Diseases in Australian frogs

The aims of this project were to investigate the diseases present in Australian amphibians and to determine the cause of the mass mortalities in wild frogs in Queensland that had resulted in population declines. Initially diagnostic pathology methods were used to examine endangered frogs collected from a mass die-off in north Queensland, as well as to survey other frogs for disease. The survey was achieved using a collaborative approach involving herpetologists, ecologists and other scientists around Australia who submitted sick and dead frogs for testing. Epidermal infections of a new species of chytrid fungus, Batrachochytrium dendrobatidis, were consistently found in frogs during mass mortalities in the wild and in captivity, including some of the last individuals of Taudactylus acutirostris. Detailed investigations of this pathogen included experimental transmissions, pathology, studies on the distribution in adults and tadpoles, epidemiology, and examination of the morphology and lifecycle. Further work was aimed at developing diagnostic tests, and treatment and disinfection protocols. A wide range of diseases were diagnosed in frogs submitted for the disease survey, including those caused by Mucor amphibiorum, Spirometra erinacei, Aphanomyces sp., Chlamydia pneumoniae, and neoplasms. Vacuolating and ulcerative dermatoses also occurred but the aetiology was not determined. However, the only disease that was common and had an obvious impact on the abundance of frogs was chytridiomycosis, which accounted for the deaths of 56.5% of frogs submitted. B. dendrobatidis was found to be widespread across Australia with a broad host range, and caused disease in 35 native Myobatrachid and Hylid species as well as the Bufonid, Bufo marinus. The incidence of chytridiomycosis showed a distinct seasonal effect with most frogs dying in winter. Most infected frogs were submitted from Queensland and New South Wales, with less from Victoria, South Australia and Western Australia. Large outbreaks occurred regularly in Litoria caerulea around Brisbane. Diseased frogs were from a variety of habitats at high and low altitudes. Sporangia of B. dendrobatidis grow within cells of the stratum granulosum and stratum corneum. As the fungus matures it is carried outwards with the epidermal cell. The entire contents of sporangia cleave into flagellated zoospores that are released from discharge tubes which protrude through the skin surface. Chytridiomycosis occurs as an extensive infection of the ventral skin and feet resulting in hyperkeratosis, hyperplasia, erosions, focal necrosis, and occasional ulceration of the epidermis, with minimal inflammation in the skin. Ultrastructural pathology demonstrated that infection of the epidermis stimulated an increased turnover of epidermal cells leading to hyperplasia. Initially disease was transmitted experimentally by exposing frogs to infected skin scrapings, and then once pure cultures of B. dendrobatidis were established, frogs were exposed to zoospores. Experimental infections in Mixophyes fasciolatus and L. caerulea at between 17°C and 24°C resulted in 100% mortality. Deaths (or terminal illness requiring euthanasia) occurred 9 - 76 days after exposure to the fungus, with most frogs dying between 18 and 48 days. Doses as low as 100 zoospores resulted in the death of 3/3 M. fasciolatus, while 3 frogs each given 10 zoospores did not succumb. At 27°C, 4/8 experimentally infected M. fasciolatus died while the rest remained healthy. Infection was confirmed in 3 of these, but was eliminated by 98 days. Attempts to infect Bufo marinus and Limnodynastes peronii were not successful. Healthy tadpoles could apparently carry infections in their mouthparts from soon after hatching until metamorphic climax when sporangia were rapidly redistributed to the skin of the body as the beaks were shed. The infected metamorphs died at about 2 – 3 weeks old. The distribution of sporangia in tadpoles and metamorphs during development followed the changes in the distribution of keratin. Accurate testing of sick frogs was achieved using histology or examination of skin scrapings. Histological examination of toe-clips was useful for ante mortem testing of healthy frogs, but was found to have low sensitivity (52.7%). Polyclonal antibodies were generated to B. dendrobatidis by inoculating rabbits and sheep. Although these antibodies cross-reacted with other chytridiomycetes, they were used in an immunoperoxidase test that increased sensitivity of diagnosis (61.8%) and ease of interpretation. A wide range of antifungal drugs and compounds were effective against B. dendrobatidis in vitro, but when fluconazole and benzalkonium chloride were selected for testing in an animal trial they were found to be ineffective for treating infected frogs. Sporangia and zoospores were sensitive to three common disinfectants when used at routine levels - 70% ethanol, 0.1 % benzalkonium chloride and 0.1 % Virkon. Cultures were also killed by drying for an hour, and by incubating at temperatures at 32°C or above. Chytridiomycosis is a highly pathogenic emerging infectious disease that has caused mass mortality of wild frogs leading to population declines and extinctions in protected montane rainforest areas. The most plausible explanation for the extreme susceptibility of some amphibian species giving rise to unsustainably high mortality rates is that they have not co-evolved with this pathogen, and that B. dendrobatidis was introduced to Australia and has spread through a naïve population