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.

Occurrence of cloacal prolapse in wild hylids in the Wet Tropics, Australia.

The idiopathic syndrome of cloacal prolapse was observed in the frog Litoria rheocola at 2 sites in the Wet Tropics of Australia; 1 of 365 individuals (0.3%) at Tully Gorge National Park and 5 of 92 individuals (5.4%) at Murray Upper National Park. The condition appeared to have resolved spontaneously with no clinical complications in 2 individuals at successive captures 14 and 155 d later. It was not observed in the sympatric frogs L. genimaculata (n = 206), L. nannotis (n = 357) or Nyctimystes dayi (n = 304) at either location. The underlying cause was not conclusively determined.

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.

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.