Survey protocol for detecting chytridiomycosis in all Australian frog populations

Abstract

Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused the decline and extinction of frogs, but the distribution of Bd is not completely known. This information is crucial to implementing appropriate quarantine strategies, preparing for outbreaks of chytridiomycosis due to introduction of Bd, and for directing conservation actions towards affected species. This survey protocol provides a simple and standard method for sampling all frog populations in Australia to maximise the chances of detecting Bd. In order to structure and prioritise the protocol, areas are divided by bioregion and frog species are allocated depending on the water bodies they utilize into 3 groups representing different levels of risk of exposure to Bd. Sixty individuals per population need to be tested to achieve 95% certainty of detecting 1 positive frog, based on the minimum apparent prevalence of ≥5% in infected Australian frog populations and using a quantitative real-time TaqMan PCR test. The appropriate season to sample varies among bioregions and will ideally incorporate temperatures favourable for chytridiomycosis (e.g. maximum air temperatures generally < 27°C). Opportunistic collection and testing of sick frogs and tadpoles with abnormal mouthparts should also be done to increase the probability of detecting Bd. The survey priorities in order are (1) threatened species that may have been exposed to Bd, (2) bioregions surrounding infected bioregions/ecological groups, and (3) species of frogs of unknown infection status in infected bioregions. Within these priority groups, sampling should first target ecological groups and species likely to be exposed to Bd, such as those associated with permanent water, and areas within bioregions that have high risk for Bd as indicated by climatic modelling. This protocol can be adapted for use in other countries and a standard protocol will enable comparison among amphibian populations globally.

Evaluation of a filtration-based method for detecting Batrachochytrium dendrobatidis in natural bodies of water

“…Following Garner et al (2009) 48 a These ponds were dropped from filter sensitivity analyses because their small swab sample size and failure to detect Bd cannot be distinguished from false negatives with > 95% certainty (Skerratt et al 2008) b Ponds that were not sampled (na) within the given time period were removed from calculation of this percentage Table 1. Batrachochytrium dendrobatidis (Bd).…”

“…This technique is designed to capture zoospores or remnant Bd DNA from the water column and then amplify this DNA using PCR. These filtration techniques have the potential to significantly reduce Bd sampling effort and costs by eliminating the need to collect and process DNA samples from the currently accepted standard of 30 to 60 animals per habitat, which can be difficult to obtain when dealing with sites with low amphibian abundance or rare or cryptic species (Skerratt et al 2008). Recent studies (Kirshtein et al 2007, Walker et al 2007) successfully detected Bd from small volumes (<1 l) of field-collected water using a highly specific PCRbased assay (Hyatt et al 2007).…”

“…Although swabs from 60 animals cannot definitively confirm the absence of Bd from these ponds, this method currently serves as the most accurate way to determine the presence of Bd within a habitat, with 95% certainty if prevalence is ≥5% (Skerratt et al 2008). Using 20 ephemeral ponds with Bd-infected populations of the boreal chorus frog Pseudacris maculata, we addressed the following questions: (1) What percentage of filters fails to detect Bd when present in swabs collected from live animals?…”

Widespread occurrence of the amphibian chytrid fungus in Kenya

“…The time of sampling targeted the beginning of the 'short rains' of the bimodal precipitation pattern. No information on the seasonality of Bd infection dynamics in Africa is available but sampling took place corresponding well with the recommendations given for sampling in Australia, for example, focusing on the cold season (Kriger & Hero, 2006;Skerratt et al, 2008). Approximately 1500 specimens were examined in situ during the survey.…”

Pre-emptive National Monitoring Plan for Detecting the Amphibian Chytrid Fungus in Madagascar

“…In spite of the significant role chytridiomycosis plays in amphibian conservation, the development of survey protocols at the national level has received little attention by infected and uninfected countries, and diagnostic primer sets do not account for lineage diversity. The only published national survey exists for Australia (Skerratt et al 2008), which details a standard method for sampling prioritized wild frog populations for infection with Bd.…”