Infection increases vulnerability to climate change via effects on host thermal tolerance.

Unprecedented global climate change and increasing rates of infectious disease emergence are occurring simultaneously. Infection with emerging pathogens may alter the thermal thresholds of hosts. However, the effects of fungal infection on host thermal limits have not been examined. Moreover, the influence of infections on the heat tolerance of hosts has rarely been investigated within the context of realistic thermal acclimation regimes and potential anthropogenic climate change. We tested for effects of fungal infection on host thermal tolerance in a model system: frogs infected with the chytrid Batrachochytrium dendrobatidis. Infection reduced the critical thermal maxima (CTmax) of hosts by up to ~4 °C. Acclimation to realistic daily heat pulses enhanced thermal tolerance among infected individuals, but the magnitude of the parasitism effect usually exceeded the magnitude of the acclimation effect. In ectotherms, behaviors that elevate body temperature may decrease parasite performance or increase immune function, thereby reducing infection risk or the intensity of existing infections. However, increased heat sensitivity from infections may discourage these protective behaviors, even at temperatures below critical maxima, tipping the balance in favor of the parasite. We conclude that infectious disease could lead to increased uncertainty in estimates of species' vulnerability to climate change.

Interventions for reducing extinction risk in chytridiomycosis-threatened amphibians.

Wildlife diseases pose an increasing threat to biodiversity and are a major management challenge. A striking example of this threat is the emergence of chytridiomycosis. Despite diagnosis of chytridiomycosis as an important driver of global amphibian declines 15 years ago, researchers have yet to devise effective large-scale management responses other than biosecurity measures to mitigate disease spread and the establishment of disease-free captive assurance colonies prior to or during disease outbreaks. We examined the development of management actions that can be implemented after an epidemic in surviving populations. We developed a conceptual framework with clear interventions to guide experimental management and applied research so that further extinctions of amphibian species threatened by chytridiomycosis might be prevented. Within our framework, there are 2 management approaches: reducing Batrachochytrium dendrobatidis (the fungus that causes chytridiomycosis) in the environment or on amphibians and increasing the capacity of populations to persist despite increased mortality from disease. The latter approach emphasizes that mitigation does not necessarily need to focus on reducing disease-associated mortality. We propose promising management actions that can be implemented and tested based on current knowledge and that include habitat manipulation, antifungal treatments, animal translocation, bioaugmentation, head starting, and selection for resistance. Case studies where these strategies are being implemented will demonstrate their potential to save critically endangered species.

Presence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in threatened corroboree frog populations in the Australian Alps.

Since the early 1980s, the southern corroboree frog Pseudophryne corroboree and northern corroboree frog P. pengilleyi have been in a state of decline from their sub-alpine and high montane bog environments on the southern tablelands of New South Wales, Australia. To date, there has been no adequate explanation as to what is causing the decline of these species. We investigated the possibility that a pathogen associated with other recent frog declines in Australia, the amphibian chytrid fungus Batrachochytrium dendrobatidis, may have been implicated in the decline of the corroboree frogs. We used histology of toe material and real-time PCR of skin swabs to investigate the presence and infection rates with B. dendrobatidis in historic and extant populations of both corroboree frog species. Using histology, we did not detect any B. dendrobatidis infections in corroboree frog populations prior to their decline. However, using the same technique, high rates of infection were observed in populations of both species after the onset of substantial population declines. The real-time PCR screening of skin swabs identified high overall infection rates in extant populations of P. corroboree (between 44 and 59%), while significantly lower rates of infection were observed in low-altitude P. pengilleyi populations (14%). These results suggest that the initial and continued decline of the corroboree frogs may well be attributed to the emergence of B. dendrobatidis in populations of these species.

A zoospore inhibition technique to evaluate the activity of antifungal compounds against Batrachochytrium dendrobatidis and unsuccessful treatment of experimentally infected green tree frogs (Litoria caerulea) by fluconazole and benzalkonium chloride.

Effective and safe treatments of chytridiomycosis in amphibians, caused by Batrachochytrium dendrobatidis, are needed to help prevent mortality in captive programs for threatened species, to reduce the risk of spread, and to better manage the disease in threatened populations. We describe a simple method to determine minimum inhibitory concentrations (MICs) of antifungal agents that involves adding zoospores to various drug concentrations in 96 well plates and microscopic observation after four days. We report results from testing 10 commercially available antifungal compounds: benzalkonium chloride (<0.78 microg/ml), povidone iodine (312.5 microg/ml), amphotericin B (3.125 microg/ml), fluconazole (<1.56 microg/ml), itraconazole (<1.56 microg/ml), enilconazole (<1.56 microg/ml), mercurochrome (6.25 microg/ml), sodium chloride (12.5mg/ml), methylene blue (<1.56 microg/ml) and Virkon (3.125 microg/ml). For treatment trials of juvenile Litoria caerulea, baths of benzalkonium chloride at 1mg/L and fluconazole at 25mg/L were used on 18 experimentally infected frogs per treatment. Although these treatments resulted in longer survival times (mean 43.7+/-11.3 days) than in the untreated controls (37.9+/-9.3 days), the mortality rate was still 100%. Higher doses of fluconazole are suggested for further animal trials.

Virulence of the amphibian chytrid fungus Batrachochytium dendrobatidis varies with the strain.

Although mortality in 3 groups of 15 green tree frogs Litoria caerulea exposed to 3 isolates of Batrachochytrium dendrobatidis was 100%, time to death varied with isolate, highlighting the importance of strain and/or passage history in pathogenicity studies and possibly in the epidemiology of chytridiomycosis. A standard naming scheme for isolates of B. dendrobatidis is proposed.

Emerging disease of amphibians cured by elevated body temperature.

The emerging infectious disease chytridiomycosis is thought to have contributed to many of the recent alarming declines in amphibian populations. Mortalities associated with these declines have often occurred during cooler seasons and at high elevations, suggesting that environmental temperature may be an important factor in disease emergence. We found that thermal environment affects the progress of the disease, and that housing frogs Litoria chloris at an environmental temperature of 37 degrees C for less than 16 h can clear them of the chytrid pathogen Batrachochytrium dendrobatidis. Our experiment demonstrated that elevated body temperatures similar to those experienced in behavioral fever and during normal thermoregulation can clear frogs of chytrid infection; therefore, variation in thermoregulatory opportunities and behaviors are likely to contribute to the differences in disease incidence observed among host species, populations, and regions. Although further refinement of the technique is needed to encompass various host species, appropriately applied thermal manipulations of amphibians and their enclosures may prove to be a safe and effective way of eliminating the fungal pathogen from captive amphibian populations and: preventing accidental spread of the pathogen when animals are translocated or released from captivity.

Production of polyclonal antibodies to Batrachochytrium dendrobatidis and their use in an immunoperoxidase test for chytridiomycosis in amphibians.

Polyclonal antibodies were produced for diagnosing chytridiomycosis in amphibians. Two sheep and 4 rabbits were inoculated with homogenized whole culture of Batrachochytrium dendrobatidis in Freund's complete adjuvant or triple adjuvant. Antisera from all animals reacted strongly with all stages of B. dendrobatidis and stained the walls, cytoplasm, rhizoids and zoospores in an indirect immunoperoxidase test. Significant cross-reactivity occurred only with some fungi in the Chytridiomycota, and there are no members of this phylum besides B. dendrobatidis that infect frogs. The immunoperoxidase stain is a useful screening test when combined with recognition of the morphology and infection site of B. dendrobatidis.