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.     

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.     

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.     

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.     

Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis

An overview of the morphology and life cycle of Batrachochytrium dendrobatidis, the cause of chytridiomycosis of amphibians, is presented. We used a range of methods to examine stages of the life cycle in culture and in frog skin, and to assess ultrastructural pathology in the skin of 2 frogs. Methods included light microscopy, transmission electron microscopy with conventional methods as well as high pressure freezing and freeze substitution, and scanning electron microscopy with critical point drying as well as examination of bulk-frozen and freeze-fractured material. Although chytridiomycosis is an emerging disease, B. dendrobatidis has adaptations that suggest it has long been evolved to live within cells in the dynamic tissue of the stratified epidermis. Sporangia developed at a rate that coincided with the maturation of the cell, and fungal discharge tubes usually opened onto the distal surface of epidermal cells of the stratum corneum. A zone of condensed, fibrillar, host cytoplasm surrounded some sporangia. Hyperkeratosis may be due to (1) a hyperplastic response that leads to an increased turnover of epidermal cells, and (2) premature keratinization and death of infected cells.     

Polymorphic repetitive loci of the amphibian pathogen Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis (Bd), the cause of a fatal fungal skin disease of amphibians that has led to massive die-offs, global declines and extinctions, has spread internationally as a pandemic clone with low genetic diversity. A need exists to develop highly polymorphic markers to determine centers of origin and patterns of spread to assist in the development of management strategies. Comparison of paralogous sequences, obtained from the 2 sequenced Bd genomes, indicates useful levels of inter-strain polymorphism in repetitive fragments. We assessed 6 repetitive loci for variation within and among Australian isolates using standard fragment analysis and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis. Confirmation of inter-isolate polymorphism was achieved for 2 marker systems, highlighting the potential of repetitive loci for the development of polymorphic markers in Bd. In addition, we found that repetitive loci in Bd include possible orthologs of virulence-related genes from pathogenic fungi.     

Batrachochytrium dendrobatidis infection patterns among Panamanian amphibian species, habitats and elevations during epizootic and enzootic stages

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) has caused declines of many amphibian populations, yet the full course of the epizootic has rarely been observed in wild populations. We determined effects of elevation, habitat, and aquatic index (AI) on prevalence of infection among Panamanian amphibians sampled along 2 elevational transects. Amphibian populations on the Santa Fé transect (SFT) had declined in 2002, while those on the El Copé transect (ECT) were healthy until September 2004. In 2004 we sampled Bd along both transects, surveying the SFT 2 yr after decline, and surveying the ECT 4 mo prior to the arrival of Bd, during the epizootic, and 2 mo later. Overall prevalence of Bd along the ECT increased from 0.0 (95% CI 0.00-0.0003) to 0.51 (95% CI 0.48-0.55) over a 3 mo period, accompanied by significant decreases in amphibian abundance and species richness in all habitats. Prevalence of infection on the ECT was highest along riparian transects and at higher elevations, but not among levels of AI. Prevalence of infection on the SFT was highest in pool transects, and at higher elevations, but not among levels of AI. Riparian amphibian abundance and species richness also declined at SFT following detection of Bd in 2002. Variation among species, microenvironmental conditions, and the length of coexistence with Bd may contribute to observed differences in prevalence of Bd and in population response.     

Batrachochytrium dendrobatidis in amphibians confiscated from illegal wildlife trade and used in an ex situ breeding program in Brazil

This paper describes an outbreak of chytridiomycosis affecting a group of Dendrobates tinctorius, a Neotropical anuran species, confiscated from the illegal wildlife trade and housed in a private zoo in Brazil as part of an ex situ breeding program. We examined histological sections of the skin of 30 D. tinctorius and 20 Adelphobates galactonotus individuals. Twenty D. tinctorius (66.7%) and none of the A. galactonotus were positive for Batrachochytrium dendrobatidis (Bd). Multiple development stages of Bd infection were observed. The reasons for the inter-specific difference in the rate of infection could not be determined, and further studies are advised. Because the examined population consisted of confiscated frogs, detailed epidemiological aspects could not be investigated, and the source of the fungus remains uncertain. The existence of ex situ amphibian populations is important for protecting species at higher risk in the wild, and ex situ amphibian conservation and breeding programs in Brazil may be established using confiscated frogs as founders. However, this paper alerts these programs to the urgency of strict quarantine procedures to prevent the introduction of potential pathogens, particularly Bd, into ex situ conservation programs.     

Strain differences in the amphibian chytrid Batrachochytrium dendrobatidis and non-permanent, sub-lethal effects of infection

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is likely the cause of numerous recent amphibian population declines worldwide. While the fungus is generally highly pathogenic to amphibians, hosts express a wide range of responses to infection, probably due to variation among hosts and environmental conditions, but possibly also due to variation in Bd. We investigated variation in Bd by exposing standardized host groups to 2 Bd strains in a uniform environment. All exposed frogs became infected, but subsequent lethal and sub-lethal (weight loss) responses differed among groups. These results demonstrate variation in Bd and suggest variation occurs even at small geographical scales, likely explaining some of the variation in host responses. With lower than expected mortality among infected frogs, we continued our study opportunistically to determine whether or not frogs could recover from chytridiomycosis. Using heat, we cleared infection from half of the surviving frogs, leaving the other half infected, then continued to monitor mortality and weight. Mortality ceased among disinfected frogs but continued among infected frogs. Disinfected frogs gained weight significantly more than infected frogs, to the point of becoming indistinguishable from controls, demonstrating that at least some of the effects of sub-lethal chytridiomycosis on hosts can be non-permanent and reversible.     

Interactions between Batrachochytrium dendrobatidis and its amphibian hosts: a review of pathogenesis and immunity

The fungus Batrachochytrium dendrobatidis (Bd) causes a lethal skin disease of amphibians, chytridiomycosis, which has caused catastrophic amphibian die-offs around the world. This review provides a summary of host characteristics, pathogen characteristics and host-pathogen responses to infection that are important for understanding disease development.     

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.     

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.     

Known and estimated distribution in Mexico of Batrachochytrium dendrobatidis,a pathogenic fungus of amphibians

Chytridiomycosis caused by fungus Batrachochytrium dendrobatidis (Bd) is one of the decline global causes of amphibians. Currently, it is distributed throughout a broad range of climates and ecosystems around the world. An epidemic wave of chytridiomycosis began in North America, resulting in population decline and local extinction of many species, reconfiguring species composition of amphibian communities in the Americas. In Mexico, Bd has caused an amphibian population decrease, and its potential distribution area has not been determined. We reviewed the number of species infected, obtaining Bd frequency of infection by land use and vegetation type, and by elevation range. We examined the known distribution of Bd, estimated the potential distribution, and obtained the bioclimate variables relevant for Bd. Our results indicate that in Mexico, Bd has been detected in 78 species of amphibians in 10 families, from 29 different land use and vegetation types, with cloud forest having the highest number of cases (139) and infected species (15). Bd occurs over an elevation range of 1–3,300 m asl and is most frequent at 1,200–1,500 m asl (36%). In addition to the regions previously described as suitable for Bd, our model included desert, coastal, and tropical forest regions, revealing an increase in the area where amphibians could be at risk of infection. Distribution of Bd is mainly associated with temperature of the wettest quarter and potential evapotranspiration of the warmer quarter. We offer an estimate of the ideal conditions for Bd in Mexico, also information for future studies on Bd and the conservation of amphibians. Abstract in Spanish is available with online material.     

Experimental infection of self-cured Leiopelma archeyi with the amphibian chytrid Batrachochytrium dendrobatidis

The susceptibility of Archey's frog Leiopelma archeyi to Batrachochytrium dendrobatidis (Bd) is unknown, although one large population is thought to have declined sharply due to chytridiomycosis. As primary infection experiments were not permitted in this endangered New Zealand species, 6 wild-caught L. archeyi that naturally cleared infections with Bd while in captivity were exposed again to Bd to assess their immunity. These frogs were from an infected population at Whareorino, which has no known declines. All 6 L. archeyi became reinfected at low intensities, but rapidly self cured, most by 2 wk. Six Litoria ewingii were used as positive controls and developed heavier infections and clinical signs by 3 wk, demonstrating that the zoospore inoculum was virulent. Six negative controls of each species remained uninfected and healthy. Our results show that L. archeyi that have self cured have resistance to chytridiomycosis when exposed. The pattern is consistent with innate or acquired immunity to Bd, and immunological studies are needed to confirm this.     

Possible modes of dissemination of the amphibian chytrid Batrachochytrium dendrobatidis in the environment

Amphibian chytridiomycosis caused by Batrachochytrium dendrobatidis has spread at an alarming rate over large distances throughout sensitive frog populations in eastern Australia, Central America and New Zealand. Infected amphibians and contaminated water are implicated in translocation, but other vectors are unknown. Through in vitro studies we show that potential means of translocation may be moist soil and bird feathers. B. dendrobatidis survived for up to 3 mo in sterile, moist river sand with no other nutrients added. B. dendrobatidis attached to and grew on sterile feathers and were able to be transported by feathers to establish new cultures in media, surviving between 1 and 3 h of drying between transfers. If these in vitro results are valid in the natural environment, the findings raise the possibilities that B. dendrobatidis may be translocated by movement of moist river sand and that birds may carry the amphibian chytrid between frog habitats. However, further studies using sand and feathers containing normal microflora are essential.     

Identification and partial characterization of an elastolytic protease in the amphibian pathogen Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis (Bd) is a fungus that causes chytridiomycosis, a disease that has been implicated as a cause of amphibian population declines worldwide. Infected animals experience hyperkeratosis and sloughing of the epidermis due to penetration of the keratinized tissues by the fungus. These symptoms have led us to postulate that Bd produces proteases that play a role in the infection process. Here, we show that Bd is capable of degrading elastin in vitro, a protein found in the extracellular matrix of the host animal. Elastolytic enzyme activity was partially purified using ion exchange chromatography and size-exclusion filtration from cultures grown in inducing media. The elastolytic activity of the purified fraction had a pH optimum of 8, was strongly inhibited by EDTA and phenylmethylsulfonyl fluoride (PMSF), and was partially inhibited by an elastase-specific inhibitor. This activity was also enhanced by the presence of Mg2+ and Ca2+ but not Zn2+. An antiserum directed against Aspergillus fumigatus serine protease (Alp) was found to react with a polypeptide of approximately 110 kDa from the purified material. Using immunofluorescence, this antiserum was also observed to react with zoospores and sporangia grown on toad skin. These observations suggest that Bd may produce proteases similar to those produced by other pathogenic fungi that are capable of degrading proteins found in the extracellular matrix. The proteolytic activity exhibited in vitro might aid the organism in its ability to colonize and destroy the epidermis of its amphibian host.     

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.     

Biotic and abiotic determinants of Batrachochytrium dendrobatidis infections in amphibians of the Brazilian Atlantic Forest

Latitudinal gradients are linked to the dynamics of infectious diseases. Both prevalence and infection intensity of the amphibian-killing fungus, Batrachochytrium dendrobatidis (Bd), vary with latitude. Here, we tested whether abiotic and biotic factors are associated with Bd infection prevalence and intensity along a large latitudinal gradient across the Brazilian Atlantic Forest. We detected a positive association between infection prevalence and infection intensity with latitude; elevation, temperature and precipitation best explained infection prevalence, while temperature best explained infection intensity. We also detected a positive association between species richness and Bd infections and associations between Bd infections with host reproductive biology and habitat type. This represents the longest and most thoroughly sampled latitudinal gradient of Bd in anuran populations. Our results corroborate earlier findings that abiotic factors are a major determinant of Bd infections and highlight the need for a better understanding of the role that species diversity plays in disease outcomes.     

Detection of Batrachochytrium dendrobatidis in amphibians inhabiting cloud forests and coffee agroecosystems in central Veracruz,Mexico

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is a threat to the survival of amphibians worldwide, a situation that is compounded by several other factors. In this study, we determined the prevalence of Bd and its relationship to biotic and abiotic variables for six amphibian communities in two cloud forest fragments and four coffee agroecosystems in central Veracruz, Mexico. A sampling effort of 768 person-hours and 109 skin swabs resulted in the detection of B. dendrobatidis in four amphibian species belonging to three families. The co-inertia model showed the following as the most important variables: tree density, fern species, temperature and elevation, fragment or site size, and structural index. Conversely, we did not find a clear relationship between Bd prevalence and the habitat management gradient. The highest prevalence was found in the second cloud forest, but a very similar result was found in one of the traditional agroecosystems; the lowest levels of prevalence were found in another second traditional agroecosystem and the first cloud forest. The degree of infection was highest in the cloud forests where the diversity of trees, orchids, and elevation was higher. Ecnomiohyla miotympanum was the most abundant species and was found to be infected in four of the five sites, presenting the highest degree of infection.     

Growth of the amphibian pathogen, Batrachochytrium dendrobatidis, in response to chemical properties of the aquatic environment

Water samples from two of 17 field sites in Arizona (USA) inhibited growth of the amphibian pathogen, Batrachochytrium dendrobatidis. Chemical analyses of samples revealed statistically significant facilitating or inhibitory activity of certain elements. Although low levels of copper were found in environmental samples demonstrating facilitated growth, growth was inhibited at concentrations of copper sulfate (CuSO(4)) at or greater than 100 ppm.