Fungal Pathogen Batrachochytrium Dendrobatidis Research Articles

Geography, Host Genetics, and Cross-Domain Microbial Networks Structure the Skin Microbiota of Fragmented Brazilian Atlantic Forest Frog Populations.

The host‐associated microbiome plays a significant role in health. However, the roles of factors such as host genetics and microbial interactions in determining microbiome diversity remain unclear. We examined these factors using amplicon‐based sequencing of 175 Thoropa taophora frog skin swabs collected from a naturally fragmented landscape in southeastern Brazil. Specifically, we examined (1) the effects of geography and host genetics on microbiome diversity and structure; (2) the structure of microbial eukaryotic and bacterial co‐occurrence networks; and (3) co‐occurrence between microeukaryotes with bacterial OTUs known to affect growth of the fungal pathogen Batrachochytrium dendrobatidis (Bd). While bacterial alpha diversity varied by both site type and host MHC IIB genotype, microeukaryotic alpha diversity varied only by site type. However, bacteria and microeukaryote composition showed variation according to both site type and host MHC IIB genotype. Our network analysis showed the highest connectivity when both eukaryotes and bacteria were included, implying that ecological interactions may occur among domains. Lastly, anti‐Bd bacteria were not broadly negatively co‐associated with the fungal microbiome and were positively associated with potential amphibian parasites. Our findings emphasize the importance of considering both domains in microbiome research and suggest that for effective probiotic strategies for amphibian disease management, considering potential interactions among all members of the microbiome is crucial.

Infection with Batrachochytrium dendrobatidis reduces salamander capacity to mount a cell-mediated immune response.

The vertebrate immune system is a costly defense system that is responsible for preventing and eliminating parasites and pathogens. Theory predicts that hosts experience tradeoffs associated with immune deployment and other physiological functions. Although empirical evidence for immune-physiology tradeoffs are well documented in the literature, fewer studies have examined tradeoffs within the immune system in wild vertebrates. We explored the topic of concomitant immune challenges in amphibians by exposing salamanders (Plethodon cinereus) to a fungal pathogen Batrachochytrium dendrobatidis (hereafter "Bd") and then to phytohemagglutinin (hereafter "PHA"). We measured Bd infection using quantitative PCR and used measurements of the tail thickness at the PHA injection site as an estimate of skin swelling. We tested whether Bd reduced the salamander's capacity to mount an immune response towards PHA or whether Bd would stimulate immune activity and thereby increase the response towards PHA. Salamanders that were infected with Bd had a reduced skin-swelling when injected with PHA compared to noninfected salamanders, a result that is consistent with the hypothesis that Bd-infected salamanders have lower immunocompetence than noninfected salamanders. We also found that PHA-induced swelling response was negatively associated with Bd infection abundance (i.e., the infection burden of all exposed salamanders, including those that were exposed but not infected), indicating that salamanders with a higher infection abundance had the lowest swelling response to PHA. Our results suggest that individuals of P. cinereus might experience an energetic tradeoff between successfully fighting off Bd and mounting an immune response towards PHA.

A new species of marsupial frog (Anura; Gastrotheca) from the Cordillera de Colán in northeastern Peru

We describe a new species of marsupial frog, genusGastrotheca, using morphological characters and molecular data as lines of evidence. The new species was discovered in the páramo and the ecotone between páramo and humid montane forest of Cordillera de Colán, at elevations between 3136 and 3179 m a.s.l., in northeastern Peru. The new species is distinguished from all its congeners by the combination of the following characters: coarsely granular skin on dorsum, a green dorsal coloration without pattern, finger I shorter than finger II, turquoise iris, and a venter without blotches, flecks or dots. Furthermore, we include a detailed osteological description of the newGastrothecaspecies based on Micro-CT scanning. Based on our phylogenetic analyses, the new species belongs to theGastrotheca marsupiataspecies group, is sister toG. oresbiosand closely related toG. psychrophila,G. spectabilis,G. stictopleuraand one undescribed species. Additionally, we test for the presence of the fungal pathogenBatrachochytrium dendrobatidis(Bd). NoBdinfection was detected forG. gemmasp. nov. specimens butBdprevalence was detected among syntopic frogs.

Chytridiomycosis in Asian Amphibians, a Global Resource for Batrachochytrium dendrobatidis (Bd) Research.

Chytridiomycosis is an emerging infectious disease affecting amphibians globally and it is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Chytridiomycosis has caused dramatic declines and even extinctions in wild amphibian populations in Europe, Australia, Central and North America. Spanning over two and a half decades, extensive research has led to discovery of epizootic and enzootic lineages of this pathogen. However, the Bd–amphibian system had garnered less attention in Asia until recently when an ancestral Bd lineage was identified in the Korean peninsula. Amphibians co-exist with the pathogen in Asia, only sub-lethal effects have been documented on hosts. Such regions are ‘coldspots’ of infection and are an important resource to understand the dynamics between the enzootic pathogen—Bd and its obligate host—amphibians. Insights into the biology of infection have provided new knowledge on the multi-faceted interaction of Bd in a hyperdiverse Asian amphibian community. We present the findings and highlight the knowledge gap that exists, and propose the ways to bridge them. We emphasize that chytridiomycosis in Asia is an important wildlife disease and it needs focussed research, as it is a dynamic front of pathogen diversity and virulence.

Time in the Laboratory, but Not Exposure to a Chytrid Fungus, Results in Rapid Change in Spring Peeper (Pseudacris crucifer) Skin Bacterial Communities

Interactions between pathogens and the symbiotic microbial communities that reside in hosts can impact disease processes. Symbiotic microbial communities can act to prevent pathogen infection in some instances, while in other instances, pathogens can disrupt these symbiotic communities. We sought to address these interactions between the fungal skin pathogen Batrachochytrium dendrobatidis (Bd) and the skin bacterial communities of adult Spring Peepers (Pseudacris crucifer). In the laboratory, frogs were exposed to Bd zoospores for a 12-hour period and then monitored for five days. Bacterial community composition on the skin was assessed upon initial collection, 24 hours post-exposure, and at the conclusion of the experiment using 16S rRNA gene amplicon sequencing. Little impact on the bacterial community was observed as a result of Bd exposure, suggesting that the timeline was either too short to observe the effects of Bd, or that the skin communities are resilient in the face of such short exposure periods. In contrast, laboratory housing was associated with changes in skin bacterial community composition, in terms of both relative abundances and bacterial taxa that were present. These findings suggest that even a short time away from their natural environment can have strong effects on the composition of amphibian skin communities and that Bd exposure may not always disrupt these skin communities.

Spatial Risk Analysis of Batrachochytrium dendrobatidis, A Global Emerging Fungal Pathogen.

Chytridiomycosis, a leading cause for the global decline in the number of amphibians, is caused by the fungal pathogen Batrachochytrium dendrobatidis. In this study, global distribution data of B. dendrobatidis were collected from January 2009 to May 2019. Space-time scan statistics and the maximum entropy (MaxEnt) model were used to analyze the epidemic trends and aggregation of the pathogen, and predict B. dendrobatidis distribution through its relationships with climate factors, wind speed, and solar radiation. The results of space-time scan statistics show seven clusters of data for the distribution of B. dendrobatidis. The time was mainly concentrated in 2009, 2013, 2015, and 2016, and the regions were primarily concentrated in southeastern Canada, southwestern France, Nigeria, Cameroon, eastern Brazil, southeastern Brazil, central Madagascar, and central and eastern Australia. MaxEnt showed that annual precipitation had the largest contribution percentage in the model, and annual mean temperature highly influenced the distribution of B. dendrobatidis. The global high-risk areas of B. dendrobatidis distribution were mainly observed in western Canada, southern Brazil, Chile, the United Kingdom, Japan, the Republic of Korea, eastern South Africa, eastern Madagascar, southeastern Australia, and southern China.

Inhibition of Batrachochytrium dendrobatidis Infection by Skin Bacterial Communities in Wild Amphibian Populations

Skin-associated bacteria are known to inhibit infection by the fungal pathogen Batrachochytrium dendrobatidis (Bd) in amphibians. It has also been postulated that skin-associated bacterial community is related to Bd infection intensity. However, our understanding of host microbial dynamics and their importance in regulating Bd intensity is limited. We analyzed Bd infection and skin-associated bacteria from two amphibian species, the salamander Ambystoma rivulare and the frog Lithobates spectabilis that co-occurred in a tropical high-altitude site in central Mexico. Sixty-three percent of sampled salamander individuals and 80% of frog individuals tested positive for Bd. Overall, we registered 622 skin-associated bacterial genera, from which 73 are known to have Bd inhibitory effects. These inhibitory taxa represented a relative abundance of 50% in relation to total relative bacterial abundance. Our results indicated that, although sharing some bacterial taxa, bacterial community from the skin of both species was different in taxonomic composition and in relative abundance. Pseudomonas spp. and Stenotrophomonas spp. were among the five most abundant bacterial taxa of both species. Both bacterial taxa inhibit Bd infection. We detected that bacterial richness and relative abundance of inhibitory Bd bacteria were negatively related to intensity of Bd infection independent of species and seasons. Despite the high Bd prevalence in both host species, no dead or sick individuals were registered during field surveys. The relatively low levels of Bd load apparently do not compromise survival of host species. Therefore, our results suggested that individuals analyzed were able to survive and thrive under a dynamic relation with enzootic infections of Bd and their microbiota.

Spatiotemporal adaptive evolution of an MHC immune gene in a frog-fungus disease system.

Genetic diversity of major histocompatibility complex (MHC) genes is linked to reduced pathogen susceptibility in amphibians, but few studies also examine broad spatial and temporal patterns of MHC and neutral genetic diversity. Here, we characterized range-wide MHC diversity in the Northern leopard frog, Rana pipiens, a species found throughout North America that is experiencing disease-related declines. We used previously sequenced neutral markers (mitochondrial DNA and microsatellites), sequenced an expressed MHC class IIß gene fragment, and measured infection prevalence and intensity of the global fungal pathogen Batrachochytrium dendrobatidis (Bd) across 14 populations. Four populations were sampled across two decades, enabling temporal comparisons of selection and demography. We recovered 37 unique MHC alleles, including 17 that were shared across populations. Phylogenetic and population genetic patterns between MHC and neutral markers were incongruent, and five MHC codon positions associated with peptide binding were under positive selection. MHC heterozygosity, but not neutral marker heterozygosity, was a significant factor explaining spatial patterns of Bd prevalence, whereas only environmental variables predicted Bd intensity. MHC allelic richness (AR) decreased significantly over time but microsatellite-based AR did not, highlighting a loss of functional immunogenetic diversity that may be associated with Bd selective pressures. MHC supertype 4 was significantly associated with an elevated risk of Bd infection, whereas one supertype 2 allele was associated with a nearly significant reduced risk of Bd. Taken together, these results provide evidence that positive selection contributes to MHC class IIß evolution in R. pipiens and suggest that functional MHC differences across populations may contribute to disease adaptation.

Wildlife pathogen detection: evaluation of alternative DNA extraction protocols.

Accurate detection of wildlife pathogens is critical in wildlife disease research. False negatives or positives can have catastrophic consequences for conservation and disease-mitigation decisions. Quantitative polymerase chain reaction is commonly used for molecular detection of wildlife pathogens. The reliability of this method depends on the effective extraction of the pathogen’s DNA from host samples. A wildlife disease that has been in the centre of conservationist’s attention is the amphibian disease Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Here, we compare the efficiency of a spin column extraction kit (QIAGEN), commonly used in Bd DNA extraction, to an alternative spin column kit (BIOKÈ) used in extractions from other types of samples, which is considerably cheaper but not typically used for Bd DNA extraction. Additionally, we explore the effect of an enzymatic pre-treatment on detection efficiency. Both methods showed similar efficiency when extracting Bd DNA from zoospores from laboratory-created cell-cultures, as well as higher efficiency when combined with the enzymatic pre-treatment. Our results indicate that selecting the optimal method for DNA extraction is essential to ensure minimal false negatives and reduce project costs.

The first detection of the fungal pathogen batrachochytrium dendrobatidis in Norway with no evidence of population declines for great crested and smooth newts based on modeling on traditional trapping data

AbstractThe pathogenic fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis in amphibians across the world, often leading to population declines and species extinctions. Here, we present the first detection of Bd in Norway, diagnosed using environmental DNA (eDNA) in water samples. In a genetic screening of 31 ponds in southeastern Norway for Bd, great crested newt (Triturus cristatus) and smooth newt (Lissotriton vulgaris), we detected Bd in five different localities. Resampling in one location revealed that time of sampling seemingly had a strong effect on the concentration of Bd eDNA in the water, which increased from May till June. In contrast, eDNA concentration of great crested newt decreased during the same period. Following a traditional sampling approach with traps, newts in the investigated ponds have been sampled for population estimates since 2012. We imposed a model on the catch data and contrasted the Bd‐positive ponds to the Bd‐negative ponds, finding no signs of population declines in the Bd‐positive ponds. This could suggest either recent infection events, with animals still having low prevalence of Bd and with little time for Bd to affect the present breeding generations of newts, and/or that the two newt species are infection‐tolerant. This study further highlights eDNA as a tool for early detection of invasive species.

Indirect terrestrial transmission of amphibian chytrid fungus from reservoir to susceptible host species leads to fatal chytridiomycosis

AbstractThe amphibian chytrid fungal pathogen (Batrachochytrium dendrobatidis, henceforth Bd) has had a devastating impact on biodiversity, causing the decline or extinction of over 500 amphibian species. Yet, our understanding of Bd transmission pathways remains incomplete, in particular for host species with weak aquatic associations, and between reservoir and susceptible host species. We examined Bd transmission from a potential reservoir host to a potentially susceptible critically endangered host; directly assessing the capacity of the former to transmit Bd, and the susceptibility of the latter as a Bd host. Using cohousing versus sequential use of the same enclosure by the two species, we distinguished the effects of direct versus indirect (environmental) transmission. Our study provides clear evidence that both direct and indirect terrestrial transmission from a reservoir to susceptible host species results in fatal chytridiomycosis. Transmission mode had no effect on overall morbidity or disease progression in the susceptible species. Our results demonstrate that reservoir and susceptible hosts do not need to be in the same place at the same time, or within an aquatic environment for transmission to occur. Our demonstration of indirect terrestrial transmission from a reservoir to susceptible host identifies mechanisms by which Bd may drive ongoing declines in populations where the pathogen is now endemic. Identifying these transmission pathways is important for understanding long‐term extinction vulnerability of remnant populations of declining species challenged by disease.

Skin microbiome correlates with bioclimate and Batrachochytrium dendrobatidis infection intensity in Brazil\u2019s Atlantic Forest treefrogs

In Brazil’s Atlantic Forest (AF) biodiversity conservation is of key importance since the fungal pathogen Batrachochytrium dendrobatidis (Bd) has led to the rapid loss of amphibian populations here and worldwide. The impact of Bd on amphibians is determined by the host's immune system, of which the skin microbiome is a critical component. The richness and diversity of such cutaneous bacterial communities are known to be shaped by abiotic factors which thus may indirectly modulate host susceptibility to Bd. This study aimed to contribute to understanding the environment-host–pathogen interaction determining skin bacterial communities in 819 treefrogs (Anura: Hylidae and Phyllomedusidae) from 71 species sampled across the AF. We investigated whether abiotic factors influence the bacterial community richness and structure on the amphibian skin. We further tested for an association between skin bacterial community structure and Bd co-occurrence. Our data revealed that temperature, precipitation, and elevation consistently correlate with richness and diversity of the skin microbiome and also predict Bd infection status. Surprisingly, our data suggest a weak but significant positive correlation of Bd infection intensity and bacterial richness. We highlight the prospect of future experimental studies on the impact of changing environmental conditions associated with global change on environment-host–pathogen interactions in the AF.

Chytridiomycosis-induced mortality in a threatened anuran

Effectively planning conservation introductions involves assessing the suitability of both donor and recipient populations, including the landscape of disease risk. Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), has caused extensive amphibian declines globally and may hamper reintroduction attempts. To determine Bd dynamics in potential source populations for conservation translocations of the threatened California red-legged frog (Rana draytonii) to Yosemite National Park, we conducted Bd sampling in two populations in the foothills of the Sierra Nevada Mountains, California, U.S.A. At one of two sites, we observed lethally high Bd loads in early post-metamorphic life stages and confirmed one chytridiomycosis-induced mortality, the first such report for this species. These results informed source population site selection for subsequent R. draytonii conservation translocations. Conservation efforts aimed at establishing new populations of R. draytonii in a landscape where Bd is ubiquitous can benefit from an improved understanding of risk through disease monitoring and ex situ infection studies.

Whole exome sequencing identifies the potential for genetic rescue in iconic and critically endangered Panamanian harlequin frogs.

Avoiding extinction in a rapidly changing environment often relies on a species' ability to quickly adapt in the face of extreme selective pressures. In Panamá, two closely related harlequin frog species (Atelopus varius and Atelopus zeteki) are threatened with extinction due to the fungal pathogen Batrachochytrium dendrobatidis (Bd). Once thought to be nearly extirpated from Panamá, A.varius have recently been rediscovered in multiple localities across their historical range; however, A.zeteki are possibly extinct in the wild. By leveraging a unique collection of 186 Atelopus tissue samples collected before and after the Bd outbreak in Panama, we describe the genetics of persistence for these species on the brink of extinction. We sequenced the transcriptome and developed an exome-capture assay to sequence the coding regions of the Atelopus genome. Using these genetic data, we evaluate the population genetic structure of historical A.varius and A.zeteki populations, describe changes in genetic diversity over time, assess the relationship between contemporary and historical individuals, and test the hypothesis that some A.varius populations have rapidly evolved to resist or tolerate Bd infection. We found a significant decrease in genetic diversity in contemporary (compared to historical) A.varius populations. We did not find strong evidence of directional allele frequency change or selection for Bd resistance genes, but we uncovered a set of candidate genes that warrant further study. Additionally, we found preliminary evidence of recent migration and gene flow in one of the largest persisting A.varius populations in Panamá, suggesting the potential for genetic rescue in this system. Finally, we propose that previous conservation units should be modified, as clear genetic breaks do not exist beyond the local population level. Our data lay the groundwork for genetically informed conservation and advance our understanding of how imperiled species might be rescued from extinction.

Female Salamanders Experience Higher Parasitism Compared to Males: A Cost of Female Reproduction?

Males tend to experience higher rates of parasitism compared to females, a phenomenon associated with ecological factors, the fact that males engage in risky behaviors, and because testosterone is known to be immunosuppressive. However, females could experience higher rates of parasitism if energy is allocated from costly immune responses towards producing eggs. We used pooled data sets from laboratory experiments to investigate sex-specific differences in salamander (Plethodon cinereus) resistance to the emerging fungal pathogen Batrachochytrium dendrobatidis (“Bd”). Contrary to our predictions, we found that female salamanders had a higher prevalence of infection (∼56%) and carried a higher Bd infection burden (455 zoospores equivalents per sample) compared to male salamanders (which had a Bd infection prevalence of ∼24% and an average infection burden of 58 zoospore equivalents per sample). We also found that female reproductive investment (i.e., mass of eggs) positively correlated with Bd infection burden, suggesting that females who previously invested more into reproduction carried a higher Bd infection burden. Collectively, our findings might indicate that female salamanders experience a cost of reproduction in the form of decreased disease resistance.

Identification of antibiotics for use in selection of the chytrid fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans.

Global amphibian populations are being decimated by chytridiomycosis, a deadly skin infection caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Although ongoing efforts are attempting to limit the spread of these infections, targeted treatments are necessary to manage the disease. Currently, no tools for genetic manipulation are available to identify and test specific drug targets in these fungi. To facilitate the development of genetic tools in Bd and Bsal, we have tested five commonly used antibiotics with available resistance genes: Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin. We have identified effective concentrations of each for selection in both liquid culture and on solid media. These concentrations are within the range of concentrations used for selecting genetically modified cells from a variety of other eukaryotic species.

Immunological Aspects of Chytridiomycosis

Amphibians are currently the most threatened vertebrate class, with the disease chytridiomycosis being a major contributor to their global declines. Chytridiomycosis is a frequently fatal skin disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). The severity and extent of the impact of the infection caused by these pathogens across modern Amphibia are unprecedented in the history of vertebrate infectious diseases. The immune system of amphibians is thought to be largely similar to that of other jawed vertebrates, such as mammals. However, amphibian hosts are both ectothermic and water-dependent, which are characteristics favouring fungal proliferation. Although amphibians possess robust constitutive host defences, Bd/Bsal replicate within host cells once these defences have been breached. Intracellular fungal localisation may contribute to evasion of the induced innate immune response. Increasing evidence suggests that once the innate defences are surpassed, fungal virulence factors suppress the targeted adaptive immune responses whilst promoting an ineffectual inflammatory cascade, resulting in immunopathology and systemic metabolic disruption. Thus, although infections are contained within the integument, crucial homeostatic processes become compromised, leading to mortality. In this paper, we present an integrated synthesis of amphibian post-metamorphic immunological responses and the corresponding outcomes of infection with Bd, focusing on recent developments within the field and highlighting future directions.

The amphibian complement system and chytridiomycosis.

Understanding host immune function and ecoimmunology is increasingly important at a time when emerging infectious diseases (EIDs) threaten wildlife. One EID that has emerged and spread widely in recent years is chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which is implicated unprecedented amphibian declines around the world. The impacts of Bd have been severe for many amphibian species, but some populations have exhibited signs of persistence, and even recovery, in some regions. Many mechanisms may underpin this pattern and amphibian immune responses are likely one key component. Although we have made great strides in understanding amphibian immunity, the complement system remains poorly understood. The complement system is a nonspecific, innate immune defense that is known to enhance other immune responses. Complement activation can occur by three different biochemical pathways and result in protective mechanisms, such as inflammation, opsonization, and pathogen lysis, thereby providing protection to the host. We currently lack an understanding of complement pathway activation for chytridiomycosis, but several studies have suggested that it may be a key part of an early and robust immune response that confers host resistance. Here, we review the available research on the complement system in general as well as amphibian complement responses to Bd infection. Additionally, we propose future research directions that will increase our understanding of the amphibian complement system and other immune responses to Bd. Finally, we suggest how a deeper understanding of amphibian immunity could enhance the conservation and management of amphibian species that are threatened by chytridiomycosis.

HIGHER INFECTION PREVALENCE IN AMPHIBIANS INHABITING HUMAN-MADE COMPARED TO NATURAL WETLANDS.

It is unclear how suitable human-made wetlands are for supporting wildlife and how they impact wildlife disease risk. Natural wetlands (those that were created without human actions) can support more diverse and resilient communities that are at lower risk of disease outbreaks. We compared frog community composition and infection with the pathogenic fungus Batrachochytrium dendrobatidis (Bd) between human-made and natural wetlands in Tippecanoe County, Indiana, US. We conducted visual encounter surveys of frog communities and quantified Bd infection prevalence at four natural and five human-made wetlands. Water parameters associated with human practices (e.g., pH, salinity) and surrounding land use were also compared across sites. We found higher Bd infection prevalence at human-made sites than at natural sites, with monthly differences showing highest infection in spring and fall, and decreasing infection with increasing water temperature. However, we found no differences between human-made and natural sites regarding amphibian community composition, water quality, or surrounding land use. Further, we found frog density increased with distance to nearest roads among both human-made and natural sites. These findings might suggest that human-made wetlands can support frog communities similar to natural wetlands, but pose a greater risk of Bd infection.

Fungal infection, decline and persistence in the only obligate troglodytic Neotropical salamander.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) is implicated in global mass die-offs and declines in amphibians. In Mesoamerica, the Bd epidemic wave hypothesis is supported by detection of Bd in historic museum specimens collected over the last century, yet the timing and impact of the early stages of the wave remain poorly understood. Chiropterotriton magnipes, the only obligate troglodytic Neotropical salamander, was abundant in its small range in the decade following its description in 1965, but subsequently disappeared from known localities and was not seen for 34 years. Its decline is roughly coincident with that of other populations of Neotropical salamanders associated with the invasion and spread of Bd. To determine the presence and infection intensity of Bd on C. magnipes and sympatric amphibian species (which are also Bd hosts), we used a noninvasive sampling technique and qPCR assay to detect Bd on museum specimens of C. magnipes collected from 1952 to 2012, and from extant populations of C. magnipes and sympatric species of amphibians. We also tested for the presence of the recently discovered Batrachochytrium salamandivorans (Bsal), another fungal chytridiomycete pathogen of salamanders, using a similar technique specific for Bsal. We did not detect Bd in populations of C. magnipes before 1969, while Bd was detected at low to moderate prevalence just prior to and during declines. This pattern is consistent with Bd-caused epizootics followed by host declines and extirpations described in other hosts. We did not detect Bsal in any extant population of C. magnipes. We obtained one of the earliest positive records of the fungus to date in Latin America, providing additional historical evidence consistent with the Bd epidemic wave hypothesis. Genotyping results show that at least one population is currently infected with the Global Panzootic Lineage of Bd, but our genotyping of the historical positive samples was unsuccessful. The lack of large samples from some years and the difficulty in genotyping historical Bd samples illustrate some of the difficulties inherent in assigning causality to historical amphibian declines. These data also provide an important historical baseline for actions to preserve the few known remaining populations of C. magnipes.