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.     

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.     

Assessing host extinction risk following exposure to Batrachochytrium dendrobatidis

Wildlife diseases are increasingly recognized as a major threat to biodiversity. Chytridiomycosis is an emerging infectious disease of amphibians caused by the fungus Batrachochytrium dendrobatidis (Bd). Using a mathematical model and simulations, we study its effects on a generic riparian host population with a tadpole and adult life stage. An analytical expression for the basic reproduction quotient, Q_o, of the pathogen is derived. By sampling the entire relevant parameter space, we perform a statistical assessment of the importance of all considered parameters in determining the risk of host extinction, upon exposure to Bd. We find that Q_o not only gives a condition for the initial invasion of the fungus, but is in fact the best predictor for host extinction. We also show that the role of tadpoles, which in some species tolerate infections, is ambivalent. While tolerant tadpoles may provide a reservoir for the fungus, thus facilitating its persistence or even amplifying its outbreaks, they can also act as a rescue buffer for a stressed host population. Our results have important implications for amphibian conservation efforts.     

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.     

Life history tradeoffs influence mortality associated with the amphibian pathogen Batrachochytrium dendrobatidis

Fatal amphibian chytridiomycosis has typically been associated with the direct costs of infection. However the relationship between exposure to the pathogen, infection and mortality may not be so straightforward. Using results from both field work and experiments we report how exposure of common toads to Batrachochytrium dendrobatidis influences development and survival and how developmental stage influences host responses. Our results show that costs are accrued in a dose dependent manner during the larval stage and are expressed at or soon after metamorphosis. Exposure to B. dendrobatidis always incurs a growth cost for tadpoles and can lead to larval mortality before or soon after metamorphosis even when individuals do not exhibit infection at time of death. In contrast, exposure after metamorphosis almost always results in infection, but body size dictates survival to a greater extent than does dose. These data show that amphibian survival in the face of challenge by an infectious agent is dependent on host condition as well as life history stage. Under current models of climate change, many species of amphibia are predicted to increasingly occur outside their environmental optima. In this case, condition-dependent traits such as we have demonstrated may weigh heavily on species survival.     

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.     

Additional disinfectants effective against the amphibian chytrid fungus Batrachochytrium dendrobatidis

Chytridiomycosis, a disease contributing to amphibian declines worldwide, is caused by the fungus Batrachochytrium dendrobatidis. Identifying efficient and practical disinfectants effective against B. dendrobatidis is important to reduce the spread of the disease both in the wild and captivity. Previous studies identified a range of suitable disinfectant strategies. We evaluated the suitability of 3 additional disinfectants: two of these (TriGene Virucidal Disinfectant Cleaner and F10 Super Concentrate Disinfectant) are mixtures of chemicals and one (Betadine Antiseptic Liquid) contains a single active ingredient, povidone iodine. The disinfectants were tested using a range of concentrations for 1,5 and 10 min to determine their ability to kill B. dendrobatidis in vitro. The measure of effectiveness was 100% kill of zoosporangia grown in multiwell plates. All disinfectants had a 100% efficacy at concentrations recommended by the manufacturers. The lowest concentrations capable of 100% kill after exposure for 1 min were 0.1 ml l(-1) for TriGene, 0.33 ml l(-1) for F10 and 100 ml l(-1) for Betadine. TriGene is the most effective disinfectant yet to be found, and both TriGene and F10 are more effective than various disinfectants tested in previous studies. TriGene and F10 are considered suitable for use in the field, as only small amounts of concentrate are needed.     

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.     

Species-level correlates of susceptibility to the pathogenic amphibian fungus Batrachochytrium dendrobatidis in the United States

Disease is often implicated as a factor in population declines of wildlife and plants. Understanding the characteristics that may predispose a species to infection by a particular pathogen can help direct conservation efforts. Recent declines in amphibian populations world-wide are a major conservation issue and may be caused in part by a fungal pathogen, Batrachochytrium dendrobatidis (Bd). We used Random Forest, a machine learning approach, to identify species-level characteristics that may be related to susceptibility to Bd. Our results suggest that body size at maturity, aspects of egg laying behavior, taxonomic order and family, and reliance on water are good predictors of documented infection for species in the continental United States. These results suggest that, whereas local-scale environmental variables are important to the spread of Bd, species-level characteristics may also influence susceptibility to Bd. The relationships identified in this study suggest future experimental tests, and may target species for conservation efforts.     

The chytrid fungus Batrachochytrium dendrobatidis is non-randomly distributed across amphibian breeding habitats

The chytrid fungus Batrachochytrium dendrobatidis has been implicated as the causative agent of mass mortalities, population declines, and the extinctions of stream-breeding amphibian species worldwide. While the factors that limit the distribution and abundance of B. dendrobatidis across large geographical regions are fairly well understood, little is known about the distribution of the fungus within localized areas such as individual catchments. The accurate identification of amphibian populations likely to be exposed to the fungus is urgently required for effective disease management. We conducted disease surveys of frogs representing five ecological guilds in south-east Queensland, Australia, and hypothesized that if B. dendrobatidis were responsible for the disappearance of stream-breeding amphibian populations, infection prevalence and intensity would be greatest in frogs breeding in permanent, flowing water. Overall, 30.3% of the 519 frogs we sampled were infected with B. dendrobatidis . However, infections were not evenly distributed across the ecological guilds, being almost completely restricted to frogs breeding at permanent waterbodies. Of these, stream breeders were significantly more likely to be infected than were pond breeders, though the intensity of frogs' infections did not differ significantly between the two guilds. Batrachochytrium dendrobatidis was detected on only one of the 117 frogs that were found at ephemeral ponds, ephemeral streams, or terrestrial sites. These findings provide strong support for the hypothesis that B. dendrobatidis was responsible for many of the unexplained disappearances of stream-breeding amphibian populations in recent decades, and will enable wildlife managers to more accurately focus conservation efforts on those species at highest risk of disease-related decline.     

Chemotaxis of the amphibian pathogen Batrachochytrium dendrobatidis and its response to a variety of attractants

Batrachochytrium dendrobatidis is a fungal pathogen of amphibians that is increasingly implicated as a major cause of large-scale mortalities of amphibian species worldwide. Previous studies indicate that motile zoospores of B. dendrobatidis colonize the keratinized tissues of susceptible amphibians. Infections spread to adults and cause destruction of epidermal tissue. In an effort to understand how the chytrid cues into its host we developed an assay to study chemotaxis in the fungus. Here we show that zoospores exhibit positive movement toward a variety of attractants including sugars, proteins and amino acids. These observations suggest that the chytrid can respond to nutritional cues, including those of host origin. Implications of these observations to amphibian susceptibility to infection and chytrid virulence are discussed.     

Infection with Batrachochytrium dendrobatidis is common in tropical lowland habitats: Implications for amphibian conservation

Numerous species of amphibians declined in Central America during the 1980s and 1990s. These declines mostly affected highland stream amphibians and have been primarily linked to chytridiomycosis, a deadly disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Since then, the majority of field studies on Bd in the Tropics have been conducted in midland and highland environments (>800 m) mainly because the environmental conditions of mountain ranges match the range of ideal abiotic conditions for Bd in the laboratory. This unbalanced sampling has led researchers to largely overlook host–pathogen dynamics in lowlands, where other amphibian species declined during the same period. We conducted a survey testing for Bd in 47 species (n = 348) in four lowland sites in Costa Rica to identify local host–pathogen dynamics and to describe the abiotic environment of these sites. We detected Bd in three sampling sites and 70% of the surveyed species. We found evidence that lowland study sites exhibit enzootic dynamics with low infection intensity and moderate to high prevalence (55% overall prevalence). Additionally, we found evidence that every study site represents an independent climatic zone, where local climatic differences may explain variations in Bd disease dynamics. We recommend more detection surveys across lowlands and other sites that have been historically considered unsuitable for Bd occurrence. These data can be used to identify sites for potential disease outbreaks and amphibian rediscoveries.     

Non-detection of mycoviruses in amphibian chytrid fungus (Batrachochytrium dendrobatidis) from Australia

Mycoviruses may influence the pathogenicity of disease-causing fungi. Although mycoviruses have been found in some chytrid fungi, limited testing has not detected them in Batrachochytrium dendrobatidis (Bd), the cause of the devastating amphibian disease, chytridiomycosis. Here we conducted a survey for mycovirus presence in 38 Bd isolates from Australia (n = 31), Brazil (n = 5) and South Korea (n = 2) with a combination of modern high-throughput sequencing and conventional dsRNA cellulose chromatography. Mycoviruses were not detected in any isolates. This result was unexpected, given the long evolutionary history of Bd, as well as the high prevalence of mycoviruses in related fungal species. Given our widespread sampling in Australia and the limited number of Bd introductions, we suggest that mycoviruses are uncommon or absent from Australian Bd. Testing more isolates from regions where Bd originated, as well as regions with high diversity or low fungal virulence may identify mycoviruses that could aid in disease control.     

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.     

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.     

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.     

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.     

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

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