Amphibian Pathogen Batrachochytrium dendrobatidis Is Inhibited by the Cutaneous Bacteria of Amphibian Species

Population declines of amphibian species in many parts of the world are associated with a lethal fungal pathogen, Batrachochytrium dendrobatidis. Using laboratory challenge assays, we describe the inhibition of B. dendrobatidis by members of eight genera of bacteria isolated from the skin of two amphibian species that exhibit parental care behavior (Plethodon cinereus and Hemidactylium scutatum). We found that members of three genera of bacteria isolated from the skins of the salamander P. cinereus and members of seven genera isolated from the salamander H. scutatum inhibited the growth of B. dendrobatidis. Understanding how B. dendrobatidis interacts with an ecological community of cutaneous flora may be important in explaining and preventing amphibian population declines.     

Germ Tube Mediated Invasion of Batrachochytrium dendrobatidis in Amphibian Skin Is Host Dependent

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis, Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro, comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea.Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus.In X. laevis, interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.     

Mapping the Global Emergence of Batrachochytrium dendrobatidis,the Amphibian Chytrid Fungus

The rapid worldwide emergence of the amphibian pathogen Batrachochytrium dendrobatidis (Bd) is having a profound negative impact on biodiversity. However, global research efforts are fragmented and an overarching synthesis of global infection data is lacking. Here, we provide results from a community tool for the compilation of worldwide Bd presence and report on the analyses of data collated over a four-year period. Using this online database, we analysed: 1) spatial and taxonomic patterns of infection, including amphibian families that appear over- and under-infected; 2) relationships between Bd occurrence and declining amphibian species, including associations among Bd occurrence, species richness, and enigmatic population declines; and 3) patterns of environmental correlates with Bd, including climate metrics for all species combined and three families (Hylidae, Bufonidae, Ranidae) separately, at both a global scale and regional (U.S.A.) scale. These associations provide new insights for downscaled hypothesis testing. The pathogen has been detected in 52 of 82 countries in which sampling was reported, and it has been detected in 516 of 1240 (42%) amphibian species. We show that detected Bd infections are related to amphibian biodiversity and locations experiencing rapid enigmatic declines, supporting the hypothesis that greater complexity of amphibian communities increases the likelihood of emergence of infection and transmission of Bd. Using a global model including all sampled species, the odds of Bd detection decreased with increasing temperature range at a site. Further consideration of temperature range, rather than maximum or minimum temperatures, may provide new insights into Bd-host ecology. Whereas caution is necessary when interpreting such a broad global dataset, the use of our pathogen database is helping to inform studies of the epidemiology of Bd, as well as enabling regional, national, and international prioritization of conservation efforts. We provide recommendations for adaptive management to enhance the database utility and relevance.     

ITS1 Copy Number Varies among Batrachochytrium dendrobatidis Strains: Implications for qPCR Estimates of Infection Intensity from Field-Collected Amphibian Skin Swabs

Genomic studies of the amphibian-killing fungus (Batrachochytrium dendrobatidis, [Bd]) identified three highly divergent genetic lineages, only one of which has a global distribution. Bd strains within these linages show variable genomic content due to differential loss of heterozygosity and recombination. The current quantitative polymerase chain reaction (qPCR) protocol to detect the fungus from amphibian skin swabs targets the intergenic transcribed spacer 1 (ITS1) region using a TaqMan fluorescent probe specific to Bd. We investigated the consequences of genomic differences in the quantification of ITS1 from eight distinct Bd strains, including representatives from North America, South America, the Caribbean, and Australia. To test for potential differences in amplification, we compared qPCR standards made from Bd zoospore counts for each strain, and showed that they differ significantly in amplification rates. To test potential mechanisms leading to strain differences in qPCR reaction parameters (slope and y-intercept), we: a) compared standard curves from the same strains made from extracted Bd genomic DNA in equimolar solutions, b) quantified the number of ITS1 copies per zoospore using a standard curve made from PCR-amplicons of the ITS1 region, and c) cloned and sequenced PCR-amplified ITS1 regions from these same strains to verify the presence of the probe site in all haplotypes. We found high strain variability in ITS1 copy number, ranging from 10 to 144 copies per single zoospore. Our results indicate that genome size might explain strain differences in ITS1 copy number, but not ITS1 sequence variation because the probe-binding site and primers were conserved across all haplotypes. For standards constructed from uncharacterized Bd strains, we recommend the use of single ITS1 PCR-amplicons as the absolute standard in conjunction with current quantitative assays to inform on copy number variation and provide universal estimates of pathogen zoospore loads from field-caught amphibians.     

Amphibian Symbiotic Bacteria Do Not Show a Universal Ability To Inhibit Growth of the Global Panzootic Lineage of Batrachochytrium dendrobatidis

Microbiomes associated with multicellular organisms influence the disease susceptibility of hosts. The potential exists for such bacteria to protect wildlife from infectious diseases, particularly in the case of the globally distributed and highly virulent fungal pathogen Batrachochytrium dendrobatidis of the global panzootic lineage (B. dendrobatidis GPL), responsible for mass extinctions and population declines of amphibians. B. dendrobatidis GPL exhibits wide genotypic and virulence variation, and the ability of candidate probiotics to restrict growth across B. dendrobatidis isolates has not previously been considered. Here we show that only a small proportion of candidate probiotics exhibited broad-spectrum inhibition across B. dendrobatidis GPL isolates. Moreover, some bacterial genera showed significantly greater inhibition than others, but overall, genus and species were not particularly reliable predictors of inhibitory capabilities. These findings indicate that bacterial consortia are likely to offer a more stable and effective approach to probiotics, particularly if related bacteria are selected from genera with greater antimicrobial capabilities. Together these results highlight a complex interaction between pathogens and host-associated symbiotic bacteria that will require consideration in the development of bacterial probiotics for wildlife conservation. Future efforts to construct protective microbiomes should incorporate bacteria that exhibit broad-spectrum inhibition of B. dendrobatidis GPL isolates.     

The Amphibian Chytrid Fungus, Batrachochytrium dendrobatidis, in Fully Aquatic Salamanders from Southeastern North America

Little is known about the impact that the pathogenic amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), has on fully aquatic salamander species of the eastern United States. As a first step in determining the impacts of Bd on these species, we aimed to determine the prevalence of Bd in wild populations of fully aquatic salamanders in the genera Amphiuma, Necturus, Pseudobranchus, and Siren. We sampled a total of 98 salamanders, representing nine species from sites in Florida, Mississippi, and Louisiana. Overall, infection prevalence was found to be 0.34, with significant differences among genera but no clear geographic pattern. We also found evidence for seasonal variation, but additional sampling throughout the year is needed to clarify this pattern. The high rate of infection discovered in this study is consistent with studies of other amphibians from the southeastern United States. Coupled with previously published data on life histories and population densities, the results presented here suggest that fully aquatic salamanders may be serving as important vectors of Bd and the interaction between these species and Bd warrants additional research.     

Trypan Blue Dye is an Effective and Inexpensive Way to Determine the Viability of Batrachochytrium dendrobatidis Zoospores

Batrachochytrium dendrobatidis (Bd) has been implicated in hundreds of amphibian declines and is the focus of a vast amount of research. Despite this, there is no reported efficient way to assess Bd viability. Discriminating between live and dead Bd would help determine the dose of live Bd zoospores and whether factors have lethal or sublethal effects on Bd. We tested whether trypan blue, a common stain to discriminate live and dead cells, could be used to assess Bd viability. We show that the proportion of live zoospores (zoospores that excluded the trypan blue dye) matched the proportion of known live zoospores added to cultures. In contrast, all of the zoosporangia stages of Bd stained blue. These results demonstrate that trypan blue can be used to determine the viability of Bd zoospores but not zoosporangia. We recommend using trypan blue to report the number of live zoospores to which hosts are exposed.     

Predicting the Distribution of the Amphibian Pathogen Batrachochytrium dendrobatidis in the New World1

One application of ecological niche modeling is predicting suitable areas for the establishment of invasive species. Herein, I model the fundamental niche of the chytrid fungus Batrachochytrium dendrobatidis, a pathogen linked to amphibian declines on several continents. Niche models were generated with the Genetic Algorithm of Rule‐Set Prediction using point distribution data of the pathogen and digital maps of environmental variables integrated in a GIS environment. The distribution of regions suitable for B. dendrobatidis in the New World is extensive and includes significant portions of: (1) Sierra Madre Occidental pine‐oak forest; (2) Sonoran and Sinaloan dry forest; (3) Veracruz moist forest; (4) Central America east from the Isthmus of Tehuantepec; (5) Caribbean Islands; (6) temperate forest in Chile and western Argentina south of latitude 30°S; (7) Andes above 1000 m of altitude in Venezuela, Colombia, and Ecuador; (8) eastern slopes of the Andes in Peru and Bolivia; (9) Brazilian Atlantic forest; (10) Uruguay, Paraguay, and northeastern Argentina; (11) southwestern and Madeira‐Tapajós Amazonian tropical rainforests. The regions with the highest suitability for B. dendrobatidis include habitats that contain the world's most diverse amphibian faunas. Models were built with New World localities, but also showed strong predictability for B. dendrobatidis localities in the Old World. Out of a total of 59 reported Old World localities for B. dendrobatidis, 56 occurred within regions with high predicted suitability. I also present analyses of the environmental envelope of B. dendrobatidis and discuss the implications of the results for the conservation of amphibians in the neotropics.     

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.     

A reservoir species for the emerging Amphibian pathogen Batrachochytrium dendrobatidis thrives in a landscape decimated by disease

Chytridiomycosis, a disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is driving amphibian declines and extinctions in protected areas globally. The introduction of invasive reservoir species has been implicated in the spread of Bd but does not explain the appearance of the pathogen in remote protected areas. In the high elevation (>1500 m) Sierra Nevada of California, the native Pacific chorus frog, Pseudacris regilla, appears unaffected by chytridiomycosis while sympatric species experience catastrophic declines. We investigated whether P. regilla is a reservoir of Bd by comparing habitat occupancy before and after a major Bd outbreak and measuring infection in P. regilla in the field, monitoring susceptibility of P. regilla to Bd in the laboratory, examining tissues with histology to determine patterns of infection, and using an innovative soak technique to determine individual output of Bd zoospores in water. Pseudacris regilla persists at 100% of sites where a sympatric species has been extirpated from 72% in synchrony with a wave of Bd. In the laboratory, P. regilla carried loads of Bd as much as an order of magnitude higher than loads found lethal to sympatric species. Histology shows heavy Bd infection in patchy areas next to normal skin, a possible mechanism for tolerance. The soak technique was 77.8% effective at detecting Bd in water and showed an average output of 68 zoospores per minute per individual. The results of this study suggest P. regilla should act as a Bd reservoir and provide evidence of a tolerance mechanism in a reservoir species.     

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.     

Rapid Response to Evaluate the Presence of Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis) and Ranavirus in Wild Amphibian Populations in Madagascar

We performed a rapid response investigation to evaluate the presence and distribution of amphibian pathogens in Madagascar following our identification of amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd) and ranavirus in commercially exported amphibians. This targeted risk-based field surveillance program was conducted from February to April 2014 encompassing 12 regions and 47 survey sites. We simultaneously collected amphibian and environmental samples to increase survey sensitivity and performed sampling both in wilderness areas and commercial amphibian trade facilities. Bd was not detected in any of 508 amphibian skin swabs or 68 water filter samples, suggesting pathogen prevalence was below 0.8%, with 95% confidence during our visit. Ranavirus was detected in 5 of 97 amphibians, including one adult Mantidactylus cowanii and three unidentified larvae from Ranomafana National Park, and one adult Mantidactylus mocquardi from Ankaratra. Ranavirus was also detected in water samples collected from two commercial amphibian export facilities. We also provide the first report of an amphibian mass-mortality event observed in wild amphibians in Madagascar. Although neither Bd nor ranavirus appeared widespread in Madagascar during this investigation, additional health surveys are required to disentangle potential seasonal variations in pathogen abundance and detectability from actual changes in pathogen distribution and rates of spread. Accordingly, our results should be conservatively interpreted until a comparable survey effort during winter months has been performed. It is imperative that biosecurity practices be immediately adopted to limit the unintentional increased spread of disease through the movement of contaminated equipment or direct disposal of contaminated material from wildlife trade facilities. The presence of potentially introduced strains of ranaviruses suggests that Madagascar''s reptile species might also be threatened by disease. Standardized population monitoring of key amphibian and reptile species should be established with urgency to enable early detection of potential impacts of disease emergence in this global biodiversity hotspot.     

First Detection of Batrachochytrium dendrobatidis in Wild Frogs from Bangladesh

EcoHealth - Global amphibian populations are facing a novel threat, chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), which is responsible for the severe decline of a...     

Spatial Assessment of Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis) in South Africa Confirms Endemic and Widespread Infection

Chytridiomycosis has been identified as a major cause of global amphibian declines. Despite widespread evidence of Batrachochytrium dendrobatidis infection in South African frogs, sampling for this disease has not focused on threatened species, or whether this pathogen poses a disease risk to these species. This study assessed the occurrence of Bd-infection in South African Red List species. In addition, all known records of infection from South Africa were used to model the ecological niche of Bd to provide a better understanding of spatial patterns and associated disease risk. Presence and prevalence of Bd was determined through quantitative real-time PCR of 360 skin swab samples from 17 threatened species from 38 sites across the country. Average prevalence was 14.8% for threatened species, with pathogen load varying considerably between species. MaxEnt was used to model the predicted distribution of Bd based on 683 positive records for South Africa. The resultant probability threshold map indicated that Bd is largely restricted to the wet eastern and coastal regions of South Africa. A lack of observed adverse impacts on wild threatened populations supports the endemic pathogen hypothesis for southern Africa. However, all threatened species occur within the limits of the predicted distribution for Bd, exposing them to potential Bd-associated risk factors. Predicting pathogen distribution patterns and potential impact is increasingly important for prioritising research and guiding management decisions.     

Quantitative PCR detection of Batrachochytrium dendrobatidis DNA from sediments and water

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease implicated in amphibian declines on 5 continents. Polymerase chain reaction (PCR) primer sets exist with which amphibians can be tested for this disease, and advances in sampling techniques allow non-invasive testing of animals. We developed filtering and PCR based quantitative methods by modifying existing PCR assays to detect Bd DNA in water and sediments, without the need for testing amphibians; we tested the methods at 4 field sites. The SYBR based assay using Boyle primers (SYBR/Boyle assay) and the Taqman based assay using Wood primers performed similarly with samples generated in the laboratory (Bd spiked filters), but the SYBR/Boyle assay detected Bd DNA in more field samples. We detected Bd DNA in water from 3 of 4 sites tested, including one pond historically negative for chytridiomycosis. Zoospore equivalents in sampled water ranged from 19 to 454 1(-1) (nominal detection limit is 10 DNA copies, or about 0.06 zoospore). We did not detect DNA of Bd from sediments collected at any sites. Our filtering and amplification methods provide a new tool to investigate critical aspects of Bd in the environment.     

Detection of Batrachochytrium dendrobatidis in Endemic Salamander Species from Central Texas

A nested PCR protocol was used to analyze five endemic salamander species from Central Texas for the presence of the emerging pathogen, chytrid fungus (Batrachochytrium dendrobatidis). Chytrid fungus was detected from samples of each of the five species sampled: with low abundance, in the Texas salamander (Eurycea neotenes) (1 positive out of 16 individuals tested; 1/16), the Blanco River Springs salamander (E. pterophila) (1/20), the threatened San Marcos salamander (E. nana) (1/17), and the endangered Barton Springs salamander (E. sosorum) (1/7); much higher abundance was obtained for the Jollyville Plateau salamander (E. tonkawae) (6/14), which has recently been petitioned for addition to the USA endangered species list. With one exception, sequences of PCR products were identical to the 5.8S rRNA gene, and nearly so for the flanking internal transcribed spacer (ITS) regions of B. dendrobatidis which confirmed the detection of chytrid fungus, and thus demonstrated the presence of this pathogen in populations of endangered species in Central Texas. These confirmations were obtained from nonconsumptive tail clippings which confirms the applicability of historically collected samples from other studies in the examination of the fungus across time.