Andrea D. Phillott

Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs

The global emergence and spread of the pathogenic, virulent, and highly transmissible fungus Batrachochytrium dendrobatidis, resulting in the disease chytridiomycosis, has caused the decline or extinction of up to about 200 species of frogs. Key postulates for this theory have been completely or partially fulfilled. In the absence of supportive evidence for alternative theories despite decades of research, it is important for the scientific community and conservation agencies to recognize and manage the threat of chytridiomycosis to remaining species of frogs, especially those that are naive to the pathogen. The impact of chytridiomycosis on frogs is the most spectacular loss of vertebrate biodiversity due to disease in recorded history.

BSA reduces inhibition in a TaqMan® assay for the detection of Batrachochytrium dendrobatidis

A TaqMan assay for the causative agent of chytridiomycosis in amphibians (Batrachochytrium dendrobatidis) can be inhibited by phenolic compounds, including humic and tannic acids, resulting in false negatives. Bovine serum albumin (BSA) is known to reduce inhibition of PCR when samples are contaminated with these inhibitors. We assessed the effect of BSA in reducing inhibition of the TaqMan assay when analyzing skin swabs for B. dendrobatidis. We found that the addition of BSA to the TaqMan reaction reduced inhibition to insignificant levels. BSA did not appreciably affect the efficiency or analytical sensitivity of the TaqMan reaction in the analysis of standard DNA solutions free from environmental inhibitors. We recommend the addition of 400 ng microl(-1) of BSA to the standard TaqMan assay to reduce inhibition associated with sampling wild amphibians.

Chytridiomycosis and Seasonal Mortality of Tropical Stream‐Associated Frogs 15 Years after Introduction of Batrachochytrium dendrobatidis

Assessing the effects of diseases on wildlife populations can be difficult in the absence of observed mortalities, but it is crucial for threat assessment and conservation. We performed an intensive capture-mark-recapture study across seasons and years to investigate the effect of chytridiomycosis on demographics in 2 populations of the threatened common mist frog (Litoria rheocola) in the lowland wet tropics of Queensland, Australia. Infection prevalence was the best predictor for apparent survival probability in adult males and varied widely with season (0-65%). Infection prevalence was highest in winter months when monthly survival probabilities were low (approximately 70%). Populations at both sites exhibited very low annual survival probabilities (12-15%) but high recruitment (71-91%), which resulted in population growth rates that fluctuated seasonally. Our results suggest that even in the absence of observed mortalities and continued declines, and despite host-pathogen co-existence for multiple host generations over almost 2 decades, chytridiomycosis continues to have substantial seasonally fluctuating population-level effects on amphibian survival, which necessitates increased recruitment for population persistence. Similarly infected populations may thus be under continued threat from chytridiomycosis which may render them vulnerable to other threatening processes, particularly those affecting recruitment success.

Minimising exposure of amphibians to pathogens during field studies

Many of the recent global amphibian mass mortalities, declines and extinctions have been attributed to the emerging infectious disease chytridiomycosis. There have been mass mortalities due to ranaviral disease but no major declines or extinctions. Controlling the transmission and spread of disease is of utmost importance, especially where there is the potential for human involvement. We have reviewed current hygiene guidelines for working with wild frogs, identified potential flaws and recommended those most suitable and effective for the field environment. Our within-site hygiene measures aim to reduce the risk of transmission among individuals. These measures encompass the capture, handling and holding of amphibians, skin disinfection before and after invasive procedures, marking frogs, sealing open wounds and treatment of accessory equipment. Our between-site hygiene measures aim to mitigate the risk of pathogen spread among populations. We have designed a risk calculator to help simplify and standardise the decision-making process for determining the level of risk and appropriate risk mitigation strategies to reduce the risk of increasing pathogen spread above background levels. Calculation of an overall risk score for pathogen spread takes into account the prior activity of field workers, the proposed activity, remoteness of the site, presence of known pathogens and the consequences of increased pathogen spread for amphibians in a given area.

The distribution and host range of the pandemic disease chytridiomycosis in Australia, spanning surveys from 1956–2007

Chytridiomycosis is the worst disease to affect vertebrate biodiversity on record. In Australia, it is thought to have caused the extinction of four frog species, and it threatens the survival of at least 10 more. We report the current distribution and host range of this invasive disease in Australia, which is essential knowledge for conservation management. We envisage that the data be used in a global and national context for predictive modeling, meta-analyses, and risk assessment. Our continent-wide data set comprises 821 sites in Australia and includes 10 183 records from >80 contributors spanning collection dates from 1956 to 2007. Sick and dead frogs from the field and apparently healthy frogs from museum collections were tested opportunistically for the presence of Batrachochytrium dendrobatidis, the fungal pathogen causing chytridiomycosis, and apparently healthy frogs and tadpoles found during surveys were tested purposively. The diagnostic tests used were histology of skin samples and quantitative PCR of skin swabs. Chytridiomycosis was found in all Australian states and the Australian Capital Territory, but not in the Northern Territory. Currently it appears to be confined to the relatively cool and wet areas of Australia, such as along the Great Dividing Range and adjacent coastal areas in the eastern mainland states of Queensland, New South Wales, and Victoria, eastern and central Tasmania, southern South Australia, and southwestern Western Australia. Batrachochytrium dendrobatidis may have been introduced into Australia via the port of Brisbane around 1978 and spread northward and southward. It did not appear to arrive in Western Australia until 1985. The earliest records from South Australia and Tasmania are from 1995 and 2004, respectively, although archival studies from these states are lacking. We also report negative findings showing that the disease does not currently occur in some areas that appear to be environmentally suitable, including Cape York Peninsula in Queensland and most of the World Heritage Area in western Tasmania. Infection with B. dendrobatidis has been recorded from 63 frog species in Australia to date, all belonging to the Hylidae, Limnodynastidae, and Myobatrachidae, with the exception of one individual of a species from the Microhylidae and the introduced cane toad of the family Bufonidae.

The distribution of failed eggs and the appearance of fungi in artificial nests of green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtles

Fungi are often found on the exterior of unhatched eggs in sea turtle nests in eastern Australia. Artificial incubation of green (Chelonia mydas) and loggerhead (Caretta caretta) turtle eggs allowed the radiation of fungi to be monitored. Fortuitous egg mortality provided a nutrient source for common soil mycobiota (Fusarium solani and Pseudallescheria boydii) and served as a focus for the progressive spread of fungal hyphae to adjacent viable eggs. In extreme cases the entire egg mass was enveloped and resulted in nil hatch success.

Global distribution of two fungal pathogens threatening endangered sea turtles.

Nascent fungal infections are currently considered as one of the main threats for biodiversity and ecosystem health, and have driven several animal species into critical risk of extinction. Sea turtles are one of the most endangered groups of animals and only seven species have survived to date. Here, we described two pathogenic species, i.e., Fusarium falciforme and Fusarium keratoplasticum, that are globally distributed in major turtle nesting areas for six sea turtle species and that are implicated in low hatch success. These two fungi possess key biological features that are similar to emerging pathogens leading to host extinction, e.g., high virulence, and a broad host range style of life. Their optimal growth temperature overlap with the optimal incubation temperature for eggs, and they are able to kill up to 90% of the embryos. Environmental forcing, e.g., tidal inundation and clay/silt content of nests, were correlated to disease development. Thus, these Fusarium species constitute a major threat to sea turtle nests, especially to those experiencing environmental stressors. These findings have serious implications for the survival of endangered sea turtle populations and the success of conservation programs worldwide.

Application of the survey protocol for chytridiomycosis to Queensland, Australia

Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), which causes chytridiomycosis, has resulted in the extinction of frogs, but the distribution of Bd is incompletely known. We trialled the survey protocol for Bd by attempting to systematically map its distribution in Queensland, Australia. Bd was easily detected in known infected areas, such as the Wet Tropics and South East Queensland. It was not detected in bioregions adjacent to, but inland from or to the north of, infected regions: Einasleigh Uplands and Cape York adjacent to the infected Wet Tropics; and Brigalow Belt South adjacent to the infected South East Queensland bioregion. These regions where Bd was not detected have bordered infected regions for between 15 yr (in northern Queensland) and 30 yr (in southern Queensland), and so they define the geographical limits of Bd with regard to the long-term environmental conditions in Queensland. The Gulf Plains, a bioregion distant from infected bioregions, was also negative. Bd was confined to rainforest and bordering habitats, such as wet eucalypt forests. Infections were largely confined to permanent water-associated species, consistent with this being an important cause of this group having the greatest declines. Our data supports biogeographic climatic models that show much of inland and northern Australia to be too hot and dry to support Bd. As there is limited opportunity for Bd to spread further in Queensland, the priority for management is reducing the impact of Bd in affected populations and assisting frogs to disperse into their former distributions. Given that the survey protocol has been applied successfully in Australia it may be useful for mapping the distribution of Bd in other parts of the world.

Influence of Diminished Respiratory Surface Area on Survival of Sea Turtle Embryos

It has been suggested that fungal presence on sea turtle eggs may impede gas exchange. To investigate the influence of diminished gas exchange surface upon embryo survivorship, flatback (Natator depressus) and green (Chelonia mydas) eggs were painted with petroleum jelly. Variable proportions of the egg surface were covered, including both respiratory and nonrespiratory domains. Embryo survival varied with site inhibited, proportion of eggshell affected, and species of turtle. If fungi on the exterior of the eggshell are able to impede respiratory gas exchange, their presence on the upper hemisphere (primary gas exchange area in early incubation) will result in the highest embryo mortality. Large eggs are likely to demonstrate a higher survivorship than small eggs, due to their larger available respiratory area and/or to variation in weight or stage-specific embryonic metabolic demands. Interspecific differences in egg size may therefore be a contributory factor to observed mortality rate differences in the natural presence of fungi.

Storage of samples at high temperatures reduces the amount of amphibian chytrid fungus Batrachochytrium dendrobatidis DNA detectable by PCR assay

Chytridiomycosis, caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), is an emerging infectious disease responsible for amphibian declines on several continents. In laboratory conditions, optimal temperatures for Bd growth and survivorship are between 17 and 25 degrees C. We investigated the effect of different storage temperatures, both in field and laboratory conditions, on detection of Bd from swabs stored for 7 d. We sampled 52 wild Litoria wilcoxii males for Bd by simultaneously running 2 cotton swabs along the skin of the frog. One group of swabs was stored in a freezer within 2 h of sampling and the other was kept in a car in an exposed environment for 7 d before being stored in the freezer. In the laboratory experiment, swabs were inoculated with zoospores of Bd and underwent one of 4 treatments: immediate DNA extraction, or storage at 27, 38 or 45 degrees C for 7 d prior to DNA extraction. Swabs from all treatments were analyzed by quantitative (real-time) PCR test. Though prevalence of Bd did not differ significantly between swabs that were frozen and those that remained in a car for 7 d (19.2 vs. 17.3%, respectively), the number of Bd zoospores detected on car swabs taken from infected frogs was, on average, 67% less than that detected on the corresponding frozen swab. In the laboratory experiment, the number of zoospore equivalents varied significantly with treatment (F(3,35) = 4.769, p = 0.007), indicating that there was reduced recovery of Bd DNA from swabs stored at higher temperatures compared with those stored at lower temperatures or processed immediately. We conclude that failure to store swabs in cool conditions can result in a significant reduction in the amount of Bd DNA detected using the PCR assay. Our results have important implications for researchers conducting field sampling of amphibians for Bd.