Batrachochytrium dendrobatidis in Darwin's frog Rhinoderma spp. in Chile

The presence of the chytrid fungus Batrachochytrium dendrobatidis in Chile was evaluated in 2 endangered frog species of the genus Rhinoderma. Specimens from a captive rearing facility, wild populations and preserved collection material were analyzed using histological and molecular techniques. The fungus was identified in the rearing facility and in wild populations, but not in the archived frogs. This study confirms, for first time, the presence of chytridiomycosis in Rhinoderma darwinii in Chile.     

Presence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in threatened corroboree frog populations in the Australian Alps

Since the early 1980s, the southern corroboree frog Pseudophryne corroboree and northern corroboree frog P. pengilleyi have been in a state of decline from their sub-alpine and high montane bog environments on the southern tablelands of New South Wales, Australia. To date, there has been no adequate explanation as to what is causing the decline of these species. We investigated the possibility that a pathogen associated with other recent frog declines in Australia, the amphibian chytrid fungus Batrachochytrium dendrobatidis, may have been implicated in the decline of the corroboree frogs. We used histology of toe material and real-time PCR of skin swabs to investigate the presence and infection rates with B. dendrobatidis in historic and extant populations of both corroboree frog species. Using histology, we did not detect any B. dendrobatidis infections in corroboree frog populations prior to their decline. However, using the same technique, high rates of infection were observed in populations of both species after the onset of substantial population declines. The real-time PCR screening of skin swabs identified high overall infection rates in extant populations of P. corroboree (between 44 and 59%), while significantly lower rates of infection were observed in low-altitude P. pengilleyi populations (14%). These results suggest that the initial and continued decline of the corroboree frogs may well be attributed to the emergence of B. dendrobatidis in populations of these species.     

Fluorescent microscopy of viable Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis ( Bd ), a chytrid fungus, is a causative agent of chytridiomycosis and amphibian population declines worldwide. The sequenced genome of Bd provides information necessary for studying the fungus and its molecular biology. Fluorescent microscopy is a technique used to image targeted molecules in live or fixed organisms to understand cellular trafficking and localization, but the use of fluorescent microscopy with Bd has not yet been demonstrated. Two fluorescent stains were tested for their use in live-cell imaging of Bd , i.e., the cell wall-specific fluorophore Solophenyl Flavine 7GFE and the DNA-specific fluorophore DRAQ5. These specific staining patterns were observed in live cultures of Bd when visualized with laser-scanning confocal microscopy.     

Retrospective Survey of Museum Specimens Reveals Historically Widespread Presence of Batrachochytrium dendrobatidis in China

Chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has been implicated in amphibian population declines worldwide. However, no amphibian declines or extinctions associated with Bd have been reported in Asia. To investigate the history of this pathogen in China, we examined 1,007 museum-preserved amphibian specimens of 80 species collected between 1933 and 2009. Bd was detected in 60 individuals (6.0%), with the earliest case of Bd infection occurring in one specimen of Bufo gargarizans and two Fejervarya limnocharis, all collected in 1933 from Chongqing, southwest China. Although mainly detected in non-threatened native amphibians, Bd was also found in four endangered species. We report the first evidence of Bd for Taiwan and the first detection of Bd in the critically endangered Chinese giant salamander (Andrias davidianus). Bd appears to have been present at a low rate of infection since at least the 1930s in China, and no significant differences in prevalence were detected between decades or provinces, suggesting that a historical steady endemic relationship between Bd and Chinese amphibians has occurred. Our results add new insights on the global emergence of Bd and suggest that this pathogen has been more widely distributed in the last century than previously believed.     

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.     

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.     

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.     

Survey for the pathogenic chytrid fungus Batrachochytrium dendrobatidis in southwestern North Carolina salamander populations

Batrachochytrium dendrobatidis is a fungal pathogen responsible for a potentially fatal disease of amphibians. We conducted a survey for B. dendrobatidis in the Appalachian Mountains of southwestern North Carolina, USA, from 10 June to 23 July 23 2009. Ventral skin swabs were collected from plethodontid salamanders (n=278) and real-time PCR was performed to test for the presence of B. dendrobatidis. We found no evidence of B. dendrobatidis, suggesting that B. dendrobatidis is absent or present in such low levels that it was undetected. If B. dendrobatidis was present at the time of our sampling, this survey supports evidence of low prevalence of B. dendrobatidis in North American headwater stream salamander populations.     

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.     

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.     

Historic occurrence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in hellbender Cryptobranchus alleganiensis populations from Missouri

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) was recently detected in Missouri hellbender Cryptobranchus alleganiensis populations that have declined precipitously for unclear reasons. The objective of this study was to determine whether Bd occurred historically in Missouri hellbender populations or is a relatively novel occurrence. Epidermal tissue was removed from 216 archived hellbenders collected from 7 Missouri streams between 1896 and 1994. Histological techniques and an immunoperoxidase stain were used to confirm historic occurrence of Bd infection in hellbenders from the North Fork of the White (1969, 1973, 1975), Meramec (1975, 1986), Big Piney (1986), and Current rivers (1988). Bd was not detected in hellbenders from the Niangua, Gasconade or Eleven Point rivers. The study detected no evidence for endemism of Bd in Missouri hellbender populations prior to 1969, despite the fact that nearly one third of the hellbenders sampled were collected earlier. Our findings are consistent with the hypothesis that Bd is a non-endemic pathogen in North America that was introduced in the second half of the twentieth century.     

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.     

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.     

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.     

Magnetic capture hybridization of Batrachochytrium dendrobatidis genomic DNA

We hybridized biotinylated probes that anneal at multiple locations throughout the Batrachochytrium dendrobatidis (Bd) genome to selectively capture Bd genomic DNA (gDNA) by binding the probe-gDNA complex to streptavidin coated magnetic beads. We then whole genome amplified the captured gDNA. This method extends the usefulness of field-collected swabs for downstream PCR-based genomic applications.     

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.     

Predators reduce Batrachochytrium dendrobatidis infection loads in their prey

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