Evaluation of tadpole mouthpart depigmentation as a diagnostic test for infection by Batrachochytrium dendrobatidis for four California anurans

The objective of this study was to evaluate the utility of gross morphologic examination of larval mouthpart defects as a diagnostic screening test to detect Batrachochytrium dendrobatidis infection in four California, USA, anuran species. We examined mouthparts of 2,034 tadpoles of Bufo boreas, Pseudacris regilla, and Rana catesbeiana collected in 2003 and 2004 and Bufo canorus collected in 2004. Data were recorded for three morphologic features: upper toothrows, lower toothrows, and combined jaw sheaths. Mouthpart defects were observed in all four species (n=757), but only two species were infected with B. dendrobatidis (n=84). Sensitivity and specificity of the mouthparts test were 76% and 58%, respectively. Forty-two percent of B. dendrobatidis-negative animals would have been designated positive based on mouthpart defects. Observed prevalence was 43%, and true prevalence was 3.0%. Tests of the null hypothesis using logistic regression analysis showed that anuran larval mouthpart defects were not associated with B. dendrobatidis infection whether mouthparts scores were tested by individual morphologic feature or in combination (P=0.37). We conclude that B. dendrobatidis infection and anuran larval mouthpart defects are two separate processes that may occur concurrently and that evaluation of tadpole oral morphology is neither an accurate nor a reliable diagnostic test for B. dendrobatidis infection for the four species tested.     

Germ Tube Mediated Invasion of Batrachochytrium dendrobatidis in Amphibian Skin Is Host Dependent

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis, Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro, comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea.Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus.In X. laevis, interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.     

Cryo-archiving of Batrachochytrium dendrobatidis and other chytridiomycetes

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide. It has been associated with catastrophic declines of frog populations including those in pristine habitats in Queensland, Australia. To facilitate genetic and disease studies of this fungus and related species, it is essential to have a reliable long-term storage method to maintain genetic integrity of isolates. We have adapted well-established techniques used for the long-term storage of tissue-culture cell lines to the preservation of B. dendrobatidis and other chytridiomycetes. This simple method has allowed us to recover these fungi from storage at -80 degrees C and in liquid nitrogen over an extended period. With this technique it is now possible to preserve saprobic and parasitic isolates from a variety of environmental and disease situations for comparative genetic and biological studies.     

Controlling wildlife fungal disease spread: in vitro efficacy of disinfectants against Batrachochytrium dendrobatidis and Mucor amphibiorum

Chytridiomycosis in amphibians, and mucormycosis in the platypus Ornithorhynchus anatinus and amphibians, are serious fungal diseases affecting these aquatic taxa. In Tasmania, Australia, the fungi that cause these diseases overlap in range along with Phytophthora cinnamomi (Pc), an invasive fungal plant pathogen. To identify disinfectants that may be useful to reduce anthropogenic spread of these fungi to uninfected wilderness areas, for example by bush walkers and forestry or fire-fighting operations, we tested 3 disinfectants and a fire-fighting foam against Mucor amphibiorum (Ma) and tested 1 disinfectant and the foam against Batrachochytrium dendrobatidis (Bd). Combining the present study with previous work we found Bd was more susceptible to all 4 chemicals than Ma. Phytoclean, a disinfectant used at 2 to 10% for 30 s to control Pc, killed cultures of Bd at 0.075% and Ma at 5%, when also applied for 30 s. The disinfectant F10sc was not effective against Ma at standard exposures, but previous work shows Bd is killed at 0.03% with a 1 min exposure. Path-X is effective against Bd at 0.001% with a 30 s exposure and killed Ma at 1% with a 5 min exposure. Forexpan S, a foam added to water at 0.1 to 1% to control forest fires, killed Bd but not Ma when used at 1% for 2 min. Therefore, Phytoclean and Path-X have broader efficacy, although Path-X has not been trialled against Pc. Interestingly a positive mating strain of Ma (from a platypus) was more resistant to disinfectants than a negative strain (from a frog). Current protocols against Pc that involve high concentrations (10%) of Phytoclean are likely to reduce spread of pathogenic wildlife fungi, which is important for protecting biodiversity.     

Host thermoregulatory constraints predict growth of an amphibian chytrid pathogen (Batrachochytrium dendrobatidis)

1. The course and outcome of many wildlife diseases are context-dependent, and therefore change depending on the behaviour of hosts and environmental response of the pathogen.2. Contemporary declines in amphibian populations are widely attributed to chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis. Despite the thermal sensitivity of the pathogen and its amphibian hosts, we do not understand how host thermal regimes experienced by frogs in the wild directly influence pathogen growth.3. We tested how thermal regimes experienced by the rainforest frog Litoria rheocola in the wild influence pathogen growth in the laboratory, and whether these responses differ from pathogen growth under available environmental thermal regimes.4. Frog thermal regimes mimicked in the laboratory accelerated pathogen growth during conditions representative of winter at high elevations more so than if temperatures matched air or stream water temperatures. By contrast, winter frog thermal regimes at low elevations slowed pathogen growth relative to air temperatures, but not water temperatures.5. The growth pattern of the fungus under frog thermal regimes matches field prevalence and intensity of infections for this species (high elevation winter > high elevation summer > low elevation winter > low elevation summer), whereas pathogen growth trajectories under environmental temperatures did not match these patterns.6. If these laboratory results translate into field responses, tropical frogs may be able to reduce disease impacts by regulating their body temperatures to limit pathogen growth (e.g., by using microhabitats that facilitate basking to reach high temperatures); in other cases, the environment may limit the ability of frogs to thermoregulate such that individuals are more vulnerable to this pathogen (e.g., in dense forests at high elevations).7. Species-specific thermoregulatory behaviour, and interactions with and constraints imposed by the environment, are therefore essential to understanding and predicting the spatial and temporal impacts of this global disease.     

Historical dynamics of Batrachochytrium dendrobatidis in Amazonia

The Amazon forest is known for its astonishing amphibian diversity, yet the potential distribution and underlying impacts of the most important amphibian pathogen is unknown for most of Amazonia. In this retrospective survey of preserved Leptodactylus frogs, collected over a 119 yr period, we used quantitative PCR to detect the fungal pathogen Batrachochytrium dendrobatidis (Bd) and performed spatial scan analyses to identify spatiotemporal clusters of Bd. We also quantified the potential effect of environmental factors on the likelihood of Bd occurrence and generated an updated suitability map for Bd in the Amazon that included our retrospective sampling. We detected Bd in lowland Amazon as early as 1935, in the state of Pará, Brazil, and we found low prevalence (～ 3.8%) over time. We identified two statistically significant spatiotemporal clusters of Bd: a recent and narrow cluster in the Amazon River delta and a spatiotemporally broad cluster in the southern edge of Amazon and Brazilian savanna. Furthermore, we found an increase in Bd‐positive samples in the southwestern Amazon after the 1990s, coinciding with reported amphibian declines in neighboring high elevation sites on Andean slopes of Peru. Spatial regressions indicated that higher human interference, higher precipitation, and lower temperatures were significant predictors of Bd occurrence. Environmental niche modeling predicted some narrow areas of suitable climates along the Amazon's periphery and generally low climatic suitability for Bd in the central Amazon; although, we found clusters of Bd‐positive samples with unexpectedly high infection loads in areas of predicted low suitability. Our study indicates that accelerated human development may put Amazonian amphibians at risk from Bd introductions, and it highlights the potential need to monitor Bd dynamics near Amazonian port cities.     

Batrachochytrium dendrobatidis: requirement for further isolate collection and archiving

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis, which is lethal to many species of amphibians worldwide. Many studies have investigated the epidemiology of chytridiomycosis in amphibian populations, but few have considered possible host-pathogen coevolution. More specifically, investigations focused on the evolution of Bd, and the link with Bd virulence, are needed. Such studies, which may be important for conservation management of amphibians, depend on access to Bd isolates. Here we provide a summary of known Bd isolates that have been collected and archived in various locations around the world. Of 257 Bd isolates, we found that 53% originate from ranids in the United States. In many cases, detailed information on isolate origin is unavailable, and it is unknown how many isolates are cryo-archived. We suggest the creation of a centralized database of isolate information, and we urge researchers and managers to isolate and archive Bd to facilitate future research on chytridiomycosis.     

Landscape epidemiology of Batrachochytrium dendrobatidis in central California

Amphibian chytridiomycosis (caused by Batrachochytrium dendrobatis; Bd) was first identified in 1998 and has since been implicated in numerous amphibian declines worldwide. Most researchers have since investigated broad‐scale geographic and taxonomic occurrences of the pathogen in tropical lotic or cool montane systems. In this study, we analyzed how environmental factors, land use practices, and landscape structure may affect the dynamics of the pathogen's distribution in a landscape dominated by lentic systems within a region of Mediterranean climate. We quantified the occurrence of Bd testing the six resident amphibian species that occur in 54 isolated perennial and ephemeral ponds in central California between May and June annually from 2004 to 2007. The geographic distribution of Bd within the landscape varied markedly between years. Inter‐annual variation in climate affected the pond landscape structure indicating that climate conditions indirectly influence the distribution of the pathogen. Fourteen ponds, 12 perennial and 2 ephemeral, were positive for Bd≥3 yr of the study and were treated as Bd hotpots for comparative purposes. Occurrences of Bd within the landscape were spatially autocorrelated and ponds within ~1000–1500 m of Bd hotspots were more likely to test positive. Local land use, (presence/absence of grazing or recreational activity and developed lands), did not influence Bd status of a pond, indicating that the most likely means of Bd transmission between ponds may be waterfowl and/or amphibians.     

A DNA-based assay identifies Batrachochytrium dendrobatidis in amphibians

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.     

Quantitative measurement of Batrachochytrium dendrobatidis in amphibian skin

The ability to quantify infections provides a tool with which to perform comparative pathological research. The need exists for a simplistic standard method to compare infection levels of Batrachochytrium dendrobatidis, a major cause of global amphibian declines. Through examination of skin sloughs of the Cape river frog Afrana fuscigula, we present an accessible method that not only provides quantitative measurements of B. dendrobatidis, but also provides information that increases the confidence of detection through histological surveys. The method relies on the availability of live animals that are actively shedding skin. By employing a direct microscopic count of sporangia, it is possible to express infection in terms of density. Micro-spatial infection in the skin of A. fuscigula is characterised by significant differences in sporangium density among the different components of the foot, and by similar differences in site infection frequency. Notably, toe tips and tubercles contain higher infection densities and are more often infected than webbing or the base of the foot. This pattern of infection might facilitate disease transmission due to the increased exposure of these components to abrasion. Density data can be used with the Poisson frequency function to approximate binomial probabilities of detecting B. dendrobatidis through histology. The probability matrix produced for A. fuscigula indicated that foot-site selection for histology markedly influenced the number of sections required to detect B. dendrobatidis at a specific level of probability. Thus, examination of a test sample of skin tissue with direct-count quantification can help in planning the sampling of tissues for histological surveys.     

New records of Batrachochytrium dendrobatidis in Chilean frogs

We used molecular techniques to examine 11 species of frogs in 6 localities in southern Chile to ascertain the incidence of the chytrid fungus Batrachochytrium dendrobatidis (Bd). We detected the fungus in 2 localities (Co?aripe and Raúl Marín Balmaceda) in 3 species: Batrachyla leptopus, Pleurodema thaul and Rhinoderma darwinii. Our findings expand the list of Bd hosts to include B. leptopus and P. thaul and extend the spatial distribution in Chile to include the southernmost Bd record at Raúl Marín Balmaceda.     

Growth Characteristics and Enzyme Activity in Batrachochytrium dendrobatidis Isolates

Eighty soil samples were collected from various sites of Bahrain and screened for presence of keratinophilic fungi using hair baiting techniques for isolation. Thirty-six isolates were recovered and identified. The cultures were identified using macro- and micromorphological features. Their identification was also confirmed by the BLAST search of sequences of ITS1-5.8S-ITS2 rDNA region against the NCBI/Gene bank data and compared with deposited sequences for confirmation. Eight species of five genera were isolated viz. Aphanoascus fulvuscence (8.75%), Aphanoascus punsolae (20.00%), Chrysosporium indicum (2.50%), Chrysosporium tropicum (2.50%), Chrysosporium zonatum (3.75%), Spiromastix warcupii (1.25%), Microsporum gypseum (3.75%), and Trichophyton mentagrophytes (2.50%). In conclusion, our study indicates that keratinophilic fungi do occur in the various soils of Bahrain. Moreover, the narrow diversity and low density of keratinophilic fungi in the investigated soils is expected and is emblematic to other hot arid environments.     

Daphnia in tadpole mesocosms: trophic links and interactions with Batrachochytrium dendrobatidis

1. Amphibians are in decline, and the disease chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has been repeatedly implicated throughout the world. This chytrid reproduces via an infectious, motile zoospore stage that remains viable for weeks in the water column. 2. Daphnia is a keystone zooplankton grazer in intact freshwater ecosystems, whose importance to amphibians may be overlooked. As an efficient grazer, Daphnia can suppress chytrid epidemics by consuming zoospores and may therefore play a role in Bd infection dynamics. Daphnia may also have important effects on tadpoles by mediating the properties of pond food webs. We tested the role of Daphnia in outdoor mesocosms containing the tadpoles of red‐legged frogs (Rana aurora) infected with Bd. We also tested the ability of Daphnia to filter Bd from the water column in laboratory microcosms. 3. In the water of microcosms, Daphnia dramatically decreased the number of Bd genomic equivalents detectable using quantitative PCR. Bd genomic equivalents fell below the limit of detection at very high (>1 Daphnia mL^?1) Daphnia densities. 4. In mesocosms, Daphnia was critical to the development of tadpoles: in the presence of Daphnia, tadpoles were twofold heavier at metamorphosis than in their absence. Daphnia and Bd interacted to affect the tadpole survival: survival was highest in the presence of Daphnia and in the absence of Bd. We were unable to detect an effect of Daphnia on the transmission of Bd in mesocosms. However, Bd transmission among the tadpoles in mesocosms was unexpectedly low, limiting our power to detect an effect of Daphnia on transmission. 5. Tadpole dissection showed that tadpoles also consumed large numbers of Daphnia. Current models of mesocosm food webs that assume no predation by tadpoles on zooplankton therefore probably overlook important features of both natural and experimental systems.     

Predators reduce Batrachochytrium dendrobatidis infection loads in their prey

相似文献   

Environmental detection of Batrachochytrium dendrobatidis in a temperate climate

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.     

Widespread historical presence of Batrachochytrium dendrobatidis in African pipid frogs

Aim Amphibian chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), is associated with global amphibian population declines and species extinctions. Current evidence indicates that the pathogen has recently spread globally from an enzootic focus, with Xenopus spp. (family Pipidae) in South Africa having been identified as a likely source. The aim of this study was to investigate further the likelihood of African Xenopus spp. as the original source of Bd. Location We examined 665 museum specimens of 20 species of African and South American pipid frogs collected between 1844 and 1994 and held in the collection of the Natural History Museum, London. Methods Skin brushings taken from adult amphibians and brushings from the mouthparts, lips and developing hind limbs of larval pipid frogs were examined for the presence of Bd using real‐time PCR. Results We found six cases of Bd infection in three Xenopus spp. (from Africa), but none of the South American pipids was positive, although only 45 South American frogs were available for examination. The earliest case of Bd infection was in a specimen of Xenopus fraseri collected from Cameroon in 1933. A consistently low prevalence of infection over time indicates that a historical equilibrium existed between Xenopus spp. and Bd infection in Africa. Main conclusions Our results suggest that Bd infection was present in Xenopus spp. across sub‐Saharan Africa by the 1930s, providing additional support for the ‘out of Africa’ hypothesis. If this hypothesis is correct, it strengthens the argument for stringent control of human‐assisted movements of amphibians and other wildlife world‐wide to minimize the likelihood of pathogen introduction and disease emergence that can threaten species globally. Our findings help inform species selection for conservation in the face of the current Bd pandemic and also guide future research directions for selecting Bd isolates for sequencing and virulence testing.     

Amphibians Testing Negative for Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans on the Qinghai-Tibetan Plateau,China

A disease caused by the fungi Batrachochytrium dendrobatidis(Bd) and Batrachochytrium salamandrivorans(Bsal) is responsible for recent worldwide declines and extinctions of amphibian populations.The Qinghai-Tibetan Plateau(QTP) is aglobal biodiversity hotspot,yet little is known about the prevalence of Bd and Bsal in this region.In this study,we collected 336 non-invasive skin swabs from wild amphibians(including an exotic amphibian species) on the QTP.In addition,to assess the historical prevalence of Bd and Bsal on the QTP,we collected 117 non-invasive skin swabs from museum-archived amphibian samples(from 1964–1982) originating from the QTP.Our results showed all samples to be negative for Bd and Bsal.The government should ban the potentially harmful introduction of non-native amphibian species to the QTP and educate the public about the impacts of releasing exotic amphibians from chytridinfected areas into native environments of the QTP.     

Transmission of Batrachochytrium dendrobatidis within and between amphibian life stages

Chytridiomycosis is an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis, which has been implicated in amphibian declines worldwide. The mountain yellow-legged frog Rana muscosa is a declining amphibian species that can be infected by B. dendrobatidis; however, transmission between conspecifics has not been documented. Here, we present experimental evidence that R. muscosa tadpoles can be infected by fungal zoospores and that they can transmit infection to each other and to postmetamorphic animals. We compared several techniques for detecting B. dendrobatidis transmission and found that histology with serial sectioning was able to detect infection before cytology or visual inspections. We also show that R. muscosa tadpoles appear healthy with B. dendrobatidis infection, while postmetamorphic animals experience mortality. In addition, we provide guidelines for visually detecting B. dendrobatidis in R. muscosa tadpoles, which may be useful in other affected species. Field surveys of infected and uninfected populations verify this identification technique.     

Early 1900s Detection of Batrachochytrium dendrobatidis in Korean Amphibians

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) is a major conservation concern because of its role in decimating amphibian populations worldwide. We used quantitative PCR to screen 244 museum specimens from the Korean Peninsula, collected between 1911 and 2004, for the presence of Bd to gain insight into its history in Asia. Three specimens of Rugosa emeljanovi (previously Rana or Glandirana rugosa), collected in 1911 from Wonsan, North Korea, tested positive for Bd. Histology of these positive specimens revealed mild hyperkeratosis – a non-specific host response commonly found in Bd-infected frogs – but no Bd zoospores or zoosporangia. Our results indicate that Bd was present in Korea more than 100 years ago, consistent with hypotheses suggesting that Korean amphibians may be infected by endemic Asian Bd strains.     

First Detection of Batrachochytrium dendrobatidis in Wild Frogs from Bangladesh

EcoHealth - Global amphibian populations are facing a novel threat, chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), which is responsible for the severe decline of a...