Bd Isolates Research Articles

No sex-dependent mortality in an amphibian upon infection with the chytrid fungus, Batrachochytrium dendrobatidis.

One of the major factors driving the currently ongoing biodiversity crisis is the anthropogenic spread of infectious diseases. Diseases can have conspicuous consequences, such as mass mortality events, but may also exert covert but similarly severe effects, such as sex ratio distortion via sex-biased mortality. Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is among the most important threats to amphibian biodiversity. Yet, whether Bd infection can skew sex ratios in amphibians is currently unknown, although such a hidden effect may cause the already dwindling amphibian populations to collapse. To investigate this possibility, we collected common toad (Bufo bufo) tadpoles from a natural habitat in Hungary and continuously treated them until metamorphosis with sterile Bd culture medium (control), or a liquid culture of a Hungarian or a Spanish Bd isolate. Bd prevalence was high in animals that died during the experiment but was almost zero in individuals that survived until the end of the experiment. Both Bd treatments significantly reduced survival after metamorphosis, but we did not observe sex-dependent mortality in either treatment. However, a small number of genotypically female individuals developed male phenotype (testes) in the Spanish Bd isolate treatment. Therefore, future research is needed to ascertain if larval Bd infection can affect sex ratio in common toads through female-to-male sex reversal.

An endogenous DNA virus in an amphibian-killing fungus associated with pathogen genotype and virulence

The global panzootic lineage (GPL) of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) has caused severe amphibian population declines, yet the drivers underlying the high frequency of GPL in regions of amphibian decline are unclear. Using publicly available Bd genome sequences, we identified multiple non-GPL Bd isolates that contain a circular Rep-encoding single-stranded (CRESS)-like DNA virus, which we named Bd DNA virus 1 (BdDV-1). We further sequenced and constructed genome assemblies with long read sequences to find that the virus is integrated into the nuclear genome in some strains. Attempts to cure virus-positive isolates were unsuccessful; however, phenotypic differences between naturally virus-positive and virus-negative Bd isolates suggested that BdDV-1 decreases the growth of its host invitro but increases the virulence of its host invivo. BdDV-1 is the first-described CRESS DNA mycovirus of zoosporic true fungi, with a distribution inversely associated with the emergence of the panzootic lineage.

Panzootic chytrid fungus exploits diverse amphibian host environments through plastic infection strategies.

While some pathogens are limited to single species, others can colonize many hosts, likely contributing to the emergence of novel disease outbreaks. Despite this biodiversity threat, traits associated with host niche expansions are not well understood in multihost pathogens. Here, we aimed to uncover functional machinery driving multihost invasion by focusing on Batrachochytrium dendrobatidis (Bd), a pathogen that infects the skin of hundreds of amphibians worldwide. We performed a meta-analysis of Bd gene expression using data from published infection experiments and newly generated profiles. We analysed Bd transcriptomic landscapes across the skin of 14 host species, reconstructed Bd isolates phylogenetic relationships, and inferred the origin and evolutionary history of differentially expressed genes under a phylogenetic framework comprising other 12 zoosporic fungi. Bd displayed plastic infection strategies when challenged by hosts with different disease susceptibility. Our analyses identified sets of differentially expressed genes under host environments with similar infection outcome. We stressed nutritional immunity and gene silencing as important processes required to overcome challenging skin environments in less susceptible hosts. Overall, Bd genes expressed during amphibian skin exploitation have arisen mainly via gene duplications with great family expansions, increasing the gene copy events previously described for this fungal species. Finally, we provide a comprehensive gene data set that can be used to further examine eco-evolutionary hypotheses for this host-pathogen system. Our study supports the idea that host environments exert contrasting selective pressures, such that gene expression plasticity could be one of the evolutionary keys leading to the success of multihost pathogens.

Genetic variation of Batrachochytrium dendrobatidis is linked to skin bacterial diversity in the Pacific treefrog Hyliola regilla (hypochondriaca).

Symbiotic bacterial communities are crucial to combating infections and contribute to host health. The amphibian skin microbiome plays an important role in protecting their hosts against pathogens such as Batrachochytrium dendrobatidis (Bd), one of the causative agents of chytridiomycosis, which is responsible for dramatic amphibian population declines worldwide. Although symbiotic skin bacteria are known to inhibit Bd growth, an understanding of the relationship between Bd genetic variability, environmental conditions, and skin bacterial communities is limited. Therefore, we examined the associations between Bd infection load, Bd genetic diversity and skin bacterial communities in five populations of Hyliola regilla (hypochondriaca) from environmentally contrasting sites in Baja California, Mexico. We observed differences in Bd genetics and infection load among sites and environments. Genetic analysis of Bd isolates revealed patterns of spatial structure corresponding to the five sites sampled. Amphibian skin bacterial diversity and community structure differed among environments and sites. Bacterial community composition was correlated with Bd genetic differences and infection load, with specific bacterial taxa enriched on infected and un-infected frogs. Our results indicate that skin-associated bacteria and Bd strains likely interact on the host skin, with consequences for microbial community structure and Bd infection intensity.

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.

Chytridiomycosis Outbreak in a Chilean Giant Frog (Calyptocephalella gayi) Captive Breeding Program: Genomic Characterization and Pathological Findings.

Emerging infectious diseases in wildlife are increasingly associated with animal mortality and species declines, but their source and genetic characterization often remains elusive. Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has been associated with catastrophic and well-documented amphibian population declines and extinctions at the global scale. We used histology and whole-genome sequencing to describe the lesions caused by, and the genetic variability of, two Bd isolates obtained from a mass mortality event in a captive population of the threatened Chilean giant frog (Calyptocephalella gayi). This was the first time an association between Bd and high mortality had been detected in this charismatic and declining frog species. Pathological examinations revealed that 30 dead metamorphosed frogs presented agnathia or brachygnathia, a condition that is reported for the first time in association with chytridiomycosis. Phylogenomic analyses revealed that Bd isolates (PA1 and PA2) from captive C. gayi group with other Bd isolates (AVS2, AVS4, and AVS7) forming a single highly supported Chilean Bd clade within the global panzootic lineage of Bd (BdGPL). These findings are important to inform the strengthening of biosecurity measures to prevent the impacts of chytridiomycosis in captive breeding programs elsewhere.

Amphibian resistance to chytridiomycosis increases following low‐virulence chytrid fungal infection or drug‐mediated clearance

Abstract Amphibian biodiversity is experiencing ongoing declines due in part to the infectious disease, chytridiomycosis. Efforts to mitigate the effects of the causal agent of chytridiomycosis, Batrachochytrium dendrobatidis (Bd), in the wild have not been wholly effective. Translocations are an important management tool for amphibians, and immunizations represent a possible strategy for preparing amphibians for release across a landscape where Bd exists. We evaluated the utility of using an isolate of Bd that was shown to be hypovirulent to the relict leopard frog Rana onca as a transmissible inoculum for promoting chytridiomycosis resistance. We conducted a co‐housing experiment to determine if the isolate we used could be passed between R. onca without increasing in virulence. We then followed with an experiment where frogs that were exposed to the hypovirulent isolate were then challenged with a virulent Bd isolate. In other experiments, we evaluated whether Bd infections followed by clearance with itraconazole (an antifungal) could increase resistance to chytridiomycosis in R. onca and Rana pipiens (northern leopard frog). We found that our hypovirulent Bd inoculation was transmissible between hosts, did not cause chytridiomycosis and was effective at imparting long‐term chytridiomycosis resistance. Rana onca inoculated with the hypovirulent Bd isolate had lower pathogen burdens and were 55 times more likely to survive infections by a virulent Bd isolate than non‐inoculated frogs. For both species, prior exposure to Bd followed by infection clearance with itraconazole resulted in significantly increased survivorship and lower pathogen burdens as compared to controls that had no prior Bd exposure. Rana onca that were previously exposed to Bd were more than 15 times more likely to survive infections. Previously exposed R. pipiens survived in higher proportions than controls, but with weaker statistical support. Synthesis and Applications. Translocations are an important management tool for many amphibian conservation programs. For amphibian species that have experienced declines due to the disease chytridiomycosis [caused by the pathogen Batrachochytrium dendrobatidis (Bd)], the success of translocations may be limited due to the persistent threat of the disease in the wild. We completed a series of experiments aimed at increasing amphibian resistance to chytridiomycosis using low‐virulence, transmissible Bd inoculations and Bd infection followed by clearance with an antifungal (itraconazole). Our low‐virulence Bd inoculations were transmissible, did not cause disease and provided long‐term resistance against chytridiomycosis. Similarly, infection with Bd and clearance with itraconazole led to increased resistance to chytridiomycosis in subsequent exposures. The use of a low‐virulence Bd inoculation could be further developed into a transmissible vaccine, propagating the protective benefits beyond individuals that can be directly inoculated. At this stage, we advise that using the alternative approach of infecting frogs with Bd from translocation sites followed by clearance of infections with itraconazole may be sensible for increasing host resistance to chytridiomycosis.

Thermal Performance Curves of Multiple Isolates of Batrachochytrium dendrobatidis, a Lethal Pathogen of Amphibians.

Emerging infectious disease is a key factor in the loss of amphibian diversity. In particular, the disease chytridiomycosis has caused severe declines around the world. The lethal fungal pathogen that causes chytridiomycosis, Batrachochytrium dendrobatidis (Bd), has affected amphibians in many different environments. One primary question for researchers grappling with disease-induced losses of amphibian biodiversity is what abiotic factors drive Bd pathogenicity in different environments. To study environmental influences on Bd pathogenicity, we quantified responses of Bd phenotypic traits (e.g., viability, zoospore densities, growth rates, and carrying capacities) over a range of environmental temperatures to generate thermal performance curves. We selected multiple Bd isolates that belong to a single genetic lineage but that were collected across a latitudinal gradient. For the population viability, we found that the isolates had similar thermal optima at 21°C, but there was considerable variation among the isolates in maximum viability at that temperature. Additionally, we found the densities of infectious zoospores varied among isolates across all temperatures. Our results suggest that temperatures across geographic point of origin (latitude) may explain some of the variation in Bd viability through vertical shifts in maximal performance. However, the same pattern was not evident for other reproductive parameters (zoospore densities, growth rates, fecundity), underscoring the importance of measuring multiple traits to understand variation in pathogen responses to environmental conditions. We suggest that variation among Bd genetic variants due to environmental factors may be an important determinant of disease dynamics for amphibians across a range of diverse environments.

Genotyping and differential bacterial inhibition of Batrachochytrium dendrobatidis in threatened amphibians in Costa Rica.

Amphibians have declined around the world in recent years, in parallel with the emergence of an epidermal disease called chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). This disease has been associated with mass mortality in amphibians worldwide, including in Costa Rica, and Bd is considered an important contributor to the disappearance of this group of vertebrates. While many species are susceptible to the disease, others show tolerance and manage to survive infection with the pathogen. We evaluated the pathogen Bd circulating in Costa Rica and the capacity of amphibian skin bacteria to inhibit the growth of the pathogen in vitro. We isolated and characterized - genetically and morphologically - several Bd isolates from areas with declining populations of amphibians. We determined that the circulating chytrid fungus in Costa Rica belongs to the virulent strain Bd-GPL-2, which has been related to massive amphibian deaths worldwide; however, the isolates obtained showed genetic and morphological variation. Furthermore, we isolated epidermal bacteria from 12 amphibian species of surviving populations, some in danger of extinction, and evaluated their inhibitory activity against the collection of chytrid isolates. Through bioassays we confirmed the presence of chytrid-inhibitory bacterial genera in Costa Rican amphibians. However, we observed that the inhibition varied between different isolates of the same bacterial genus, and each bacterial isolation inhibited fungal isolation differently. In total, 14 bacterial isolates belonging to the genera Stenotrophomonas, Streptomyces, Enterobacter, Pseudomonas and Klebsiella showed inhibitory activity against all Bd isolates. Given the observed variation both in the pathogen and in the bacterial inhibition capacity, it is highly relevant to include local isolates and to consider the origin of the microorganisms when performing in vivo infection tests aimed at developing and implementing mitigation strategies for chytridiomycosis.

Designing Probiotic Therapies With Broad-Spectrum Activity Against a Wildlife Pathogen.

Host-associated microbes form an important component of immunity that protect against infection by pathogens. Treating wild individuals with these protective microbes, known as probiotics, can reduce rates of infection and disease in both wild and captive settings. However, the utility of probiotics for tackling wildlife disease requires that they offer consistent protection across the broad genomic variation of the pathogen that hosts can encounter in natural settings. Here we develop multi-isolate probiotic consortia with the aim of effecting broad-spectrum inhibition of growth of the lethal amphibian pathogen Batrachochytrium dendrobatidis (Bd) when tested against nine Bd isolates from two distinct lineages. Though we achieved strong growth inhibition between 70 and 100% for seven Bd isolates, two isolates appeared consistently resistant to inhibition, irrespective of probiotic strategy employed. We found no evidence that genomic relatedness of the chytrid predicted similarity of inhibition scores, nor that increasing the genetic diversity of the bacterial consortia could offer stronger inhibition of pathogen growth, even for the two resistant isolates. Our findings have important consequences for the application of probiotics to mitigate wildlife diseases in the face of extensive pathogen genomic variation.

Population-Level Resistance to Chytridiomycosis is Life-Stage Dependent in an Imperiled Anuran.

Amphibian declines caused by chytridiomycosis have been severe, but some susceptible populations have persisted or even recovered. Resistance to the causal agent Batrachochytrium dendrobatidis (Bd) could result from alleles of the adaptive immune system. During metamorphosis, however, immune systems may not be fully functional, implying that an effective immune response to Bd may be life-stage dependent. We evaluated the susceptibility of the relict leopard frog (Rana onca) sourced from two areas where Bd was present or absent, and where the populations appeared to show differences in pathogen resistance. We evaluated whether population-level resistance manifested across life stages using challenge experiments with late-stage tadpoles (Gosner stage 31-38), metamorphs (stage 45-46), and juvenile frogs. We used three different Bd isolates including one from wild R. onca to challenge juvenile frogs and focused on the isolate from R. onca to challenge tadpoles and resulting metamorphs. We found that juveniles from the Bd exposed population were 5.5 times more likely to survive Bd infection and 10 times more likely to clear infections than those from the area without Bd. In contrast, and regardless of the source area, we observed 98% survivorship of tadpoles, but only 19% survivorship of resulting metamorphs following re-exposure. Given the low survivorship of exposed metamorphs in the laboratory, we speculate on how resistance characteristics, whether adaptive or innate, that do not manifest at each life stage could develop in the wild. We suggest that seasonal high temperatures during times when metamorphosis appears common may modulate the effects of the pathogen during this most susceptible life stage.

Gene expression varies within and between enzootic and epizootic lineages of Batrachochytrium dendrobatidis (Bd) in the Americas

While much research focus is paid to hypervirulent fungal lineages during emerging infectious disease outbreaks, examining enzootic pathogen isolates can be equally fruitful in delineating infection dynamics and determining pathogenesis. The fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd), exhibits markedly different patterns of disease in natural populations, where it has caused massive amphibian declines in some regions, yet persists enzootically in others. Here we compare in vitro gene expression profiles of a panel of Bd isolates representing both the enzootic Bd-Brazil lineage, and the more recently diverged, panzootic lineage, Bd-GPL. We document significantly different lineage-specific and intralineage gene expression patterns, with Bd-Brazil upregulating genes with aspartic-type peptidase activity, and Bd-GPL upregulating CBM18 chitin-binding genes, among others. We also find pronounced intralineage variation in membrane integrity and transmembrane transport ability within our Bd-GPL isolates. Finally, we highlight unexpectedly divergent expression profiles in sympatric panzootic isolates, underscoring microgeographic functional variation in a largely clonal lineage. This variation in gene expression likely plays an important role in the relative pathogenesis and host range of Bd-Brazil and Bd-GPL isolates. Together, our results demonstrate that functional genomics approaches can provide information relevant to studies of virulence evolution within the Bd clade.

Skin-associated lactic acid bacteria from North American bullfrogs as potential control agents of Batrachochytrium dendrobatidis.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis and has been a key driver in the catastrophic decline of amphibians globally. While many strategies have been proposed to mitigate Bd outbreaks, few have been successful. In recent years, the use of probiotic formulations that protect an amphibian host by killing or inhibiting Bd have shown promise as an effective chytridiomycosis control strategy. The North American bullfrog (Lithobates catesbeianus) is a common carrier of Bd and harbours a diverse skin microbiota that includes lactic acid bacteria (LAB), a microbial group containing species classified as safe and conferring host benefits. We investigated beneficial/probiotic properties: anti-Bd activity, and adhesion and colonisation characteristics (hydrophobicity, biofilm formation and exopolysaccharide-EPS production) in two confirmed LAB (cLAB-Enterococcus gallinarum CRL 1826, Lactococcus garvieae CRL 1828) and 60 presumptive LAB (pLAB) [together named as LABs] isolated from bullfrog skin.We challenged LABs against eight genetically diverse Bd isolates and found that 32% of the LABs inhibited at least one Bd isolate with varying rates of inhibition. Thus, we established a score of sensitivity from highest (BdGPL AVS7) to lowest (BdGPL C2A) for the studied Bd isolates. We further reveal key factors underlying host adhesion and colonisation of LABs. Specifically, 90.3% of LABs exhibited hydrophilic properties that may promote adhesion to the cutaneous mucus, with the remaining isolates (9.7%) being hydrophobic in nature with a surface polarity compatible with colonisation of acidic, basic or both substrate types. We also found that 59.7% of LABs showed EPS synthesis and 66.1% produced biofilm at different levels: 21% weak, 29% moderate, and 16.1% strong. Together all these properties enhance colonisation of the host surface (mucus or epithelial cells) and may confer protective benefits against Bd through competitive exclusion. Correspondence analysis indicated that biofilm synthesis was LABs specific with high aggregating bacteria correlating with strong biofilm producers, and EPS producers being correlated to negative biofilm producing LABs. We performed Random Amplified Polymorphic DNA (RAPD)-PCR analysis and demonstrated a higher degree of genetic diversity among rod-shaped pLAB than cocci. Based on the LAB genetic analysis and specific probiotic selection criteria that involve beneficial properties, we sequenced 16 pLAB which were identified as Pediococcus pentosaceus, Enterococcus thailandicus, Lactobacillus pentosus/L. plantarum, L. brevis, and L. curvatus. Compatibility assays performed with cLAB and the 16 species described above indicate that all tested LAB can be included in a mixed probiotic formula. Based on our analyses, we suggest that E. gallinarum CRL 1826, L. garvieae CRL 1828, and P. pentosaceus 15 and 18B represent optimal probiotic candidates for Bd control and mitigation.

Bullfrog farms release virulent zoospores of the frog-killing fungus into the natural environment

Bullfrog farming and trade practices are well-established, globally distributed, and economically valuable, but pose risks for biodiversity conservation. Besides their negative impacts on native amphibian populations as an invasive species, bullfrogs play a key role in spreading the frog-killing fungus Batrachochytrium dendrobatidis (Bd) in the natural environment. Bullfrogs are tolerant to Bd, meaning that they can carry high infection loads without developing chytridiomycosis. To test the potential of bullfrog farms as reservoirs for diverse and virulent chytrid genotypes, we quantified Bd presence, prevalence and infection loads across approximately 1,500 farmed bullfrogs and in the water that is released from farms into the environment. We also described Bd genotypic diversity within frog farms by isolating Bd from dozens of infected tadpoles. We observed individuals infected with Bd in all sampled farms, with high prevalence (reaching 100%) and high infection loads (average 71,029 zoospore genomic equivalents). Average outflow water volume from farms was high (60,000 L/day), with Bd zoospore concentration reaching approximately 50 million zoospores/L. Because virulent pathogen strains are often selected when growing in tolerant hosts, we experimentally tested whether Bd genotypes isolated from bullfrogs are more virulent in native anuran hosts compared to genotypes isolated from native host species. We genotyped 36 Bd isolates from two genetic lineages and found that Bd genotypes cultured from bullfrogs showed similar virulence in native toads when compared to genotypes isolated from native hosts. Our results indicate that bullfrog farms can harbor high Bd genotypic diversity and virulence and may be contributing to the spread of virulent genotypes in the natural environment. We highlight the urgent need to implement Bd monitoring and mitigation strategies in bullfrog farms to aid in the conservation of native amphibians.

Double trouble: co-infections of chytrid fungi will severely impact widely distributed newts

Invasive fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) are causing mortality events and population declines in amphibians around the world. B. salamandrivorans has not been found in the Americas but is predicted to emerge there given the presence of competent hosts, high volume import pathways, and lack of effective biosecurity measures. Eastern newts (Notophthalmus viridescens) are the most abundant and widespread salamanders in United States with known susceptibility to both fungi. However, little is known about how their current Bd infections will interact with novel Bsal infections. Here, we ran a series of experiments in which we exposed newts to each fungal pathogen, combinations of both fungi, and three Bd isolates to: (1) determine if natural exposure to Bd provides protection against Bsal, (2) quantify the effect of co-infections on newt survival, and (3) examine if resistance responses are general to multiple lineages. We found that hosts exposed under simultaneous co-infections experienced 78% mortality over a period of 18 weeks, which was driven by the persistence of Bsal because newts cleared Bd infection within a month. In a subsequent experiment, resistant newts also resolved infections of three Bd genotypes, suggesting that the defense mechanism is effective across all lineages. However, re-exposing these individuals with a tenfold higher dose (106Bd zoospores) increased their susceptibility to Bsal, and 93% of them sustained Bsal infections. Our findings will improve the estimates of decline risk for a species that could serve as a potential superspreader of Bsal in North America.

Genetic characterization of chytrids isolated from larval amphibians collected in central and east Texas

Abstract Chytridiomycosis, an emerging infectious disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), has caused amphibian population declines worldwide. Bd was first described in the 1990s and there are still geographic gaps in the genetic analysis of this globally distributed pathogen. Relatively few genetic studies have focused on regions where Bd exhibits low virulence, potentially creating a bias in our current knowledge of the pathogen's genetic diversity. Disease-associated declines have not been recorded in Texas (USA), yet Bd has been detected on amphibians in the state. These strains have not been isolated and characterized genetically; therefore, we isolated, cultured, and genotyped Bd from central Texas and compared isolates to a panel of previously genotyped strains distributed across the Western Hemisphere. We also isolated other chytrids from east Texas not known to infect amphibians. To identify larval amphibian hosts, we sequenced part of the COI gene. Among 37 Bd isolates from Texas, we detected 19 unique multi-locus genotypes, but found no genetic structure associated with host species, Texas localities, or across North America. Isolates from central Texas exhibit high diversity and genetically cluster with BdGPL isolates from the western U.S. that have caused amphibian population declines. This study genetically characterizes isolates of Bd from the south central U.S. and adds to the global knowledge of Bd genotypes.

Globally invasive genotypes of the amphibian chytrid outcompete an enzootic lineage in coinfections.

Competition between genotypes is likely to be a key driver of pathogen evolution, particularly following a geographical invasion by distant strains. Theory predicts that competition between disease strains will result in the most virulent strain persisting. Despite its evolutionary implications, the role of strain competition in shaping populations remains untested for most pathogens. We experimentally investigated the in vivo competitive differences between two divergent lineages of the amphibian-killing chytrid fungus ( Batrachochytrium dendrobatidis, Bd). These Bd lineages are hypothesized to have diverged in allopatry but been recently brought back into secondary contact by human introduction. Prior studies indicate that a panzootically-distributed, global lineage of Bd was recently introduced into southern Brazil, and is competitively excluding enzootic lineages in the southern Atlantic Forest. To test for differences in competitive ability between invasive and enzootic Brazilian Bd isolates, we coinfected a model host frog system which we developed for this study ( Hymenochirus curtipes). We tracked isolate-specific zoospore production over the course of the coinfection experiment with chip-based digital PCR (dPCR). The globally invasive panzootic lineage had a competitive advantage in spore production especially during the first one to four weeks of infection, and on frogs that eventually succumbed to Bd infection. Our study provides new evidence that competitive pressure resulting from the human movement of pathogen strains can rapidly alter the genetics, community dynamics and spatial epidemiology of pathogens in the wild.

Host–pathogen dynamics among the invasive American bullfrog (Lithobates catesbeianus) and chytrid fungus (Batrachochytrium dendrobatidis)

The combination of introduced host species and emerging pathogens can result in unanticipated disease dynamics and novel host–pathogen interactions. The American bullfrog (Lithobates catesbeianus) is a successful invasive amphibian in the western U.S. that can act as a host to the emerging fungal pathogen, Batrachochytrium dendrobatidis (Bd) implicated in the decline of amphibian populations worldwide. We examined if wild-caught invasive bullfrogs were differentially susceptible to two regionally distinct isolates of Bd. Newly metamorphosed bullfrog individuals were exposed to either a Bd strain originally isolated from bullfrogs in their endemic range or a strain from the invaded range in the western U.S. We quantified initial infection load and compared mortality rates and changes in infection load after 30 days to determine strain-specific susceptibility. Wild-caught bullfrogs from the western U.S. were particularly susceptible to an eastern Bd strain. Although infection loads were not different between strains, individuals exposed to the western strain survived, suggesting the ability to reduce their infection. Our findings highlight differences between strains and response variation of an invasive species host.

Amphibian chytridiomycosis outbreak dynamics are linked with host skin bacterial community structure

Host-associated microbes are vital for combatting infections and maintaining health. In amphibians, certain skin-associated bacteria inhibit the fungal pathogen Batrachochytrium dendrobatidis (Bd), yet our understanding of host microbial ecology and its role in disease outbreaks is limited. We sampled skin-associated bacteria and Bd from Pyrenean midwife toad populations exhibiting enzootic or epizootic disease dynamics. We demonstrate that bacterial communities differ between life stages with few shared taxa, indicative of restructuring at metamorphosis. We detected a significant effect of infection history on metamorph skin microbiota, with reduced bacterial diversity in epizootic populations and differences in community structure and predicted function. Genome sequencing of Bd isolates supports a single introduction to the Pyrenees and reveals no association between pathogen genetics and epidemiological trends. Our findings provide an ecologically relevant insight into the microbial ecology of amphibian skin and highlight the relative importance of host microbiota and pathogen genetics in predicting disease outcome.

Host size influences the effects of four isolates of an amphibian chytrid fungus.

Understanding factors that influence host–pathogen interactions is key to predicting outbreaks in natural systems experiencing environmental change. Many amphibian population declines have been attributed to an amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). While this fungus is widespread, not all Bd‐positive populations have been associated with declines, which could be attributed to differences in pathogen virulence or host susceptibility. In a laboratory experiment, we examined the effects of Bd isolate origin, two from areas with Bd‐associated amphibian population declines (El Copé, Panama, and California, USA) and two from areas without Bd‐related population declines (Ohio and Maine, USA), on the terrestrial growth and survival of American toad (Anaxyrus americanus) metamorphs reared in larval environments with low or high intraspecific density. We predicted that (1) Bd isolates from areas experiencing declines would have greater negative effects than Bd isolates from areas without declines, and (2) across all isolates, growth and survival of smaller toads from high‐density larval conditions would be reduced by Bd exposure compared to larger toads from low‐density larval conditions. Our results showed that terrestrial survival was reduced for smaller toads exposed to Bd with variation in the response to different isolates, suggesting that smaller size increased susceptibility to Bd. Toads exposed to Bd gained less mass, which varied by isolate. Bd isolates from areas with population declines, however, did not have more negative effects than isolates from areas without recorded declines. Most strikingly, our study supports that host condition, measured by size, can be indicative of the negative effects of Bd exposure. Further, Bd isolates’ impact may vary in ways not predictable from place of origin or occurrence of disease‐related population declines. This research suggests that amphibian populations outside of areas experiencing Bd‐associated declines could be impacted by this pathogen and that the size of individuals could influence the magnitude of Bd's impact.