Search

Many microorganisms are able to cause diseases in amphibians, and in the past few years one of the most reported has been Batrachochytrium dendrobatidis. This fungus was first reported in Brazil in 2005; following this, other reports were made in specimens deposited in museum collections, captive and free-living frogs. The aim of this study was to compare singleplex and nested-PCR techniques to detect B. dendrobatidis in free-living and apparently healthy adult frogs from the Brazilian Atlantic Forest. The sample collection area was a protected government park, with no general entrance permitted and no management of the animals there. Swabs were taken from the skin of 107 animals without macroscopic lesions and they were maintained in ethanol p.a. Fungal DNA was extracted and identification of B. dendrobatidis was performed using singleplex and nested-PCR techniques, employing specific primers sequences. B. dendrobatidis was detected in 61/107 (57%) and 18/107 (17%) animals, respectively by nested and singleplex-PCR. Nested-PCR was statistically more sensible than the conventional for the detection of B. dendrobatidis (Chi-square = 37.1; α = 1%) and the agreement between both techniques was considered just fair (Kappa = 0.27). The high prevalence obtained confirms that these fungi occur in free-living frogs from the Brazilian Atlantic Forest with no macroscopic lesions, characterizing the state of asymptomatic carrier. We concluded that the nested-PCR technique, due to its ease of execution and reproducibility, can be recommended as one of the alternatives in epidemiological surveys to detect B. dendrobatidis in healthy free-living frog populations.

All anuran specimens in the Wildlife Conservation Society's collections testing positive for Batrachochytrium dendrobatidis (Bd) were treated with itraconazole and then studied after treatment to assess the long-term effects of itraconazole and the drug's effectiveness in eliminating Bd carriers. Twenty-four individuals and eight colonies of 11 different species (75 total specimens) tested positive for Bd via polymerase chain reaction (PCR) on multicollection survey. All positive individuals and colonies were treated with a 0.01% itraconazole bath solution and retested for Bd via one of two PCR methodologies within 14 days of treatment completion, and all were negative for Bd. A total of 64 animals received secondary follow-up PCR testing at the time of death, 6-8 mo, or 12-15 mo post-treatment. Fourteen animals (14/64, 21.9%) were PCR positive for Bd on second follow-up. The highest percentage positive at second recheck were green-and-black poison dart frogs (Dendrobates auratus; 5/5 specimens, 100%), followed by red-eyed tree frogs (Agalychnis callidryas; 4/11, 36.4%), grey tree frogs (Hyla versicolor; 1/3, 33.3%), and green tree frogs (Hyla cinera; 3/11, 27.3%). Re-testing by PCR performed on 26/28 individuals that died during the study indicated 11/26 (42.3%) were positive (all via DNA extracted from formalin-fixed paraffin-embedded skin sections). However, there was no histologic evidence of chytridiomycosis in any of 27/28 individuals. The small number of deceased animals and effects of postmortem autolysis limited the ability to determine statistical trends in the pathology data, but none of the necropsied specimens showed evidence of itraconazole toxicity. Problems with itraconazole may be species dependent, and this report expands the list of species that can tolerate treatment. Although itraconazole is effective for clearance of most individuals infected with Bd, results of the study suggest that repeat itraconazole treatment and follow-up diagnostics may be required to ensure that subclinical infections are eliminated in amphibian collections.

Chytridiomycosis is an amphibian disease of global conservation concern that is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Since the discovery of Bd in 1998, several methods have been used for detection of Bd; among these polymerase chain reaction (PCR) from skin swabs is accepted as the best method due to its noninvasiveness, high sensitivity and ease of use. However, PCR is not without problems - to be successful, this technique is dependent upon the presence of nondegraded DNA template and reaction contents that are free from inhibitors. Here, we report on an investigation of several techniques aimed at improving the reliability of the Bd PCR assay by minimizing the effects of humic acid (HA), a potent PCR inhibitor. We compared the effectiveness of four DNA extraction kits (DNeasy, QIAamp DNA Stool, PowerLyzer Power Soil and PrepMan Ultra) and four PCR methods (Amplitaq Gold, bovine serum albumin, PowerClean DNA Clean-up and inhibitor resistant Taq Polymerase). The results of this and previous studies indicate that chytridiomycosis studies that use PCR methods for disease detection may be significantly underestimating the occurrence of Bd. Our results suggest that to minimize the inhibitory effects of HA, DNeasy should be used for sample DNA extraction and Amplitaq Gold with bovine serum albumin should be used for the Bd PCR assay. We also outline protocols tested, show the results of our methods comparisons and discuss the pros and cons of each method., (© 2012 Blackwell Publishing Ltd.)

The fungal pathogen Batrachochytrium dendrobatidis threatens amphibian populations around the world. The ability to detect this pathogen on infected animals and in the environment is critical for understanding and controlling this pandemic. We tested several advances in quantitative PCR (qPCR) techniques to detect B. dendrobatidis DNA. We used a fast PCR thermocycler and enzymes that reduced the volume and the duration of the reaction. We also compared a conventional TaqMan minor groove binding (MGB) probe to an identical locked nucleic acid (LNA) counterpart. The fast qPCR reaction had a high degree of sensitivity to B. dendrobatidis DNA. The LNA probe was effective for detecting B. dendrobatidis DNA and produced results -similar to those of the MGB probe. The modifications that we tested can improve the cost, time efficiency and specificity of quantitative PCR as a tool for detecting pathogen DNA.

A sensitive and quantitative TaqMan assay for the causative agent of chytridiomycosis in amphibians (Batrachochytrium dendrobatidis) has been developed and is routinely used in diagnostic laboratories. We assessed whether the real time detection of the TaqMan assay was as sensitive as the detection of the PCR product by agarose gel electrophoresis and ethidium bromide staining. We found, for practical purposes, that gel-based detection of the diagnostic fragment produced by means of the TaqMan assay or by conventional PCR that used a different polymerase and reaction mix was as sensitive as the real-time detection of the TaqMan assay. We recommend the qualified use of conventional PCR amplification combined with agarose gel electrophoresis and ethidium bromide staining for studies where only prevalence data are required, funding for equipment is limited or the acquisition of a real-time system is not cost effective.

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.

The aetiological agent of amphibian chytridiomycosis Batrachochytrium dendrobatidis is a primary cause of amphibian population declines. Current surveillance is based on the detection of B. dendrobatidis in its host but in vitro work suggests infective stages may survive in the abiotic environment for at least 3 mo. We describe here a surveillance system using filtration and quantitative PCR that can detect B. dendrobatidis in small (< 1 l) volumes of water. After assessing the analytical sensitivity of the protocol for both water and sediment samples in the laboratory, we analyzed environmental samples from the Sierra de Guadarrama mountain range in Spain at locations associated with chytrid-related die-offs and at other sites across Spain. B. dendrobatidis was detected in samples from 64% of the ponds in the Sierra de Guadarrama and at 2 sites outside this region, showing that levels of amphibian exposure to B. dendrobatidis are spatially heterogeneous. In experimental microcosms, we detected B. dendrobatidis for up to 12 wk, though we found no evidence for an overall increase in biomass. Our results emphasise the need to further investigate the life cycle of B. dendrobatidis to more completely understand the epidemiology of this emerging pathogen.

Batrachochytrium dendrobatidis is a fungus belonging to the Phylum Chytridiomycota, Class Chytridiomycetes, Order Chytridiales, and is the highly infectious aetiological agent responsible for a potentially fatal disease, chytridiomycosis, which is currently decimating many of the world's amphibian populations. The fungus infects 2 amphibian orders (Anura and Caudata), 14 families and at least 200 species and is responsible for at least 1 species extinction. Whilst the origin of the agent and routes of transmission are being debated, it has been recognised that successful management of the disease will require effective sampling regimes and detection assays. We have developed a range of unique sampling protocols together with diagnostic assays for the detection of B. dendrobatidis in both living and deceased tadpoles and adults. Here, we formally present our data and discuss them in respect to assay sensitivity, specificity, repeatability and reproducibility. We suggest that compliance with the recommended protocols will avoid the generation of spurious results, thereby providing the international scientific and regulatory community with a set of validated procedures which will assist in the successful management of chytridiomycosis in the future.

Batrachochytrium dendrobatidis (Bd) infection on post-metamorphic frogs and salamanders is commonly diagnosed using polymerase chain reaction (PCR) of skin scrapings taken with mildly abrasive swabs. The technique is sensitive, non-lethal, and repeatable for live animals. Tadpoles are generally not sampled by swabbing but are usually killed and their mouthparts excised to test for the pathogen. We evaluated a technique for non-lethal Bd diagnosis using quantitative PCR (qPCR) on swabs scraped over the mouthparts of live tadpoles. The sensitivity of non-lethal (swabbing) and lethal (removal of mouthparts) sampling was assessed using 150 Bd-infected Rana subaquavocalis tadpoles. Swabbing was consistently less sensitive than lethal sampling, but still detected Bd. Experimental Bd prevalence was 41.1% when estimated by destructively sampling mouthparts and 4.7 to 36.6% (mean = 21.4%) when estimated with swabs. Detection rates from swabbing varied with investigator and time since infection. The likelihood of detecting Bd-infected tadpoles was similar regardless of size and developmental stage. Swabbing mouthparts of live tadpoles is a feasible and effective survey technique for Bd, but, because it is less sensitive, more tadpoles must be sampled to estimate prevalence at a confidence level comparable to destructive sampling.

Chytridiomycosis is an emerging infectious disease of amphibians associated with mass mortalities and population declines worldwide. Recent technological advances have resulted in a highly sensitive, non-invasive technique for diagnosing the disease based on a quantitative (real-time) polymerase chain reaction (qPCR) assay. The qPCR assay yields the most accurate and informative data of any available detection technique. However, due to the relatively high costs involved, it has yet to attain widespread use by chytridiomycosis researchers. Using the results of a disease survey of 467 wild frogs from eastern Queensland, Australia, we examine the necessity of triplicate assays in qPCR detection of chytridiomycosis. We describe a singlicate qPCR assay that can be used to substantially decrease costs, with no significant decrease in sensitivity. We also demonstrate that detection of chytridiomycosis by use of the conventional PCR assay may lead to appreciable underestimations in disease prevalence. We recommend that amphibian disease researchers adopt the singlicate qPCR assay as the primary means of chytridiomycosis detection.

Chytridiomycosis is a lethal disease of amphibians associated with mass mortalities and population declines worldwide. An accurate, non-invasive technique for detecting chytridiomycosis is urgently needed to determine the current geographical distribution of the disease, and its prevalence in wild amphibian populations. Herein we evaluate a recently devised, rapid, non-invasive, swab-PCR assay. We sampled 101 wild juvenile Mixophyes iteratus by both a skin swab for use in PCR analysis, and a toe-clip for examination by histological methods. The swab-PCR assay detected chytridiomycosis infection in a minimum of 14.9% of frogs, whereas histology detected infection in no more than 6.9% of frogs. We conclude that the swab-PCR technique is the more reliable means of detecting chytridiomycosis in wild amphibians, and that it precludes the need for toe-clipping as a means of sampling for the presence of the disease in future surveys. Further, we document a significant negative relationship between a juvenile frog's snout-vent length and its likelihood of being infected with the disease.

Chytridiomycosis caused by Batrachochytrium dendrobatidis (Chytridiomycota) has been implicated in declines of amphibian populations on four continents. We have developed a sensitive and specific polymerase chain reaction-based assay to detect this pathogen. We isolated B. dendrobatidis from captive and wild amphibians collected across North America and sequenced the internal transcribed spacer regions of the rDNA cassette of multiple isolates. We identified two primers (Bd1a and Bd2a) that are specific to B. dendrobatidis under amplification conditions described in this study. DNA amplification with Bd1a/Bd2a primers produced a fragment of approximately 300 bp from B. dendrobatidis DNA but not from DNA of other species of chytrids or common soil fungi. The assay detected 10 zoospores or 10 pg of DNA from B. dendrobatidis and detected infections in skin samples from a tiger salamander (Ambystoma tigrinum), boreal toads (Bufo boreas), Wyoming toads (Bufo baxteri), and smooth-sided toads (Bufo guttatus). This assay required only small samples of skin and can be used to process a large number of samples.