Academic literature on the topic 'Amphibian fungal pathogen'

AbstractChytridiomycosis, a disease in amphibians caused by Batrachochytrium dendrobatidis (Bd), has led to a population decline and extinction of frog species since 1996. The objective of this study was to determine the prevalence of and the need for establishing a surveillance system for monitoring chytridiomycosis in five national zoos and five free ranging protected areas across Thailand. A total of 492 skin swab samples were collected from live and dead animals and tested by polymerase chain reaction (PCR) for the presence of Bd. The positive specimens were confi rmed by amplicon sequencing and examined by histopathology and immunohistochemistry. From July 2009 to August 2012, the prevalence of Bd from frog skin samples was low (4.27%), monitored by PCR. All samples from live amphibians were negative. The positive cases were only from dead specimens (21/168, 12.5% dead samples) of two non-native captive species, poison dart frog (Dendrobates tinctorius) and tomato frog (Dyscophus antongilii) in one zoo. Immunohistochemistry and histopathology revealed the typical feature of fl ask-shaped zoosporangia and septate thalli, supporting the PCR-based evidence of chytridiomycosis in captive amphibians in Thailand, but detected Bd in only 7/21 of the PCR-positive samples. Although the introduction of a pathogenic strain of Bd from imported carriers might have a serious impact on the native amphibian populations in Thailand, chytridiomycosis has not currently been detected in native Thai amphibians. An active surveillance system is needed for close monitoring of the fungus crossing into Thai amphibian populations

Host-associated microbiomes perform many beneficial functions including resisting pathogens and training the immune system. Here, we show that amphibians developing in captivity lose substantial skin bacterial diversity, primarily due to reduced ongoing input from environmental sources. We combined studies of wild and captive amphibians with a database of over 1 000 strains that allows us to examine antifungal function of the skin microbiome. We tracked skin bacterial communities of 62 endangered boreal toads, Anaxyrus boreas , across 18 time points, four probiotic treatments, and two exposures to the lethal fungal pathogen Batrachochytrium dendrobatidis ( Bd ) in captivity, and compared these to 33 samples collected from wild populations at the same life stage. As the amphibians in captivity lost the Bd -inhibitory bacteria through time, the proportion of individuals exposed to Bd that became infected rose from 33% to 100% in subsequent exposures. Inoculations of the Bd -inhibitory probiotic Janthinobacterium lividum resulted in a 40% increase in survival during the second Bd challenge, indicating that the effect of microbiome depletion was reversible by restoring Bd -inhibitory bacteria. Taken together, this study highlights the functional role of ongoing environmental inputs of skin-associated bacteria in mitigating a devastating amphibian pathogen, and that long-term captivity decreases this defensive function.

SUMMARYOutbreaks of cutaneous infectious disease in amphibians are increasingly being attributed to an overlooked group of fungal-like pathogens, the Dermocystids. During the last 10 years on the Isle of Rum, Scotland, palmate newts (Lissotriton helveticus) have been reportedly afflicted by unusual skin lesions. Here we present pathological and molecular findings confirming that the pathogen associated with these lesions is a novel organism of the order Dermocystida, and represents the first formally reported, and potentially lethal, case of amphibian Dermocystid infection in the UK. Whilst the gross pathology and the parasite cyst morphology were synonymous to those described in a study from infectedL. helveticusin France, we observed a more extreme clinical outcome on Rum involving severe subcutaneous oedema. Phylogenetic topologies supported synonymy between Dermocystid sequences from Rum and France and as well as their distinction fromAmphibiocystidiumspp. Phylogenetic analysis also suggested that the amphibian-infecting Dermocystids are not monophyletic. We conclude that theL. helveticus-infecting pathogen represents a single, novel species;Amphibiothecum meredithae.

Batrachochytrium dendrobatidisis a fungal pathogen in the phylum Chytridiomycota that causes the skin disease chytridiomycosis. Chytridiomycosis is considered an emerging infectious disease linked to worldwide amphibian declines and extinctions. Although amphibians have well-developed immune defenses, clearance of this pathogen from the skin is often impaired. Previously, we showed that the adaptive immune system is involved in the control of the pathogen, butB. dendrobatidisreleases factors that inhibitin vitroandin vivolymphocyte responses and induce lymphocyte apoptosis. Little is known about the nature of the inhibitory factors released by this fungus. Here, we describe the isolation and characterization of three fungal metabolites produced byB. dendrobatidisbut not by the closely related nonpathogenic chytridHomolaphlyctis polyrhiza. These metabolites are methylthioadenosine (MTA), tryptophan, and an oxidized product of tryptophan, kynurenine (Kyn). Independently, both MTA and Kyn inhibit the survival and proliferation of amphibian lymphocytes and the Jurkat human T cell leukemia cell line. However, working together, they become effective at much lower concentrations. We hypothesize thatB. dendrobatidiscan adapt its metabolism to release products that alter the local environment in the skin to inhibit immunity and enhance the survival of the pathogen.

Infectious diseases rarely end in extinction. Yet the mechanisms that explain how epidemics subside are difficult to pinpoint. We investigated host-pathogen interactions after the emergence of a lethal fungal pathogen in a tropical amphibian assemblage. Some amphibian host species are recovering, but the pathogen is still present and is as pathogenic today as it was almost a decade ago. In addition, some species have defenses that are more effective now than they were before the epidemic. These results suggest that host recoveries are not caused by pathogen attenuation and may be due to shifts in host responses. Our findings provide insights into the mechanisms underlying disease transitions, which are increasingly important to understand in an era of emerging infectious diseases and unprecedented global pandemics.

Emerging infectious diseases are a pressing threat to global biological diversity. Increased incidence and severity of novel pathogens underscores the need for methodological advances to understand pathogen emergence and spread. Here, we use genetic epidemiology to test, and challenge, key hypotheses about a devastating zoonotic disease impacting amphibians globally. Using an amplicon-based sequencing method and non-invasive samples we retrospectively explore the history of the fungal pathogen Batrachochytrium dendrobatidis ( Bd ) in two emblematic amphibian systems: the Sierra Nevada of California and Central Panama. The hypothesis in both regions is the hypervirulent Global Panzootic Lineage of Bd ( Bd GPL) was recently introduced and spread rapidly in a wave-like pattern. Our data challenge this hypothesis by demonstrating similar epizootic signatures can have radically different underlying evolutionary histories. In Central Panama, our genetic data confirm a recent and rapid pathogen spread. However, Bd GPL in the Sierra Nevada has remarkable spatial structuring, high genetic diversity and a relatively older history inferred from time-dated phylogenies. Thus, this deadly pathogen lineage may have a longer history in some regions than assumed, providing insights into its origin and spread. Overall, our results highlight the importance of integrating observed wildlife die-offs with genetic data to more accurately reconstruct pathogen outbreaks.

Biodiversity is declining at unprecedented rates worldwide. Yet cascading effects of biodiversity loss on other taxa are largely unknown because baseline data are often unavailable. We document the collapse of a Neotropical snake community after the invasive fungal pathogen Batrachochytrium dendrobatidis caused a chytridiomycosis epizootic leading to the catastrophic loss of amphibians, a food source for snakes. After mass mortality of amphibians, the snake community contained fewer species and was more homogeneous across the study site, with several species in poorer body condition, despite no other systematic changes in the environment. The demise of the snake community after amphibian loss demonstrates the repercussive and often unnoticed consequences of the biodiversity crisis and calls attention to the invisible declines of rare and data-deficient species.

Batrachochytrium dendrobatidis (Bd), the chytrid fungus which infects keratinized amphibian skin and causes the lethal disease chytridiomycosis, has been linked to population declines and extinctions worldwide (Lips et al. 2006). Amphibians infected with Bd may suffer a variety of outcomes. Individuals of some species have been killed by :S 100 Bd zoospores, while other species, such as the North American bullfrog Rana catesbiana, are highly resistant (Daszak et al. 2004). Within an amphibian species, populations may also respond differently to Bd, with some declining et al. remaining stable (Kriger and Hero 2006). Divergent outcomes among species and populations with respect to Bd may arise from at least three factors, or their interaction.

ABSTRACTChytridiomycosis, an amphibian skin disease caused by the emerging fungal pathogenBatrachochytrium dendrobatidis, has been implicated in catastrophic global amphibian declines. The result is an alarming decrease in amphibian diversity that is a great concern for the scientific community. Clinical trials testing potential antifungal drugs are needed to identify alternative treatments for amphibians infected with this pathogen. In this study, we quantified the MICs of chloramphenicol (800 μg/ml), amphotericin B (0.8 to 1.6 μg/ml), and itraconazole (Sporanox) (20 ng/ml) againstB. dendrobatidis. Both chloramphenicol and amphotericin B significantly reducedB. dendrobatidisinfection in naturally infected southern leopard frogs (Rana[Lithobates]sphenocephala), although neither drug was capable of complete fungal clearance. Long-term exposure ofR. sphenocephalato these drugs did not inhibit antimicrobial peptide (AMP) synthesis, indicating that neither drug is detrimental to this important innate skin defense. However, we observed that chloramphenicol, but not amphotericin B or itraconazole, inhibited the growth of multipleR. sphenocephalaskin bacterial isolatesin vitroat concentrations below the MIC againstB. dendrobatidis. These results indicate that treatment with chloramphenicol might dramatically alter the protective natural skin microbiome when used as an antifungal agent. This study represents the first examination of the effects of alternative antifungal drug treatments on amphibian innate skin defenses, a crucial step to validating these treatments for practical applications.

Chytridiomycosis is a fungal skin disease in amphibians that is primarily caused by Batrachochytrium dendrobatidis (Bd). We analyzed whole genome sequences of 40 bacterial isolates that had been previously cultured from the skin of four amphibian species from Virginia, USA, and tested for their ability to inhibit Bd growth via an in vitro challenge assay. These 40 isolates spanned 11 families and 13 genera. The aim of this study was to identify genomic differences among the amphibian skin bacterial isolates and generate hypotheses about possible differences that could contribute to variation in their ability to inhibit the growth of Bd. We identified sixty-five gene families that were present in all 40 isolates. We also looked for the presence of biosynthetic gene clusters. While this set of isolates contained a wide variety of biosynthetic gene clusters, the two most abundant clusters with potential anti-fungal activity were siderophores (N=17) and Type III polyketide synthases (N=20). We then analyzed the isolates belonging to the phylum Proteobacteria in more detail. We identified 197 gene families that were present in all 22 Proteobacteria. We examined various subsets of the Proteobacteria for genes for specific compounds with known activity against fungi, including chitinase and violacein. We identified a difference in the number, as well as amino acid sequences, of predicted chitinases found in two isolates belonging to the genus Agrobacterium that varied in their inhibition of Bd. After examining the annotated genomes, we identified a predicted chitinase in a Sphingomonas isolate that inhibited the growth of Bd that was absent from the five Sphingomonas isolates that did not inhibit Bd growth. The genes vioA, vioB, vioC, vioD and vioE are necessary to produce violacein, a compound which inhibits the growth of Bd. Differences in these genes were identified in three out of the four Janthinobacterium isolates. Of these three isolates, two showed strong inhibition of Bd growth, while the third inhibited Bd growth to a lesser extent. Using comparative genomics, we generated several testable hypotheses about differences among bacterial isolates that could contribute to variation in ability to inhibit Bd growth. Further work is necessary to test the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd.
Master of Science
Many amphibian population declines around the world have been caused by chytridiomycosis, a skin disease. This disease is caused by the fungus Batrachochytrium dendrobatidis (Bd). The skin of amphibians is also home to many bacteria that can provide important functions for the amphibian host, like preventing infection by Bd. To understand how these bacteria might provide protection, we examined the entire genomes of 40 bacterial isolates that reside on the skin of four amphibian species from Virginia, USA. These bacteria were previously tested for their ability to prevent Bd growth and 40 of them were chosen for sequencing based on selecting closely related isolates that varied in their ability to inhibit Bd growth. This allowed us to compare their genomes and generate hypotheses about possible genomic differences that could contribute to the variation in Bd growth inhibition. We identified sixty-five gene families that were present in all 40 bacteria. We also looked for sets of genes (biosynthetic gene clusters) that are known to produce secondary metabolites, which are compounds that can include antifungals. The two most abundant clusters we identified that had the potential to produce compounds that inhibit fungal growth were siderophores and Type III polyketide synthases. We then looked for genes that were not part of biosynthetic gene clusters that could produce specific compounds that can inhibit Bd growth, such as chitinase and violacein. We found variation in chitinase genes in several isolates that seemed to be associated with the ability to inhibit Bd growth. In addition, there were some differences in violacein genes that should be examined more in future studies. Overall, we suggest that using comparative genomic approaches can be valuable for identifying key bacterial functions in the microbiome.

As part of the ongoing loss of global biodiversity, amphibian populations are experiencing declines and extinctions. A primary factor in these declines is the skin disease chytridiomycosis, which is caused by the fungus Batrachochytrium dendrobatidis (Bd). Recent research suggests that the amphibian skin microbiota has anti-Bd activity and may be an important factor in host disease resistance. However, little is known about the basic ecology of this host-microbe symbiosis, such as how much variation there is in microbial symbionts among host species and populations, and the nature of symbiont transmission, culturability, and function. My dissertation research addressed these basic questions in microbial ecology, as well as used a novel system to examine the long-standing ecological theory of community structure-function relationships. First, host-specificity, population-level variation and potential environmental transmission of the microbiota were examined by conducting a field survey of bacterial communities from bullfrogs, newts, pond water, and pond substrate at a single pond, and newts from multiple ponds. There was variation among amphibian host species and populations in their skin symbionts, and, in a host species-specific manner, amphibian skin may select for microbes that are generally in low abundance in the environment. Second, the culturability of amphibian skin bacteria was assessed by directly comparing culture-dependent and -independent bacterial sequences from the same individuals. Although less than 7% of the amphibian skin microbes were captured using R2A medium, most of the dominant bacteria were represented in our cultures, and similar patterns of diversity among four amphibian species were captured with both approaches. Third, the relationship between microbial community structure and function and selective forces shaping structure and function were examined in bullfrogs by tracking microbial community structure and function following experimental manipulation of the skin microbiota and pathogen exposure. Results of this study demonstrated that Bd is a selective force on cutaneous bacterial community structure and function, and suggest that beneficial states of bacterial structure and function may serve to limit infection and negative fitness consequences of Bd exposure. Using a combination of observational and experimental approaches, my dissertation contributes to understanding structure-function relationships of these complex symbiotic communities of vertebrates.
Ph. D.

Amphibians are the most threatened taxon on the planet. Declines have been associated with over-exploitation, habitat loss, pollution, and pathogenic diseases, but of these factors, pollution and disease have been relatively under-studied. Here, I investigated: 1) the impacts of commonly used pesticides on aquatic communities, 2) the effect of these pesticides on amphibian susceptibility to the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), and 3) whether there are non-amphibian hosts of Bd and 4) how to best quantify the survival of Bd through ontogeny of the host. In my first research chapter, I quantified the effects of environmentally relevant concentrations of the mot commonly used synthetic fungicide in the US, chlorothalonil, on 34 species-, 2 community- and 11 ecosystem-level responses in a multitrophic-level system. Chlorothalonil increased mortality of amphibians, gastropods, zooplankton, algae, and a macrophyte (reducing taxonomic richness), reduced decomposition and water clarity, and elevated dissolved oxygen and net primary productivity. These ecosystem effects were indirect but were predictable based on changes in taxonomic richness. A path analysis suggests that chlorothalonil-induced reductions in biodiversity and top-down and bottom-up effects facilitated algal blooms that shifted ecosystem functions. In my second chapter, I investigated how a wide range of ecologically relevant concentrations of chlorothalonil affected four species of amphibians (Osteopilus septentrionalis, Rana sphenocephala, Hyla squirella and H. cinerea). I also evaluated the effects of chlorothalonil on liver tissue, immune cell density, and the stress hormone, corticosterone. Chlorothalonil killed nearly every amphibian at the expected environmental concentration (EEC) and, at concentrations to which humans are commonly exposed (up to the EEC), it was associated with elevated corticosterone levels and changes in immune cells. Three species (O. septentrionalis, R. sphenocephala, and H. cinerea) showed a non-monotonic dose-response, with low and high concentrations causing significantly greater mortality than intermediate concentrations and controls. Corticosterone exhibited a similar non-monotonic dose response and chlorothalonil concentration was inversely associated with liver tissue and immune cell densities. These studies on chlorothalonil emphasize the need to re-evaluate its safety and to further link anthropogenic-induced changes in biodiversity to altered ecosystem functions. In my third research chapter, I investigated the effects of chlorothalonil and atrazine, one of the most commonly used herbicides in the US, on amphibian susceptibility to Bd, a leading cause of amphibian extinctions. Relative to controls, atrazine monotonically reduced Bd growth in culture and on tadpoles. In contrast, chlorothalonil non-monotonically reduced Bd growth in culture and on tadpoles, with low and high concentrations causing significantly greater mortality than intermediate concentrations and controls. This study is one of only a handful of studies to document a non-monotonic dose response of an invertebrate (Bd) to a pesticide. Although both pesticides reduced Bd growth on tadpoles and in culture, neither eliminated Bd entirely, and because we know little about the long-term effects of the pesticides on hosts (e.g., immunosuppression), I do not recommend using these chemicals to control Bd. In my fourth research chapter, I investigated whether there are non-amphibian hosts for Bd. Non-amphibian hosts could explain how Bd is able to persist in the environment after amphibians are extirpated, and the extreme virulence and distribution of Bd. In laboratory and field studies, I found that crayfish, but not mosquitofish, were hosts for Bd. I found that crayfish could be infected with Bd, could maintain that infection long term (at least 3 months) and could transfer that infection to susceptible amphibians. I also revealed that exposure to water that previously held Bd caused significant crayfish mortality and gill recession, suggesting that Bd releases a chemical that can cause host pathology in the absence of infection. Most efforts to conserve and restore amphibian populations challenged by Bd have been unsuccessful, but managing alternative hosts offers a new and potentially more effective approach to managing Bd. Likewise, identifying the specific pathology-inducing chemical released by Bd might facilitate the development of new strategies to reduce the risk posed by this pathogen. The fifth and sixth research chapters are aimed to improve the quality and efficiency of Bd research. During amphibian development, Bd infections transition from the mouthparts of tadpoles to the skin of post-metamorphic frogs but this transition has never been quantified and thus researchers might be sampling the wrong parts of amphibian bodies to detect Bd. I showed that Bd abundance in O. septentrionalis mouthparts declined from Gosner stages 35-42 and increased on epidermis from Gosner stages 38-46. Assuming our findings are general across species, I recommend sampling mouthparts of amphibians less than Gosner stage 41 and hind limbs of amphibians greater than Gosner stage 41. This should provide researchers with guidance on where to sample to maximize detection of Bd. I also investigated whether Trypan blue dye could be used to determine the viability of Bd. I showed that the proportion of zoospores stained with Trypan blue dye matched the proportion of known dead zoospores added to cultures. In contrast, all of the zoosporangia stage (including known dead zoosporangia) of Bd stained blue. These results demonstrate that Trypan blue can be used to determine the viability of Bd zoospores but not zoosporangia. I recommend using Trypan blue to report the number of live zoospores to which hosts are exposed and to help determine whether factors have lethal or sublethal effects on Bd. My work demonstrates that managing exposure to contaminants and biological reservoirs for Bd might provide new hope for imperiled amphibians. Further exploring how pesticides and pathogens are contributing to amphibian declines will allow us to formulate crucial management and conservation plans to begin remediation.

abstract: Emerging infectious diseases (EIDs) in vulnerable populations are a proposed cause of reduced global biodiversity due to local and regional extinctions. Chytridiomycosis, a fungal disease caused by Batrachochytrium dendrobatidis (Bd), is affecting amphibian populations worldwide. Chapter 1 of this thesis reports using lab-raised larval tiger salamanders (Ambystoma tigrinum nebulosum), collected as eggs, to test if Bd infects them. Bd infects metamorphosed tiger salamanders; however, it is currently unknown if larvae can be infected by Bd. Adult frogs tend to host Bd on ventral surfaces and hind legs while tadpoles host Bd in keratinized mouthparts. No research has considered differences in infection between life stages of salamanders. It was hypothesized that Bd can colonize larvae in the same manner as metamorphosed animals. Larval salamanders were inoculated to test if Bd concentrations differ among body regions in larvae compared to metamorphosed salamanders. Larvae can carry Bd with the concentration of Bd varying between body region. Chapter 2 report using native tiger salamanders (Ambystoma tigrinum nebulosum), from northern Arizona and Bd as a study system to test if Bd is native or introduced to Arizona. It was hypothesized that Bd is not endemic to Arizona, but is introduced. There are multiple hypotheses regarding potential routes Bd may have traveled through Arizona and into Mexico. These hypotheses were tested using the Kaibab Plateau in Coconino County, Arizona, as a study site. The plateau is isolated from surrounding areas by the Grand Canyon to the south and the Vermillion Cliffs to the north serving as major biogeographical barriers. It is hypothesized that tiger salamanders are not dispersing into or out of the Kaibab Plateau due to geological restrictions. Bd, therefore, should not be present on salamanders on the Kaibab Plateau due to geological restriction. Tiger salamanders in stock tanks located on the Kaibab as well as preserved museum specimens housed in the Arizona State University Natural History Collection were sampled. The results indicate that Bd occurs at low levels on Kaibab Plateau tiger salamanders.
Dissertation/Thesis
Masters Thesis Biology 2019

Pathology of Australian Native Wildlife brings together in one volume available information on the pathology of Australian native vertebrate wildlife, excluding fish. It provides rapid access to documented information on diseases in Australian wildlife, domiciled either in Australia or overseas. The book comprises 45 chapters, each detailing pathological changes caused by specific pathogens including viruses, bacteria, fungi, protozoa, helminths and ectoparasites, and other injurious agents and conditions such as toxins and neoplasia affecting terrestrial and marine mammals, birds, reptiles and amphibians. Although the aim is to describe morphological (gross and microscopic) changes, the author also indicates history and clinical signs, thus providing guidance as to which lesions should be specifically searched for, and what ancillary testing might be needed to confirm a diagnosis. Illustrated throughout with colour photographs, this will be the essential reference for veterinary pathologists and clinicians, as well as wildlife researchers, zoos, wildlife parks, environmentalists, conservationists and students. Awarded a 2010 Whitley Certificate of Commendation for Zoological Resource.

The amphibian disease chytridiomycosis, caused by two fungal pathogens in the genus Batrachochytrium, has caused the greatest vertebrate biodiversity loss due to disease in recorded history. Both the pathogens and their amphibian hosts are impacted by biotic and abiotic conditions that are rapidly changing due to anthropogenic causes, challenging our understanding of how the host–pathogen relationship will shift in the future. By examining this problem through a physiological lens, we can elucidate the mechanisms driving increased susceptibility to disease. This chapter first examines the physiological tools that can be used by amphibian biologists to measure aspects of immune function, stress physiology, and energy expenditure, and the main environmental drivers of these physiological shifts. Then, we explore case studies that have linked environmental change, immune function, and shifts in disease susceptibility to inform amphibian conservation and management.

Batrachochytrium dendrobatidis (Bd) is a fungal pathogen that infects many different amphibian species, driving some of them to extinction. ‘Batrachochytrium dendrobatidis’ considers the physiology and natural history of this emerging pathogen; its discovery in the late 1990s in Australia and Central America; and the concepts and strategies used to try to determine its origins. Has it arrived relatively recently in the communities it was destroying, or did it lie dormant in those communities for millennia before suddenly beginning to cause harm? The debate between the novel pathogen hypothesis and the endemic pathogen hypothesis, versions of which apply to most emerging diseases of wildlife, is ongoing.