frog populations in pristine environments.

Many questions remain to be answered, and investigations into the origins, distribution, and spread of the amphibian chytrid are ongoing.

Diagnosis and management in captive amphibians

Chytridiomycosis can cause high mortality in captive amphibian collections. A group at the Washington zoo working independently of our group also identified the amphibian chytrid as the cause of death in several species of frogs (Pessier et al, 1999).

Clinical signs of chytridiomycosis vary between species, and include lethargy, reddening of ventral skin, convulsions with extension of hindlimbs, accumulations of sloughed skin over the body and occasional ulcers. Death usually occurs a few days after the onset of signs of disease. Suspect chytridiomycosis if frogs develop any of these signs. A high mortality rate in two to three week old metamorphs is also suggestive of this disease. None of these signs are pathognomonic for chytridiomycosis and diagnostic tests are required to confirm an outbreak.

Examination of unstained pieces of the excess skin slough from fresh, fixed or frozen specimens, under a light microscope, is a quick diagnostic method. The round or oval fungal bodies (8-20 µ m) have a distinct refractile wall, and may be empty or contain zoospores (Fig 1). Occasional empty sporangia are divided into two, four or more internal compartments by thin membranes.

Figure 1: Unstained skin slough from a green tree frog examined by light microscopy. Note refractile, round and oval fungi. Most are empty, but one contains developing zoospore (arrow). E = uninfected epidermal cells

Figure 2: Histological section of skin from a heavily infected green tree frog. The fungus does not invade through the epidermis, but occurs in the superficial keratinised layer which becomes thickened in severe infections. Solid, immature sporangia are present as well as mature sporangia containing zoospores (Z). Zoospores swim out through a discharge tube (T), and the empty sporangia remain. The amphibian chytrid does not grow hyphae. E = epidermis, D = dermis

No drugs have yet been tested for treatment, but antifungals currently used for amphibians or fish may prove useful in treating chytridiomycosis. Benzalkonium chloride is a disinfectant that has been used at 2 mg/l to successfully treat a similar superficial mycotic dermatitis in amphibians caused by Basidiobolus ranarum (Groff et al., 1991). The regime used experimentally was 30 minutes of bath treatment, on three alternate days. This was repeated in 8 days (i.e. 6 treatments in total). Oral itraconazole has also been used to treat Basidiobolus ranarum infections (Taylor, 1999). Other possible antifungal treatments include oral Ketoconazole (10mg/kg once a day) or copper sulphate baths (500mg/l dip 2 minutes daily to effect) (Raphael, 1993).

Routine quarantine procedures have been adequate in restricting outbreaks to certain tanks, and no airborne transmission has been observed (G. Marantelli, unpublished). Every group of frogs should be kept completely separate to ensure no water borne transmission of disease can occur. By changing and discarding gloves between every tank, avoiding splashing water between tanks, and disinfection of tanks and implements using 2% hypochlorite before reusing, many frogs have been housed in close proximity without transmission of disease.


References

Barr, D. J. S. (1990). Phylum Chytridiomycota. In: Handbook of Protoctista. (Eds. L. Margulis, J. O. Corliss, M. Melkonian and D. J. Chapman). Lubrecht & Kramer, Monticello, New York. pp 454-466.

Berger, L. Speare, R. Daszak, P. , Green, D. E., Cunningham, A. A., Goggin, C. L., Slocombe, R. Ragan, M. A. Hyatt, A. D., McDonald, K. R. Hines, H. B., Lips, K. R., Marantelli, G. and Parkes, H. (1998). Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proceedings of the National Academy of Science, USA 95: 9031-9036.

Groff, J. M., Mughannam, A., McDowell, T. S., Wong, A. Dykstra, M. J., Frye, F. L. and Hedrick, R. P. (1991). An epidemic of cutaneous zygomycosis in cultured dwarf African clawed frogs (Hymenochirus curtipes) due to Basidiobolus ranarum. Journal of Medical and Veterinary Mycology 29: 215-223.

Laurance, W. F., McDonald, K. R. and Speare, R. (1996). Epidemic disease and catastrophic declines of Australian rain forest frogs. Conservation Biology 10: 1-9.

Laurance, W. F., McDonald, K. R. and Speare, R. (1997). In defense of the epidemic disease hypothesis. Conservation Biology 11: 1030-1034.

Lips, K. R. (1998). Decline of a tropical montane fauna. Conservation Biology 12: 106-117.

Mahony, M. (1996). The declines of the green and golden bell frog Litoria aurea viewed in the context of declines and disappearances of other Australian frogs. Australian Zoologist 30: 237-247.

Pessier, A.P., Nichols, D.K., Longcore, J.E., and Fuller, M.S. (1999). Cutaneous chytridiomycosis in poison dart frogs (Dendrobates spp.) and White's tree frogs (Litoria caerulea). Journal of Veterinary Diagnostic Investigation (in press).

Raphael, B. L. (1993). Amphibians. Veterinary Clinics of North America: Small Animal Practice 23:1271-1285.

Richards, S. J., McDonald, K. R. and Alford, R. A. (1993). Declines in populations of Australia's endemic tropical rainforest frogs. Pacific Conservation Biology 1: 66-77.

Williams, S. E. and Hero, J. M. (1998). Rainforest frogs of the Australian Wet Tropics: guild classification and the ecological similarity of declining species. Proceedings of the Royal Society of London B. 265: 597-602.


NB: We have made available an amphibian disease site on the world wide web - http://www.jcu.edu.au/dept/PHTM/frogs/ampdis.htm This contains instructions on how to collect skin and toes to test for the chytrid, how to collect frogs for pathology, how to prevent transmission of amphibian pathogens between locations, a bibliography of amphibian declines and disease, and a list of people with expertise in frog disease.

Updated 21 December 1998
Rick Speare