Prevalence of Batrachochytrium dendrobatidis in American bullfrog and southern leopard frog larvae from wetlands on the Savannah River Site, South Carolina

Batrachochytrium dendrobatidis, an aquatic fungus, has been linked to recent amphibian population declines. Few surveys have assessed B. dendrobatidis infections in areas where the disease is suggested to be less virulent and population declines have not been observed, such as southeastern North America. Although adult Rana catesbeiana and Rana sphenocephala from the Savannah River Site, South Carolina collected in 1979 and 1982 were identified as having B. dendrobatidis, it is unknown whether the fungus is currently present at the site or if susceptibility to infection varies among species or wetlands with different histories of environmental contamination. From 15 May through 15 August 2004, we collected R. catesbeiana and R. sphenocephala tadpoles from three wetlands with differing contamination histories on the Savannah River Site, South Carolina. We found B. dendrobatidis in only one of the wetlands we surveyed. Batrachochytrium dendrobatidis infection was identified in 64% of the R. catesbeiana tadpoles sampled and histologically assessed (n=50) from a wetland contaminated with mercury, copper, and zinc. No R. sphenocephala tadpoles from this site (n=50) were infected. In combination with a recently published report, our data suggest that B. dendrobatidis has been present at the Savannah River Site for over 25 yr but has not caused any apparent population declines. This time period is similar to the known presence of 30 yr of B. dendrobatidis in northeastern North America. Our data suggest that R. sphenocephala larvae might be resistant to infection, even when occupying the same wetland as the infected R. catesbeiana. Our survey did not clarify the effects of environmental contamination on infection severity, but our study stresses the importance of additional field surveys to document how this pathogen is affecting amphibians globally.     

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.     

Reptiles as potential vectors and hosts of the amphibian pathogen Batrachochytrium dendrobatidis in Panama

Chytridiomycosis, the disease caused by Batrachochytrium dendrobatidis, is considered to be a disease exclusively of amphibians. However, B. dendrobatidis may also be capable of persisting in the environment, and non-amphibian vectors or hosts may contribute to disease transmission. Reptiles living in close proximity to amphibians and sharing similar ecological traits could serve as vectors or reservoir hosts for B. dendrobatidis, harbouring the organism on their skin without succumbing to disease. We surveyed for the presence of B. dendrobatidis DNA among 211 lizards and 8 snakes at 8 sites at varying elevations in Panama where the syntopic amphibians were at pre-epizootic, epizootic or post-epizootic stages of chytridiomycosis. Detection of B. dendrobatidis DNA was done using qPCR analysis. Evidence of the amphibian pathogen was present at varying intensities in 29 of 79 examined Anolis humilis lizards (32%) and 9 of 101 A. lionotus lizards (9%), and in one individual each of the snakes Pliocercus euryzonus, Imantodes cenchoa, and Nothopsis rugosus. In general, B. dendrobatidis DNA prevalence among reptiles was positively correlated with the infection prevalence among co-occurring anuran amphibians at any particular site (r = 0.88, p = 0.004). These reptiles, therefore, may likely be vectors or reservoir hosts for B. dendrobatidis and could serve as disease transmission agents. Although there is no evidence of B. dendrobatidis disease-induced declines in reptiles, cases of coincidence of reptile and amphibian declines suggest this potentiality. Our study is the first to provide evidence of non-amphibian carriers for B. dendrobatidis in a natural Neotropical environment.     

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.     

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.     

Surveillance for batrachochytrium dendrobatidis using Mixophyes (Anura: Myobatrachidae) larvae

Fourteen populations of anuran larvae (tadpoles), including three populations of the endangered Fleay's Barred Frog (Mixophyes fleayi) and 11 populations of the common Great Barred Frog (Mixophyes fasciolatus), in creek sites in the southeast region of Queensland were selected. Site selection was based on a history (within the district) of adult frog population declines and/or disappearances or records of infection of adult frogs or larvae by Batrachochytrium dendrobatidis. Larvae were collected once from each creek site between October 2002 and October 2004, and were between Gosner developmental stages 25 and 40. Total body length ranged from 18 mm to 100 mm. Mouthparts were examined under a dissecting microscope for grossly visible abnormalities, and then examined for histologic evidence of B. dendrobatidis. The most consistent mouthpart abnormalities found were multifocal depigmentation of the jaw sheaths and loss or shortening of the tooth rows. At the individual larva level, presence of mouthpart abnormalities was strongly associated with histologic diagnosis of B. dendrobatidis (93%). At least one larva with abnormal mouthparts was detected at 12 of the 14 sites and histologic evidence of B. dendrobatidis was detected at 13 of the 14 sites. These findings suggest that larvae of Mixophyes species are suitable for surveillance for B. dendrobatidis. We conclude that surveillance of B. dendrobatidis where individual larva prevalences of mouthpart abnormalities and histologic evidence of B. dendrobatidis are as high as those observed in this study (66% and 78%, respectively), relatively small numbers of larvae are required to detect these infections. Medium to large larvae (body length >30 mm) were much more likely to be affected than small larvae (body length < or =30 mm), suggesting that larger individuals should be targeted for surveillance.     

Early-spring survey for Batrachochytrium dendrobatidis in wild Rana dybowskii in Heilongjiang Province, China

Batrachochytrium dendrobatidis has been investigated worldwide because of its importance in population declines in multiple species of amphibians; however, little is known regarding the disease status of all native amphibian species in China. The present study is the first survey of chytridiomycosis in free-ranging amphibian populations in China, and it examined the possible presence of B. dendrobatidis in Rana dybowskii in northeastern China (Heilongjiang Province). R. dybowskii is mainly distributed in the northeast part of China and is intensively hunted for human consumption, making populations vulnerable to extirpation in the event of additional stresses from disease epidemics. The survey was performed in early spring of 2008, using a PCR assay, histological examination of skin samples, and zoospore culture. In total, 191 frogs were examined; thus, a 95% confidence limit for prevalence of 1.57% was selected. Our results demonstrate that R. dybowskii is currently free from chytridiomycosis in Heilongjiang, even though the natural conditions of the sampling sites are suitable for the occurrence of B. dendrobatidis. Central and local governments should implement strict management measures to prevent the escape of non-native commercial amphibian species into this area, which might endanger local populations of native species.     

Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians

Batrachochytrium dendrobatidis is a pathogen of amphibians that has been implicated in severe population declines on several continents. We investigated the zoospore activity, physiology and protease production of B. dendrobatidis to help understand the epidemiology of this pathogen. More than 95% of zoospores stopped moving within 24 h and swam less than 2 cm before encysting. Isolates of B. dendrobatidis grew and reproduced at temperatures of 4-25 C and at pH 4-8. Growth was maximal at 17-25 C and at pH 6-7. Exposure of cultures to 30 C for 8 d killed 50% of the replicates. B. dendrobatidis cultures grew on autoclaved snakeskin and 1% keratin agar, but they grew best in tryptone or peptonized milk and did not require additional sugars when grown in tryptone. B. dendrobatidis produced extracellular proteases that degraded casein and gelatin but had no measurable activity against keratin azure. The proteases were active against azocasein at temperatures of 6-37 C and in a pH range of 6-8, with the highest activity at temperatures of 23-30 C and at pH 8. The implications of these observations on disease transmission and development are discussed.     

Transmission of Batrachochytrium dendrobatidis to wood frogs (Lithobates sylvaticus) via a bullfrog (L. catesbeianus) vector

Chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis, threatens anuran populations worldwide. Effects of B. dendrobatidis on frog species are variable. Some species typically develop nonlethal infections and may function as carriers; others typically develop lethal infections that can lead to population declines. Nonlethal infections in the bullfrog (Lithobates catesbeianus) are well-documented. In contrast, recently metamorphosed wood frogs (L. sylvaticus) can die from chytridiomycosis. We conducted an ex-situ experiment between May and July 2010 to determine whether B. dendrobatidis-infected bullfrogs could transmit the fungus to wood frog tadpoles when the two species shared a body of water. We tested for B. dendrobatidis infections with quantitative polymerase chain reactions (qPCR) in a subsample of the wood frog tadpoles and in all metamorphosed wood frogs and compared risk of death of froglets exposed and unexposed to infected bullfrogs. We detected B. dendrobatidis sporadically in subsampled treatment tadpoles (nine of 90, 10%) and frequently in treatment froglets (112 of 113, 99.1%). Pooled risk of froglet death was higher (P<0.001) in treatment enclosures than in control enclosures. Our results indicate that, at the low infection loads bullfrogs tend to carry, swabbing for PCR analyses may underestimate prevalence of B. dendrobatidis in this species. We highlight bullfrog disease screening as a management challenge, especially in light of exotic bullfrog colonies on multiple continents and large-scale global trade in this species. We document the importance of quantifying lethal and sublethal effects of bullfrog vectors on B. dendrobatidis-susceptible species.     

The emerging amphibian pathogen Batrachochytrium dendrobatidis globally infects introduced populations of the North American bullfrog, Rana catesbeiana

Batrachochytrium dendrobatidis is the chytridiomycete fungus which has been implicated in global amphibian declines and numerous species extinctions. Here, we show that introduced North American bullfrogs (Rana catesbeiana) consistently carry this emerging pathogenic fungus. We detected infections by this fungus on introduced bullfrogs from seven of eight countries using both PCR and microscopic techniques. Only native bullfrogs from eastern Canada and introduced bullfrogs from Japan showed no sign of infection. The bullfrog is the most commonly farmed amphibian, and escapes and subsequent establishment of feral populations regularly occur. These factors taken together with our study suggest that the global threat of B. dendrobatidis disease transmission posed by bullfrogs is significant.     

Relationships among size, development, and Batrachochytrium dendrobatidis infection in African tadpoles

The fungal pathogen Batrachochytrium dendrobatidis contributes to the global decline of amphibians. Although mortality from B. dendrobatidis infections occurs primarily in postmetamorphic individuals, infected tadpoles may suffer reduced growth and developmental rates as a result of oral chytridiomycosis, possibly affecting adult fitness. We conducted a field study in which we examined South African tadpoles for oral chytridiomycosis and compared the body sizes of infected and uninfected individuals of 2 species, Heleophryne natalensis and Strongylopus hymenopus. Presence of B. dendrobatidis was determined by microscopic inspection of mouthparts. Infection prevalence was high in both species, 62.5 and 38.6%, respectively, and infected individuals were significantly larger in both species. The inclusion of developmental stage in the analysis of S. hymenopus body size eliminated the relationship between body size and infection status, suggesting that differences in body size were not due to differences in growth, but to differences in developmental stage of infected larvae. These results suggest that larvae at more advanced developmental stages are more likely to be infected with B. dendrobatidis and that infection in larval amphibians may be dependent on time or developmental status of larvae. Contrary to the results of past studies, there was no evidence that oral chytridiomycosis resulted in decreased growth of tadpoles, despite the occurrence of oral abnormalities in infected individuals of 1 species. Because tadpole performance can subsequently affect the health of anuran populations and because tadpoles can act as reservoirs of infection, the study of B. dendrobatidis in larval amphibians is important to understanding the effects of this emerging disease.     

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.     

Fast quantitative PCR, locked nucleic acid probes and reduced volume reactions are effective tools for detecting Batrachochytrium dendrobatidis DNA

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.     

Persistence of the emerging pathogen Batrachochytrium dendrobatidis outside the amphibian host greatly increases the probability of host extinction

Pathogens do not normally drive their hosts to extinction; however, Batrachochytrium dendrobatidis, which causes amphibian chytridiomycosis, has been able to do so. Theory predicts that extinction can be caused by long-lived or saprobic free-living stages. The hypothesis that such a stage occurs in B. dendrobatidis is supported by the recent discovery of an apparently encysted form of the pathogen. To investigate the effect of a free-living stage of B. dendrobatidis on host population dynamics, a mathematical model was developed to describe the introduction of chytridiomycosis into a breeding population of Bufo bufo, parametrized from laboratory infection and transmission experiments. The model predicted that the longer that B. dendrobatidis was able to persist in water, either due to an increased zoospore lifespan or saprobic reproduction, the more likely it was that it could cause local B. bufo extinction (defined as decrease below a threshold level). Establishment of endemic B. dendrobatidis infection in B. bufo, with severe host population depression, was also possible, in agreement with field observations. Although this model is able to predict clear trends, more precise predictions will only be possible when the life history of B. dendrobatidis, including free-living stages of the life cycle, is better understood.     

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.     

Waterfowl: potential environmental reservoirs of the chytrid fungus Batrachochytrium dendrobatidis

Infections with Batrachochytrium dendrobatidis (B. dendrobatidis), the causal agent of chytridiomycosis, have been shown to play an important role in the decline of amphibians worldwide. Spread of the fungus is poorly understood. Bird movement might possibly contribute to the spread of B. dendrobatidis in the environment. Therefore, 397 wild geese in Belgium were screened for presence of B. dendrobatidis on their toes using real-time quantitative PCR (qPCR). In addition, chemotaxis towards, adhesion, survival after desiccation and proliferation of B. dendrobatidis on keratinous toe scales from waterfowl were examined in vitro. qPCR revealed that 76 geese (15%) were positive for B. dendrobatidis. Results of the in vitro tests showed that B. dendrobatidis is attracted to the keratinous toes of aquatic birds on which they can adhere and even proliferate. However, desiccation is poorly tolerated. This suggests waterfowl are potential environmental reservoirs for B. dendrobatidis.     

Effects of an infectious fungus, Batrachochytrium dendrobatidis, on amphibian predator-prey interactions

The effects of parasites and pathogens on host behaviors may be particularly important in predator-prey contexts, since few animal behaviors are more crucial for ensuring immediate survival than the avoidance of lethal predators in nature. We examined the effects of an emerging fungal pathogen of amphibians, Batrachochytrium dendrobatidis, on anti-predator behaviors of tadpoles of four frog species. We also investigated whether amphibian predators consumed infected prey, and whether B. dendrobatidis caused differences in predation rates among prey in laboratory feeding trials. We found differences in anti-predator behaviors among larvae of four amphibian species, and show that infected tadpoles of one species (Anaxyrus boreas) were more active and sought refuge more frequently when exposed to predator chemical cues. Salamander predators consumed infected and uninfected tadpoles of three other prey species at similar rates in feeding trials, and predation risk among prey was unaffected by B. dendrobatidis. Collectively, our results show that even sub-lethal exposure to B. dendrobatidis can alter fundamental anti-predator behaviors in some amphibian prey species, and suggest the unexplored possibility that indiscriminate predation between infected and uninfected prey (i.e., non-selective predation) could increase the prevalence of this widely distributed pathogen in amphibian populations. Because one of the most prominent types of predators in many amphibian systems is salamanders, and because salamanders are susceptible to B. dendrobatidis, our work suggests the importance of considering host susceptibility and behavioral changes that could arise from infection in both predators and prey.     

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.     

Sodium hypochlorite denatures the DNA of the amphibian chytrid fungus Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis, an aquatic amphibian fungus, has been implicated in many amphibian declines and extinctions. A real-time polymerase chain reaction (PCR) TaqMan assay is now used to detect and quantify B. dendrobatidis on amphibians and other substrates via tissue samples, swabbing and filtration. The extreme sensitivity of this diagnostic test makes it necessary to rigorously avoid cross-contamination of samples, which can produce false positives. One technique used to eliminate contamination is to destroy the contaminating DNA by chemical means. We tested 3 concentrations of sodium hypochlorite (NaOCl) (1, 6 and 12%) over 4 time periods (1, 6, 15 and 24 h) to determine if NaOCl denatures B. dendrobatidis DNA sufficiently to prevent its recognition and amplification in PCR tests for the fungus. Soaking in 12% NaOCl denatured 100% of DNA within 1 h. Six percent NaOCl was on average 99.999% effective across all exposure periods, with only very low numbers of zoospores detected following treatment. One percent NaOCl was ineffective across all treatment periods. Under ideal, clean conditions treatment with 6% NaOCl may be sufficient to destroy DNA and prevent cross-contamination of samples; however, we recommend treatment with 12% NaOCl for 1 h to be confident all B. dendrobatidis DNA is destroyed.