Amphibian Chytridiomycosis Research Articles

Epidermal galactose spurs chytrid virulence and predicts amphibian colonization

The chytrid fungal pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans cause the skin disease chytridiomycosis in amphibians, which is driving a substantial proportion of an entire vertebrate class to extinction. Mitigation of its impact is largely unsuccessful and requires a thorough understanding of the mechanisms underpinning the disease ecology. By identifying skin factors that mediate key events during the early interaction with B. salamandrivorans zoospores, we discovered a marker for host colonization. Amphibian skin associated beta-galactose mediated fungal chemotaxis and adhesion to the skin and initiated a virulent fungal response. Fungal colonization correlated with the skin glycosylation pattern, with cutaneous galactose content effectively predicting variation in host susceptibility to fungal colonization between amphibian species. Ontogenetic galactose patterns correlated with low level and asymptomatic infections in salamander larvae that were carried over through metamorphosis, resulting in juvenile mortality. Pronounced variation of galactose content within some, but not all species, may promote the selection for more colonization resistant host lineages, opening new avenues for disease mitigation.

Alternative food sources interfere with removal of a fungal amphibian pathogen by zooplankton

Abstract While the amphibian disease chytridiomycosis is causing ongoing population declines and biodiversity losses around the globe, efficient mitigation strategies are lacking. The free‐living zoospores of the causative agents of this disease, the chytrid pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), are a potential food source for filter‐feeding micropredators as part of the aquatic food web. While consumption of zoospores can lower environmental pathogen loads, alternative food sources may interfere with pathogen removal rates. We compared the ability of three filter‐feeding zooplankton species, the cladoceran Daphnia magna, the rotifer Brachionus calyciflorus and the ostracod Heterocypris incongruens, to remove Bd zoospores in water and investigated the effect of alternative food sources, the green algae Pseudokirchneriella subcapitata and Chlorella vulgaris, on zoospore ingestion by D. magna. Daphnia magna was the only micropredator candidate that effectively removed Bd zoospores from its environment, with an average removal rate of 1,012 ± 542 GE ind.−1 hr−1 within our test system. High concentrations (1 × 105 cells/ml) of large and easily ingestible P. subcapitata reduced pathogen removal rates, whereas the small and less edible C. vulgaris did not interfere with pathogen removal. Synthesis and applications. We showed that Daphnia spp., which are keystone species in all sorts of aquatic habitats worldwide, are promising target agents for biologically mitigating chytridiomycosis infections and how natural food sources may interfere with this strategy. We also suggest potential management actions for biological disease mitigation, aiming to optimize environmental conditions for these target filter feeders, thereby reducing pathogen densities and eventually infection pressure in amphibian hosts. Examples of such management actions include, but are not limited to, removal of planktivorous fish, habitat restoration, nutrient control or agrochemical regulation in the vicinity of amphibian breeding ponds. Further studies, including field trials, are needed to confirm the effects of pathogen consumption on infection dynamics in natural situations and investigate the impact of intervention actions.

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.

Bioclimatic and anthropogenic variables shape the occurrence of Batrachochytrium dendrobatidis over a large latitudinal gradient

Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has caused the greatest known loss of biodiversity due to an infectious disease. We used Bd infection data from quantitative real-time PCR (qPCR) assays of amphibian skin swabs collected across Chile during 2008–2018 to model Bd occurrence with the aim to determine bioclimatic and anthropogenic variables associated with Bd infection. Also, we used Bd presence/absence records to identify geographical Bd high-risk areas and compare Bd prevalence and infection loads between amphibian families, ecoregions, and host ecology. Data comprised 4155 Bd-specific qPCR assays from 162 locations across a latitudinal gradient of 3700 km (18º to 51ºS). Results showed a significant clustering of Bd associated with urban centres and anthropogenically highly disturbed ecosystems in central-south Chile. Both Bd prevalence and Bd infection loads were higher in aquatic than terrestrial amphibian species. Our model indicated positive associations of Bd prevalence with altitude, temperature, precipitation and human-modified landscapes. Also, we found that macroscale drivers, such as land use change and climate, shape the occurrence of Bd at the landscape level. Our study provides with new evidence that can improve the effectiveness of strategies to mitigate biodiversity loss due to amphibian chytridiomycosis.

Rapid spread of a virulent amphibian pathogen in nature

Considering that emerging infectious diseases are one of the major drivers of global amphibian decline, controlling the spread of infections are even more challenging. Amphibian skin disease chytridiomycosis, which is caused by two species of fungi belonging to the Batrachochytrium genus, has been detected in at least 700 amphibian species causing mass mortalities in all continents where amphibians occur. Most Alytes species, including the Betic midwife toad (A. dickhilleni), are highly susceptible to B. dendrobatidis (Bd) with lethal consequences. The presence of Bd infection in A. dickhilleni was confirmed ten years ago in just three localities across the entire distribution range of this threatened species. Here we report the extraordinary Bd expansion through the entire distribution range of A. dickhilleni and analyse if former infected populations acted as the source of transmission events to current infected populations. Currently, Bd infection is broadly distributed across the entire distribution range of the species and the increase of infection prevalence reached 30–50% during a decade. The populations where the infection was detected a decade ago could be identified as likely sources of infection for some locations where the pathogen is now present. The introduction of infected amphibian hosts into previously naive A. dickhilleni breeding sites, and other anthropogenic processes, are seeming to be the most plausible way of Bd range expansion, motivating mass mortalities, population declines and extirpation events of this threatened amphibian species.

Ranavirus is widespread in Costa Rica and co-occurs with threatened amphibians.

Amphibians are globally threatened by emerging infectious diseases, and ranaviruses are among the most concerning pathogens to threaten species in the wild. We sampled for ranaviruses in wild amphibians at 8 sites in Costa Rica, spanning broad climatic zones and taxonomic associations. Seven of these sites are inhabited by highly threatened amphibian species that persist at low global population sizes after population declines due to amphibian chytridiomycosis. One of the surveyed sites is occupied by an introduced amphibian species, which is relatively rare in Central America but may be an important pathway for long-distance transport of ranaviruses. We detected ranavirus using quantitative polymerase chain reaction in 16.3% of the 243 individuals and among 5 of our 8 sites, but not at the site with the introduced species. Infection prevalence varied among species and sites, but not with mean annual temperature or mean annual precipitation. Infection intensity did not vary with species, site, temperature, or precipitation. Our results show that ranavirus infection is spatially widespread in Costa Rica, affecting a broad range of host species, and occurs across climatic zones-though we encountered no mortality or morbidity in our sampled species. Ranaviruses are known to cause intermittent mass mortality in amphibian populations, and the threatened species sampled here are likely vulnerable to population impacts from emerging ranaviruses. Therefore, we believe the potential impacts of ranaviruses on amphibian populations in tropical regions have likely been underestimated, and that they should be viewed as a potential major stressor to threatened amphibians in tropical regions.

Amphibian chytridiomycosis, a lethal pandemic disease caused by the killer fungus Batrachochytrium dendrobatidis: New approaches to host defense mechanisms and techniques for detection and monitoring

Chytridiomycosis is a catastrophic disease currently decimating worldwide amphibian populations, caused by the panzootic chytrid fungus Batrachochytrium dendrobatidis. Massive species decline to extinction catalyzes radical changes in ecosystems globally, including the largest continuous rainforest ecosystem on Earth, the Amazon rainforest. Innovative research that aims to propose feasible mechanisms of mitigation and the origins of the disease is vital, including studies addressing climatic effects on the expansion of chytridiomycosis. Thus, this publication aims to provide a comprehensive review of: i) the current technologies used for B. dendrobatidis detection and monitoring, and ii) the known Neotropical amphibian's skin microbiota with anti-fungal properties against B. dendrobatidis. Several immunologic and DNA-based methods are discussed to understand the emerging fungal pathogens and their effects on the biosphere, which can help to mitigate the devastating ecological impacts of mass amphibian morbidity. The establishment of rapid and highly accurate B. dendrobatidis detection techniques and methods for monitoring amphibian's cutaneous microbiome is crucial in the fight against chytridiomycosis.

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.

Post-epizootic microbiome associations across communities of neotropical amphibians.

Microbiome-pathogen interactions are increasingly recognized as an important element of host immunity. While these host-level interactions will have consequences for community disease dynamics, the factors which influence host microbiomes at larger scales are poorly understood. We here describe landscape-scale pathogen-microbiome associations within the context of post-epizootic amphibian chytridiomycosis, a disease caused by the panzootic chytrid fungus Batrachochytrium dendrobatidis. We undertook a survey of Neotropical amphibians across altitudinal gradients in Ecuador ~30years following the observed amphibian declines and collected skin swab-samples which were metabarcoded using both fungal (ITS-2) and bacterial (r16S) amplicons. The data revealed marked variation in patterns of both B.dendrobatidis infection and microbiome structure that are associated with host life history. Stream breeding amphibians were most likely to be infected with B.dendrobatidis. This increased probability of infection was further associated with increased abundance and diversity of non-Batrachochytrium chytrid fungi in the skin and environmental microbiome. We also show that increased alpha diversity and the relative abundance of fungi are lower in the skin microbiome of adult stream amphibians compared to adult pond-breeding amphibians, an association not seen for bacteria. Finally, stream tadpoles exhibit lower proportions of predicted protective microbial taxa than pond tadpoles, suggesting reduced biotic resistance. Our analyses show that host breeding ecology strongly shapes pathogen-microbiome associations at a landscape scale, a trait that may influence resilience in the face of emerging infectious diseases.

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.

One Health aspects & priority roadmap for fungal diseases : A mini-review.

Fungal diseases have not been taken seriously in public health agendas as well as research priorities, despite of globally causing an estimated two million deaths every year, and the emergence of many troublesome fungal pathogens like Candida auris, azole resistant Aspergillus fumigatus, terbinafine and azole resistant dermatophytes, and zoonotic sporotrichosis in humans. Fungi are also responsible for huge losses of agricultural products and stored crops as well as recent massive and unexpected mortality in animals caused by white-nose syndrome in the bats and Chytridiomycosis in amphibians. This review aims to underscore the need for collaborative, multisectoral, and trans-disciplinary approach to include the One Health approach as an essential component of surveillance, prevention, and control of globally emerging fungal diseases. Rigorous evidence based surveillance of the environment as well as strengthening rapid and quality diagnosis of fungal diseases can save millions of lives and reduce significant morbidity.

Surveying for Batrachochytrium salamandrivorans presence in Spanish captive collections of amphibians.

Batrachochytrium salamandrivorans (Bsal), a pathogenic fungus causing the fatal disease chytridiomycosis in amphibians, was likely introduced to Europe through the trade in pet salamanders from Asia and then escaped into wild populations. Among European countries, Spain has a large number of private breeders and keepers of pet salamanders, and cases of Bsal in wild and captive populations already have been confirmed there. However, surveillance for the pathogen in Spanish collections of amphibians is sparse. Therefore, assisted by private owners and breeders, we surveyed 10 amphibian collections and analysed a total of 317 samples for presence of Bsal. All of our analyses yielded negative results. However, this apparent lack of Bsal cases in captivity should not encourage relaxation of vigilance, quarantine efforts or good practices. Because amphibian collections represent highly dynamic environments (animals are coming in and out), the pathogen could easily be introduced into a collection by new individuals. Any case of Bsal infection in captive animals could lead to its further spread to wild populations of susceptible species, potentially decimating them, and thus should be prevented.

Significant reductions of host abundance weakly impact infection intensity of Batrachochytrium dendrobatidis.

Infectious diseases are considered major threats to biodiversity, however strategies to mitigate their impacts in the natural world are scarce and largely unsuccessful. Chytridiomycosis is responsible for the decline of hundreds of amphibian species worldwide, but an effective disease management strategy that could be applied across natural habitats is still lacking. In general amphibian larvae can be easily captured, offering opportunities to ascertain the impact of altering the abundance of hosts, considered to be a key parameter affecting the severity of the disease. Here, we report the results of two experiments to investigate how altering host abundance affects infection intensity in amphibian populations of a montane area of Central Spain suffering from lethal amphibian chytridiomycosis. Our laboratory-based experiment supported the conclusion that varying density had a significant effect on infection intensity when salamander larvae were housed at low densities. Our field experiment showed that reducing the abundance of salamander larvae in the field also had a significant, but weak, impact on infection the following year, but only when removals were extreme. While this suggests adjusting host abundance as a mitigation strategy to reduce infection intensity could be useful, our evidence suggests only heavy culling efforts will succeed, which may run contrary to objectives for conservation.

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.

A historical overview of Batrachochytrium dendrobatidis infection from specimens at the National Zoological Collection Suriname.

The amphibian skin disease chytridiomycosis, caused by the pathogenetic fungus Batrachochytrium dendrobatidis (Bd) has become one of the major contributors to global amphibian population declines and extinctions. This fungus has spread globally and has caused mortalities in nearly every continent. In South America, Suriname, Guyana and Paraguay are among the remaining three countries where Bd has not been detected to date. To complete the assessment of the possible presence of Bd in Suriname, 205 specimens from the Zoological Collection of Suriname, compromising 6 frog families and 15 genera were sampled for chytrid fungus. No specimens were found to be infected by this fungus and as such the outcome strengthens the previous result of field sampling that there is no support that Bd has spread to Suriname.

After the epizootic: Host–pathogen dynamics in montane tropical amphibian communities with high prevalence of chytridiomycosis

AbstractThe amphibian fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), poses a great threat to global amphibian biodiversity. In Peruvian cloud forests of the Kosñipata Valley of Manu National Park where chytrid infection is highly prevalent, we have monitored species‐rich amphibian communities since 1996. An epizootic of chytridiomycosis is thought to have caused the disappearance of 35% of species richness in the early 2000s. We investigated the post‐epizootic Bd prevalence and infection intensity within the remnant amphibian community from 2008 to 2015, and modeled Bd dynamics as a function of species, season, reproductive mode, life stage, and elevation. Prevalence was higher in 2012–2015 than in 2008–2009, but overall prevalence has remained fairly constant (~50%) post‐epizootic. We also found that while prevalence decreased with elevation during the wet season, it generally increased with elevation during the dry season, potentially due to seasonal changes in temperature and precipitation. In aquatic habitats, Bd is likely maintained through a single, stream‐breeding, putative reservoir species (which survived epizootics, in contrast to other aquatic‐breeding species). The now‐dominant terrestrial‐breeding species allow Bd to persist and spread in terrestrial habitats, possibly through individual dispersal into naïve areas. We conclude that Bd prevalence in the Kosñipata Valley has stabilized over time, suggesting that Bd is now enzootic. Long‐term monitoring of host infection is important because temporal changes in prevalence and infection intensity can cause changes in host species richness and abundance, which in turn may alter the trajectory of host–pathogen dynamics.Abstract in Spanish is available with online material.

High Variability in Infection Mechanisms and Host Responses: A Review of Functional Genomic Studies of Amphibian Chytridiomycosis

The emergence of Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), two fungal pathogens responsible for amphibian declines and extinctions worldwide, led to 20 years of intensive research focused on revealing mechanisms underlying infection, disease progression, and host outcome. Genetic and genomic studies in this system have contributed to our understanding of the origin and genetic diversity within these pathogens, their genetic architecture, and mechanisms used for host exploitation and host immune response. Functional genomic studies, specifically differential gene expression studies, have been used to characterize both pathogen and host mechanisms of exploitation and defense as well as host–pathogen interactions and the influence of biotic and abiotic environmental factors on disease outcomes. Here, we summarize the results of functional genomic studies in the amphibian–chytrid host–pathogen system, with a focus on identifying common patterns, and also examine the factors that contribute to the high variability in disease outcomes observed in nature. We found substantial variation in differential gene expression among lineages of Bd and between Bd and Bsal, pointing to some potential virulence factors for these pathogens. On the host side, we found variation in immune response among species with different levels of resistance to chytrid infections. Finally, we found significant effects of temperature and coinfection on host responses, underscoring the important role that abiotic and biotic factors play in modulating immune response.

Identification of toxic metabolites produced by Batrachochytrium dendrobatidis fungus

Batrachochytrium dendrobatidis, or Bd, is a fungus that causes chytridiomycosis in amphibians, a disease responsible for the rapid decline in amphibian populations globally. The pervasiveness and mortality rates of the Bd fungus has spurred researchers to discover why the fungus is so lethal in various species of frogs, salamanders, and caecilians. While it is known that the fungus affects the skin of amphibians and causes death through suffocation via populating in the pores of adult amphibians, little is known about the active compounds in the fungus. An understanding of the methods of infection will aid in the creation of safe ways to treat amphibians and prevent further outbreaks. To determine the metabolites excreted by the fungus, the culture medium was filtered, dried, and analyzed by LCMS. A Daphnia assay was developed in order to aid bioactivity‐guided fractionation. Preliminary data suggests the presence of several compounds of interest in the dried metabolites. The masses of the peaks of interest were used to guide further isolation and identification of compounds.Support or Funding InformationFunded by a SURF grant from the University of Tampa

Chytrid fungi and global amphibian declines.

Discovering that chytrid fungi cause chytridiomycosis in amphibians represented a paradigm shift in our understanding of how emerging infectious diseases contribute to global patterns of biodiversity loss. In this Review we describe how the use of multidisciplinary biological approaches has been essential to pinpointing the origins of amphibian-parasitizing chytrid fungi, including Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans, as well as to timing their emergence, tracking their cycles of expansion and identifying the core mechanisms that underpin their pathogenicity. We discuss the development of the experimental methods and bioinformatics toolkits that have provided a fuller understanding of batrachochytrid biology and informed policy and control measures.

Amphibian chytrid prevalence on boreal toads in SE Alaska and NW British Columbia: tests of habitat, life stages, and temporal trends.

Tracking and understanding variation in pathogens such as Batrachochytrium dendrobatidis (Bd), the agent of amphibian chytridiomycosis which has caused population declines globally, is a priority for many land managers. However, relatively little sampling of amphibian communities has occurred at high latitudes. We used skin swabs collected during 2005-2017 from boreal toads Anaxyrus boreas (n = 248), in southeast Alaska (USA; primarily in and near Klondike Gold Rush National Historical Park [KLGO]) and northwest British Columbia (Canada) to determine how Bd prevalence varied across life stages, habitat characteristics, local species richness, and time. Across all years, Bd prevalence peaked in June and was >3 times greater for adult toads (37.5%) vs. juveniles and metamorphs (11.2%). Bd prevalence for toads in the KLGO area, where other amphibian species are rare or absent, was highest from river habitats (55.0%), followed by human-modified upland wetlands (32.3%) and natural upland wetlands (12.7%)-the same rank-order these habitats are used for toad breeding. None of the 12 Columbia spotted frogs Rana luteiventris or 2 wood frogs R. sylvatica from the study area tested Bd-positive, although all were from an area of low host density where Bd has not been detected. Prevalence of Bd on toads in the KLGO area decreased during 2005-2015. This trend from a largely single-species system may be encouraging or concerning, depending on how Bd is affecting vital rates, and emphasizes the need to understand effects of pathogens before translating disease prevalence into management actions.