Distribution of Batrachochytrium dendrobatidis and pathology in the skin of green tree frogs Litoria caerulea with severe chytridiomycosis

Although histopathology is used routinely for diagnosis of chytridiomycosis in live and dead amphibians, there are no quantitative data on the distribution of the causative fungus, Batrachochytrium dendrobatidis, in the skin. We performed quantitative histological examinations on 6 sites on the body and 4 toes of 10 free-ranging adult green tree frogs Litoria caerulea found recently dead or dying from chytridiomycosis. Large numbers of sporangia occurred in all areas of ventral skin and toes; on average there were 94.3 sporangia mm(-1) of superficial epidermis. The number of sporangia was highly variable and this appeared to be related to the stage in the cycle of sloughing. The stratum corneum tends to build up with high intensities of infection and then sheds entirely rather than being shed continuously. Very few or no sporangia occurred on dorsal skin. This distribution could be explained by the dryness of the dorsal skin or possibly by the greater number of serous glands, which produce antifungal peptides, on the dorsum. In some frogs, ulceration and erosions occurred on skin on the back in the absence of sporangia. Other pathological changes such as hyperkeratosis and congestion occurred much more frequently on ventral surfaces.     

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.     

Elevated temperature as a treatment for Batrachochytrium dendrobatidis infection in captive frogs

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines worldwide. In vitro laboratory studies and those done on wild populations indicate that Bd grows best at cool temperatures between 17 and 25 degrees C. In the present study, we tested whether moderately elevating the ambient temperature to 30 degrees C could be an effective treatment for frogs infected with Bd. We acquired 35 bullfrogs Rana catesbeiana from breeding facilities and 36 northern cricket frogs Acris crepitans from the wild and acclimated them to either 23 or 26 degrees C for 1 mo. Following the acclimation period, frogs were tested for the presence of Bd using qPCR TaqMan assays. The 12 R. catesbeiana and 16 A. crepitans that tested positive for Bd were subjected to 30 degrees C for 10 consecutive days before returning frogs to their starting temperatures. Post-treatment testing revealed that 27 of the 28 frogs that had tested positive were no longer infected with Bd; only a single A. crepitans remained infected following treatment. This result indicates that elevating ambient temperature to a moderate 30 degrees C can be effective as a treatment for Bd infection in captive amphibians, and suggests that heat may be a superior alternative to antifungal drugs.     

Seasonality of Batrachochytrium dendrobatidis infection in direct-developing frogs suggests a mechanism for persistence

Batrachochytrium dendrobatidis (Bd), a disease-causing amphibian-specific fungus, is widely distributed in Puerto Rico, but is restricted to elevations above 600 m. The effect of this pathogen in the wild was studied by monitoring Eleutherodactylus coqui and E. portoricensis in 2 upland forests at El Yunque, a site characterized by historic population declines in the presence of chytridiomycosis. We tested a potential synergistic interaction between climate and Bd by measuring prevalence of infection and level of infection per individual sampled (number of zoospores), across the dry and wet seasons for 2 yr (between 2005 and 2007). Infection levels in adult frogs were significantly higher during the dry season in both species studied, suggesting a cyclic pattern of dry/ cool-wet/warm climate-driven synergistic interaction. These results are consistent with ex situ experiments in which E. coqui infected with Bd were more susceptible to chytridiomycosis when subjected to limited water treatments resembling drought. Long-term data on the prevalence of Bd in the populations studied versus intensity of infection in individual frogs provided contradictory information. However, the conflicting nature of these data was essential to understand the status of Bd in the species and geographical area studied. We conclude that in Puerto Rico, Bd is enzootic, and vulnerability of eleutherodactylid frogs to this pathogen is related to seasonal climatic variables. Our data suggest a mechanism by which this disease can persist in tropical frog communities without decimation of its hosts, but that complex interactions during severe droughts may lead to population crashes.     

Production of polyclonal antibodies to Batrachochytrium dendrobatidis and their use in an immunoperoxidase test for chytridiomycosis in amphibians

Polyclonal antibodies were produced for diagnosing chytridiomycosis in amphibians. Two sheep and 4 rabbits were inoculated with homogenized whole culture of Batrachochytrium dendrobatidis in Freund's complete adjuvant or triple adjuvant. Antisera from all animals reacted strongly with all stages of B. dendrobatidis and stained the walls, cytoplasm, rhizoids and zoospores in an indirect immunoperoxidase test. Significant cross-reactivity occurred only with some fungi in the Chytridiomycota, and there are no members of this phylum besides B. dendrobatidis that infect frogs. The immunoperoxidase stain is a useful screening test when combined with recognition of the morphology and infection site of B. dendrobatidis.     

Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide, associated with declines in amphibian populations. Diagnosis of chytridiomycosis to date has largely relied upon histological and immunohistochemical examination of toe clips. This technique is invasive and insensitive particularly at early stages of infection when treatment may be possible. We have developed a real-time PCR Taqman assay that can accurately detect and quantify one zoospore in a diagnostic sample. This assay will assist the early detection of B. dendrobatidis in both captive and wild populations, with a high degree of sensitivity and specificity, thus facilitating treatment and protection of endangered populations, monitoring of pristine environments and preventing further global spread via amphibian trade.     

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.     

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.     

Prevalence of Batrachochytrium dendrobatidis in three species of wild frogs on Prince Edward Island, Canada

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has resulted in the decline or extinction of approximately 200 frog species worldwide. It has been reported throughout much of North America, but its presence on Prince Edward Island (PEI), on the eastern coast of Canada, was unknown. To determine the presence and prevalence of Bd on PEI, skin swabs were collected from 115 frogs from 18 separate sites across the province during the summer of 2009. The swabs were tested through single round end-point PCR for the presence of Bd DNA. Thirty-one frogs were positive, including 25/93 (27%) green frogs Lithobates (Rana) clamitans, 5/20 (25%) northern leopard frogs L. (R.) pipiens, and 1/2 (50%) wood frogs L. sylvaticus (formerly R. sylvatica); 12 of the 18 (67%) sites had at least 1 positive frog. The overall prevalence of Bd infection was estimated at 26.9% (7.2-46.7%, 95% CI). Prevalence amongst green frogs and leopard frogs was similar, but green frogs had a stronger PCR signal when compared to leopard frogs, regardless of age (p < 0.001) and body length (p = 0.476). Amongst green frogs, juveniles were more frequently positive than adults (p = 0.001). Green frogs may be the most reliable species to sample when looking for Bd in eastern North America. The 1 wood frog positive for Bd was found dead from chytridiomycosis; none of the other frogs that were positive for Bd by PCR showed any obvious signs of illness. Further monitoring will be required to determine what effect Bd infection has on amphibian population health on PEI.     

Retreat sites of rain forest stream frogs are not a reservoir for Batrachochytrium dendrobatidis in northern Queensland, Australia

Chytridiomycosis is a potentially fatal disease of amphibians caused by Batrachochytrium dendrobatidis, and is implicated in declines and extinctions of amphibian populations and species around the world. To cause local host extinction, a disease organism must persist at low host densities. One mechanism that could facilitate this is the ability to persist in the environment. In the laboratory, B. dendrobatidis spreads by both frog-to-frog and environment-to-frog transmission, and can persist on a number of biotic substrates. In the field, B. dendrobatidis has been detected on environmental samples taken during an epidemic, but it is not known if it persists in the environment when endemic. Retreat sites of 2 species of Australian rain forest stream frogs Litoria lesueuri and L. nannotis were sampled 0 to 3 d after occupation during the wet and dry seasons in northern Queensland, Australia, where chytridiomycosis has been endemic for at least 10 yr. The intensity and prevalence of infection in frogs during sampling were comparatively low compared with epidemics. Diagnostic quantitative polymerase chain reaction did not detect B. dendrobatidis in any retreat site samples. It thus appears that retreat sites are not a major environmental source of infection when B. dendrobatidis occurs at low prevalence and intensity on frogs. This suggests that control efforts may not need to eliminate the organism from the environment, at least when prevalence and intensity of infection are low in frogs. Simply treating hosts may be effective at controlling the disease in the wild.     

Detection of chytridiomycosis caused by Batrachochytrium dendrobatidis in the endangered Sardinian newt (Euproctus platycephalus) in southern Sardinia, Italy

The chytridiomycete fungus Batrachochytrium dendrobatidis is known to be focally distributed across Europe, but has only been linked to "chytridiomycosis at a few locations in Spain. Here we report the second occurrence of chytridiomycosis in European amphibians. We found a population of endangered Sardinian newts (Euproctus platycephalus) exhibiting clinical signs of disease including loss of digits and patchy, discolored skin. Molecular examination of skin samples tested positive for B. dendrobatidis. The population of E. platycephalus has been in decline on a timescale consistent with the global emergence of chytridiomycosis, and the ecology of this salamander suggests that the disease in this species warrants concern.     

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.     

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.     

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.     

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.     

Experimental transmission of cutaneous chytridiomycosis in dendrobatid frogs

In a series of three experiments during March-October, 1998, two species of captive-bred poison dart frogs (Dendrobates tinctorius and D. auratus) were exposed to Batrachochytrium dendrobatidis, a recently-described chytridiomycete fungus (chytrid) that was originally isolated from a blue poison dart frog (D. azureus). All frogs exposed to the chytrids developed a fatal skin disease, whereas none of the control frogs developed skin lesions. The most consistent clinical sign in chytrid-exposed frogs was excessive shedding of skin. Gross lesions were subtle, usually affected the legs and ventrum, and consisted of mild skin thickening and discoloration. Microscopic examination of shed skin pieces and/or skin imprints demonstrated the presence of chytrids and was used for ante mortem and post mortem confirmation of chytrid infection. Histologically, there was epidermal hyperkeratosis, hyperplasia, and hypertrophy associated with low to moderate numbers of chytrids in the keratinized layers. These experiments demonstrated that Batrachochytrium dendrobatidis can be a fatal pathogen in poison dart frogs. The experimentally-induced disease in these frogs resembled cases of cutaneous chytridiomycosis that have recently been described in several other species of captive and wild amphibians.     

Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis

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.     

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.     

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.     

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.