Proteomic and phenotypic profiling of the amphibian pathogen Batrachochytrium dendrobatidis shows that genotype is linked to virulence

Population genetics of the amphibian pathogen Batrachochytrium dendrobatidis ( Bd ) show that isolates are highly related and globally homogenous, data that are consistent with the recent epidemic spread of a previously endemic organism. Highly related isolates are predicted to be functionally similar due to low levels of heritable genetic diversity. To test this hypothesis, we took a global panel of Bd isolates and measured (i) the genetic relatedness among isolates, (ii) proteomic profiles of isolates, (iii) the susceptibility of isolates to the antifungal drug caspofungin, (iv) the variation among isolates in growth and phenotypic characteristics, and (v) the virulence of isolates against the European common toad Bufo bufo . Our results show (i) genotypic differentiation among isolates, (ii) proteomic differentiation among isolates, (iii) no significant differences in susceptibility to caspofungin, (iv) differentiation in growth and phenotypic/morphological characters, and (v) differential virulence in B. bufo . Specifically, our data show that Bd isolates can be profiled by their genotypic and proteomic characteristics, as well as by the size of their sporangia. Bd genotypic and phenotypic distance matrices are significantly correlated, showing that less-related isolates are more biologically unique. Mass spectrometry has identified a set of candidate genes associated with inter-isolate variation. Our data show that, despite its rapid global emergence, Bd isolates are not identical and differ in several important characters that are linked to virulence. We argue that future studies need to clarify the mechanism(s) and rate at which Bd is evolving, and the impact that such variation has on the host–pathogen dynamic.     

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.     

Quantitative Proteomics of an Amphibian Pathogen,Batrachochytrium dendrobatidis,following Exposure to Thyroid Hormone

Batrachochytrium dendrobatidis (Bd), a chytrid fungus, has increasingly been implicated as a major factor in the worldwide decline of amphibian populations. The fungus causes chytridiomycosis in susceptible species leading to massive die-offs of adult amphibians. Although Bd infects the keratinized mouthparts of tadpoles and negatively affects foraging behavior, these infections are non-lethal. An important morphogen controlling amphibian metamorphosis is thyroid hormone (T₃). Tadpoles may be infected with Bd and the fungus may be exposed to T₃ during metamorphosis. We hypothesize that exposure of Bd to T₃ may induce the expression of factors associated with host colonization and pathogenicity. We utilized a proteomics approach to better understand the dynamics of the Bd-T₃ interaction. Using liquid chromatography-mass spectrometry (LC-MS), we generated a data set of a large number of cytoplasmic and membrane proteins following exposure of Bd to T₃. From these data, we identified a total of 263 proteins whose expression was significantly changed following T₃ exposure. We provide evidence for expression of an array of proteins that may play key roles in both genomic and non-genomic actions of T₃ in Bd. Additionally, our proteomics study shows an increase in several proteins including proteases and a class of uncommon crinkler and crinkler-like effector proteins suggesting their importance in Bd pathogenicity as well as those involved in metabolism and energy transfer, protein fate, transport and stress responses. This approach provides insights into the mechanistic basis of the Bd-amphibian interaction following T₃ exposure.     

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.     

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.     

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.     

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.     

Survival of three species of anuran metamorphs exposed to UV-B radiation and the pathogenic fungus Batrachochytrium dendrobatidis

When exploring the possible factors contributing to population declines, it is necessary to consider multiple, interacting environmental stressors. Here, we investigate the impact of 2 factors, ultraviolet radiation and disease, on the survival of anuran amphibians. Exposure to ultraviolet-B (UV-B) radiation increases mortality and results in various sub-lethal effects for many amphibian species. Infectious diseases can also negatively impact amphibian populations. In this study, we exposed metamorphic individuals (metamorphs) to both UV-B and Batrachochytrium dendrobatidis (BD), a fungal pathogen and cause of the disease chytridiomycosis, and monitored survival for 3 wk. We tested for possible interactions between UV-B and BD in 3 species: the Cascades frog Rana cascadae; the Western toad Bufo boreas; and the Pacific treefrog Hyla regilla. We found strong interspecific differences in susceptibility to BD. For example, R. cascadae suffered a large increase in mortality when exposed to BD; B. boreas also experienced mortality, but this effect was small relative to the R. cascadae response. H. regilla did not show any decrease in survival when exposed to either factor. No synergistic interactions between UV-B and BD were found for any of the test species. A previous study investigating the impact of BD on larval amphibians showed different species responses (Blaustein et al. 2005a). Our results highlight the importance of studying multiple life history stages when determining the impact of environmental stressors. The contrast between these 2 studies emphasizes how vulnerability to a pathogen can vary between life history stages within a single species.     

Cell Counting and Viability Assessment of 2D and 3D Cell Cultures: Expected Reliability of the Trypan Blue Assay

Background

Whatever the target of an experiment in cell biology, cell counting and viability assessment are always computed. The Trypan Blue (TB) assay was proposed about a century ago and is still the most widely used method to perform cell viability analysis. Furthermore, the combined use of TB with a haemocytometer is also considered the standard approach to estimate cell population density. There are numerous research articles reporting the use of TB assays to compute cell number and viability of 2D and 3D cultures. However, the literature still lacks studies regarding the reliability of the TB assay in terms of assessment of its repeatability and reproducibility.

Methods

We compared the TB assay's measurements obtained by two biologists who analysed 105 different samples in double-blind for a total of 210 counts performed. We measured: (a) the repeatability of the count performed by the same operator; (b) the reproducibility of counts performed by the two operators.

Results

There were no significant differences in the results obtained with 2D and 3D cell cultures: we estimated an approximate variability of 5% when the TB assay was used to assess the viability of the culture, and a variability of around 20% when it was used to determine the cell population density.

Conclusions

The main aim of this study was to make researchers aware of potential measurement errors when TB is used with a haemocytometer for counting and viability measurements in 2D and 3D cultures. We believe that these results can help researchers to determine whether the expected reliability of the TB assay is compliant with their applications.

     

Qualitative risk analysis of introducing Batrachochytrium dendrobatidis to the UK through the importation of live amphibians

The international amphibian trade is implicated in the emergence and spread of the amphibian fungal disease chytridiomycosis, which has resulted in amphibian declines and extinctions globally. The establishment of the causal pathogen, Batrachochytrium dendrobatidis (Bd), in the UK could negatively affect the survival of native amphibian populations. In recognition of the ongoing threat that it poses to amphibians, Bd was recently included in the World Organisation for Animal Health Aquatic Animal Health Code, and therefore is in the list of international notifiable diseases. Using standardised risk analysis guidelines, we investigated the likelihood that Bd would be introduced to and become established in wild amphibians in the UK through the importation of live amphibians. We obtained data on the volume and origin of the amphibian trade entering the UK and detected Bd infection in amphibians being imported for the pet and private collection trade and also in amphibians already held in captive pet, laboratory and zoological collections. We found that current systems for recording amphibian trade into the UK underestimate the volume of non-European Union trade by almost 10-fold. We identified high likelihoods of entry, establishment and spread of Bd in the UK and the resulting major overall impact. Despite uncertainties, we determined that the overall risk estimation for the introduction of Bd to the UK through the importation of live amphibians is high and that risk management measures are required, whilst ensuring that negative effects on legal trade are minimised.     

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.     

Terrestrial Dispersal and Potential Environmental Transmission of the Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis)

Dispersal and exposure to amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd) is not confined to the aquatic habitat, but little is known about pathways that facilitate exposure to wild terrestrial amphibians that do not typically enter bodies of water. We explored the possible spread of Bd from an aquatic reservoir to terrestrial substrates by the emergence of recently metamorphosed infected amphibians and potential deposition of Bd-positive residue on riparian vegetation in Cusuco National Park, Honduras (CNP). Amphibians and their respective leaf perches were both sampled for Bd presence and the pathogen was detected on 76.1% (35/46) of leaves where a Bd-positive frog had rested. Although the viability of Bd detected on these leaves cannot be discerned from our quantitative PCR results, the cool air temperature, closed canopy, and high humidity of this cloud forest environment in CNP is expected to encourage pathogen persistence. High prevalence of infection (88.5%) detected in the recently metamorphosed amphibians and frequent shedding of Bd-positive residue on foliage demonstrates a pathway of Bd dispersal between aquatic and terrestrial habitats. This pathway provides the opportunity for environmental transmission of Bd among and between amphibian species without direct physical contact or exposure to an aquatic habitat.     

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.     

Field and laboratory studies of the susceptibility of the green treefrog (Hyla cinerea) to Batrachochytrium dendrobatidis infection

Amphibians worldwide are experiencing devastating declines, some of which are due to the amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd). Populations in the southeastern United States, however, have not been noticeably affected by the pathogen. The green treefrog (Hyla cinerea) is abundant and widespread in the southeastern United States, but has not been documented to harbor Bd infection. This study examined the susceptibility of H. cinerea to two strains of Bd in the lab and the prevalence of infection in wild populations of this species in southeastern Louisiana. Although we were able to infect H. cinerea with Bd in the lab, we did not observe any clinical signs of chytridiomycosis. Furthermore, infection by Bd does not appear to negatively affect body condition or growth rate of post-metamorphic individuals. We found no evidence of infection in surveys of wild H. cinerea. Our results suggest that H. cinerea is not susceptible to chytridiomycosis post-metamorphosis and probably is not an important carrier of the fungal pathogen Bd in the southeastern United States, although susceptibility at the larval stage remains unknown.     

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.     

Heterogeneous Occupancy and Density Estimates of the Pathogenic Fungus Batrachochytrium dendrobatidis in Waters of North America

Biodiversity losses are occurring worldwide due to a combination of stressors. For example, by one estimate, 40% of amphibian species are vulnerable to extinction, and disease is one threat to amphibian populations. The emerging infectious disease chytridiomycosis, caused by the aquatic fungus Batrachochytrium dendrobatidis (Bd), is a contributor to amphibian declines worldwide. Bd research has focused on the dynamics of the pathogen in its amphibian hosts, with little emphasis on investigating the dynamics of free-living Bd. Therefore, we investigated patterns of Bd occupancy and density in amphibian habitats using occupancy models, powerful tools for estimating site occupancy and detection probability. Occupancy models have been used to investigate diseases where the focus was on pathogen occurrence in the host. We applied occupancy models to investigate free-living Bd in North American surface waters to determine Bd seasonality, relationships between Bd site occupancy and habitat attributes, and probability of detection from water samples as a function of the number of samples, sample volume, and water quality. We also report on the temporal patterns of Bd density from a 4-year case study of a Bd-positive wetland. We provide evidence that Bd occurs in the environment year-round. Bd exhibited temporal and spatial heterogeneity in density, but did not exhibit seasonality in occupancy. Bd was detected in all months, typically at less than 100 zoospores L⁻¹. The highest density observed was ∼3 million zoospores L⁻¹. We detected Bd in 47% of sites sampled, but estimated that Bd occupied 61% of sites, highlighting the importance of accounting for imperfect detection. When Bd was present, there was a 95% chance of detecting it with four samples of 600 ml of water or five samples of 60 mL. Our findings provide important baseline information to advance the study of Bd disease ecology, and advance our understanding of amphibian exposure to free-living Bd in aquatic habitats over time.     

Effect of temperature on host response to Batrachochytrium dendrobatidis infection in the mountain yellow-legged frog (Rana muscosa)

The pathogenic chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis, has been implicated in declines of amphibian populations throughout the world, including declines and extinctions of local populations of mountain yellow-legged frogs, Rana muscosa, in the California Sierra Nevada. Previous studies have shown B. dendrobatidis achieves its maximum growth rate in culture in the temperature range of 17-25 C, and exposure to very high temperatures can clear frogs of B. dendrobatidis infection. Here we present the results of a laboratory experiment in which experimentally infected R. muscosa tadpoles were followed through metamorphosis at temperatures of 17 and 22 C. All infected animals developed clinical disease within a similar time frame. However, frogs housed at 22 C exhibited a significantly lower mortality than those housed at 17 C. Within 35 days after metamorphosis, 50% of the frogs housed at 22 C died, while 95% of the frogs housed at 17 C died. Clinical signs subsided in the surviving frogs at 22 C, despite persistent infection. Because both temperatures are within the optimal thermal range for growth of B. dendrobatidis, we propose that the difference in outcome indicates the effect of temperature on the host's resistance to chytridiomycosis, rather than an effect on the fungus alone.