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

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

Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin

Global amphibian declines are in part driven by the chytrid fungus Batrachochytrium dendrobatidis, causing superficial dermatomycosis with epidermal hyperplasia and hyperkeratosis in infected amphibians. The susceptibility to chytridiomycosis and the severity of epidermal lesions in amphibians with chytridiomycosis are not consistent across species or even among individuals. Severe infections cause death of the animal most likely through disturbance of ion homeostasis. The mechanism by which this superficial skin infection results in epidermal lesions has so far eluded precise definition. It was the aim of this study to unravel how B. dendrobatidis causes alterations that affect skin integrity. Exposure of Xenopus laevis skin to B. dendrobatidis zoospore supernatant using skin explants and Ussing chambers caused rapid disruption of intercellular junctions, demonstrated using histology and transmission electron microscopy. The loss of intercellular junctions led to detachment-induced cell apoptosis, or anoikis. The zoospore supernatant induced neither apoptosis nor necrosis in isolated primary keratinocytes of X. laevis. This supports the idea that the loss of cell contacts triggered apoptosis in the skin explants. Mass spectrometric analysis of the protein composition of the supernatant revealed a complex mixture, including several new virulence associated proteins, such as proteases, biofilm-associated proteins and a carotenoid ester lipase. Protease and lipase activity of the supernatant was confirmed with a protease and lipase assay.In conclusion, B. dendrobatidis zoospores produce a complex mixture of proteins that quickly disturbs epidermal intercellular junctions leading to anoikis in the anuran skin. The role of the identified proteins in this process remains to be determined.     

Host invasion by Batrachochytrium dendrobatidis: fungal and epidermal ultrastructure in model anurans

The chytridiomycete fungus Batrachochytrium dendrobatidis (Bd) colonizes mouthparts of amphibian larvae and superficial epidermis of post-metamorphic amphibians, causing the disease chytridiomycosis. Fungal growth within host cells has been documented by light and transmission electron microscopy; however, entry of the fungus into host cells has not. Our objective was to document how Bd enters host cells in the wood frog Lithobates sylvaticus, a species at high mortality risk for chytridiomycosis, and the bullfrog L. catesbeianus, a species at low mortality risk for chytridiomycosis. We inoculated frogs and documented infection with transmission electron microscopy. Zoospores encysted on the skin surface and produced morphologically similar germination tubes in both host species that penetrated host cell membranes and enabled transfer of zoospore contents into host cells. Documenting fungal and epidermal ultrastructure during host invasion furthers our understanding of Bd development and the pathogenesis of chytridiomycosis.     

Interactions between Batrachochytrium dendrobatidis and its amphibian hosts: a review of pathogenesis and immunity

The fungus Batrachochytrium dendrobatidis (Bd) causes a lethal skin disease of amphibians, chytridiomycosis, which has caused catastrophic amphibian die-offs around the world. This review provides a summary of host characteristics, pathogen characteristics and host-pathogen responses to infection that are important for understanding disease development.     

Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses

Innate immune mechanisms of defense are especially important to ectothermic vertebrates in which adaptive immune responses may be slow to develop. One innate defense in amphibian skin is the release of abundant quantities of antimicrobial peptides. Chytridiomycosis is an emerging infectious disease of amphibians caused by the skin fungus, Batrachochytrium dendrobatidis . Susceptibility to chytridiomycosis varies among species, and mechanisms of disease resistance are not well understood. Previously, we have shown that Australian and Panamanian amphibian species that possess skin peptides that effectively inhibit the growth of B. dendrobatidis in vitro tend to survive better in the wild or are predicted to survive the first encounter with this lethal pathogen. For most species, it has been difficult to experimentally infect individuals with B. dendrobatidis and directly evaluate both survival and antimicrobial peptide defenses. Here, we demonstrate differences in susceptibility to chytridiomycosis among four Australian species ( Litoria caerulea, Litoria chloris, Mixophyes fasciolatus and Limnodynastes tasmaniensis ) after experimental infection with B. dendrobatidis , and show that the survival rate increases with the in vitro effectiveness of the skin peptides. We also observed that circulating granulocyte, but not lymphocyte, counts differed between infected and uninfected Lit. chloris . This suggests that innate granulocyte defenses may be activated by pathogen exposure. Taken together, our data suggest that multiple innate defense mechanisms are involved in resistance to chytridiomycosis, and the efficacy of these defenses varies by amphibian species.     

Early-spring survey for Batrachochytrium dendrobatidis in wild Rana dybowskii in Heilongjiang Province, China

Batrachochytrium dendrobatidis has been investigated worldwide because of its importance in population declines in multiple species of amphibians; however, little is known regarding the disease status of all native amphibian species in China. The present study is the first survey of chytridiomycosis in free-ranging amphibian populations in China, and it examined the possible presence of B. dendrobatidis in Rana dybowskii in northeastern China (Heilongjiang Province). R. dybowskii is mainly distributed in the northeast part of China and is intensively hunted for human consumption, making populations vulnerable to extirpation in the event of additional stresses from disease epidemics. The survey was performed in early spring of 2008, using a PCR assay, histological examination of skin samples, and zoospore culture. In total, 191 frogs were examined; thus, a 95% confidence limit for prevalence of 1.57% was selected. Our results demonstrate that R. dybowskii is currently free from chytridiomycosis in Heilongjiang, even though the natural conditions of the sampling sites are suitable for the occurrence of B. dendrobatidis. Central and local governments should implement strict management measures to prevent the escape of non-native commercial amphibian species into this area, which might endanger local populations of native species.     

Large-scale seasonal variation in the prevalence and severity of chytridiomycosis

The chytrid fungus Batrachochytrium dendrobatidis has been implicated as the causative agent of mass mortalities, population declines and the extinctions of amphibian species worldwide. Although several studies have shown that the prevalence of chytridiomycosis (the disease caused by the fungus) increases in cooler months, the magnitude and timing of these seasonal fluctuations have yet to be accurately quantified. We conducted disease sampling in a single population of stony creek frogs Litoria wilcoxii on 13 occasions over a 21-month period and used quantitative real-time polymerase chain reaction to detect and quantify the number of B. dendrobatidis zoospores present on samples. Disease prevalence varied significantly across sampling sessions, peaking at 58.3% (in early spring) and dropping to as low as 0% on two occasions (late summer and early autumn). There was a significant negative relationship between disease prevalence and mean air temperature in the 30 days prior to sampling. These large-scale seasonal fluctuations in chytridiomycosis levels will strongly influence conservation programs and amphibian disease research.     

Amphibian immune defenses against chytridiomycosis: impacts of changing environments

Eco-immunology is the field of study that attempts to understand the functions of the immune system in the context of the host's environment. Amphibians are currently suffering devastating declines and extinctions in nearly all parts of the world due to the emerging infectious disease chytridiomycosis caused by the chytrid fungus, Batrachochytrium dendrobatidis. Because chytridiomycosis is a skin infection and remains confined to the skin, immune defenses of the skin are critical for survival. Skin defenses include secreted antimicrobial peptides and immunoglobulins as well as antifungal metabolites produced by symbiotic skin bacteria. Low temperatures, toxic chemicals, and stress inhibit the immune system and may impair natural defenses against B. dendrobatidis. Tadpoles' mouth parts can be infected by B. dendrobatidis. Damage to the mouth parts can impair growth, and the affected tadpoles maintain the pathogen in the environment even when adults have dispersed. Newly metamorphosing frogs appear to be especially vulnerable to infection and to the lethal effects of this pathogen because the immune system undergoes a dramatic reorganization at metamorphosis, and postmetamorphic defenses are not yet mature. Here we review our current understanding of amphibian immune defenses against B. dendrobatidis and the ability of the pathogen to resist those defenses. We also briefly review what is known about the impacts of temperature, environmental chemicals, and stress on the host-pathogen interactions and suggest future directions for research.     

Pathophysiology in mountain yellow-legged frogs (Rana muscosa) during a chytridiomycosis outbreak

The disease chytridiomycosis is responsible for declines and extirpations of amphibians worldwide. Chytridiomycosis is caused by a fungal pathogen (Batrachochytrium dendrobatidis) that infects amphibian skin. Although we have a basic understanding of the pathophysiology from laboratory experiments, many mechanistic details remain unresolved and it is unknown if disease development is similar in wild amphibian populations. To gain a better understanding of chytridiomycosis pathophysiology in wild amphibian populations, we collected blood biochemistry measurements during an outbreak in mountain yellow-legged frogs (Rana muscosa) in the Sierra Nevada Mountains of California. We found that pathogen load is associated with disruptions in fluid and electrolyte balance, yet is not associated with fluctuations acid-base balance. These findings enhance our knowledge of the pathophysiology of this disease and indicate that disease development is consistent across multiple species and in both laboratory and natural conditions. We recommend integrating an understanding of chytridiomycosis pathophysiology with mitigation practices to improve amphibian conservation.     

Effect of temperature on host response to Batrachochytrium dendrobatidis infection in the mountain yellow-legged frog (Rana muscosa)

The pathogenic chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis, has been implicated in declines of amphibian populations throughout the world, including declines and extinctions of local populations of mountain yellow-legged frogs, Rana muscosa, in the California Sierra Nevada. Previous studies have shown B. dendrobatidis achieves its maximum growth rate in culture in the temperature range of 17-25 C, and exposure to very high temperatures can clear frogs of B. dendrobatidis infection. Here we present the results of a laboratory experiment in which experimentally infected R. muscosa tadpoles were followed through metamorphosis at temperatures of 17 and 22 C. All infected animals developed clinical disease within a similar time frame. However, frogs housed at 22 C exhibited a significantly lower mortality than those housed at 17 C. Within 35 days after metamorphosis, 50% of the frogs housed at 22 C died, while 95% of the frogs housed at 17 C died. Clinical signs subsided in the surviving frogs at 22 C, despite persistent infection. Because both temperatures are within the optimal thermal range for growth of B. dendrobatidis, we propose that the difference in outcome indicates the effect of temperature on the host's resistance to chytridiomycosis, rather than an effect on the fungus alone.     

Frog skin epithelium: electrolyte transport and chytridiomycosis

One unique physiological characteristic of frogs is that their main route for intake of water is across the skin. In these animals, the skin acts in concert with the kidney and urinary bladder to maintain electrolyte homeostasis. Water absorption across the skin is driven by the osmotic gradient that develops as a consequence of solute transport. Our recent study demonstrated that chytridiomycosis, an infection of amphibian skin by the fungal pathogen, Batrachochytrium dendrobatidis, inhibits epithelial Na(+) channels, attenuating Na(+) absorption through the skin. In frogs that become severely affected by this fungus, systemic depletion of Na(+), K(+) and Cl(-) is thought to cause deterioration of cardiac electrical function, leading to cardiac arrest. Here we review the ion transport mechanisms of frog skin, and discuss the effect of chytridiomycosis on these mechanisms.     

Environmental detection of Batrachochytrium dendrobatidis in a temperate climate

The aetiological agent of amphibian chytridiomycosis Batrachochytrium dendrobatidis is a primary cause of amphibian population declines. Current surveillance is based on the detection of B. dendrobatidis in its host but in vitro work suggests infective stages may survive in the abiotic environment for at least 3 mo. We describe here a surveillance system using filtration and quantitative PCR that can detect B. dendrobatidis in small (< 1 l) volumes of water. After assessing the analytical sensitivity of the protocol for both water and sediment samples in the laboratory, we analyzed environmental samples from the Sierra de Guadarrama mountain range in Spain at locations associated with chytrid-related die-offs and at other sites across Spain. B. dendrobatidis was detected in samples from 64% of the ponds in the Sierra de Guadarrama and at 2 sites outside this region, showing that levels of amphibian exposure to B. dendrobatidis are spatially heterogeneous. In experimental microcosms, we detected B. dendrobatidis for up to 12 wk, though we found no evidence for an overall increase in biomass. Our results emphasise the need to further investigate the life cycle of B. dendrobatidis to more completely understand the epidemiology of this emerging pathogen.     

Chytrid fungus in frogs from an equatorial African montane forest in western Uganda

Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis, was found in 24 of 109 (22%) frogs from Kibale National Park, western Uganda, in January and June 2006, representing the first account of the fungus in six species and in Uganda. The presence of B. dendrobatidis in an equatorial African montane forest raises conservation concerns, considering the high amphibian diversity and endemism characteristic of such areas and their ecological similarity to other regions of the world experiencing anuran declines linked to chytridiomycosis.     

Reptiles as potential vectors and hosts of the amphibian pathogen Batrachochytrium dendrobatidis in Panama

Chytridiomycosis, the disease caused by Batrachochytrium dendrobatidis, is considered to be a disease exclusively of amphibians. However, B. dendrobatidis may also be capable of persisting in the environment, and non-amphibian vectors or hosts may contribute to disease transmission. Reptiles living in close proximity to amphibians and sharing similar ecological traits could serve as vectors or reservoir hosts for B. dendrobatidis, harbouring the organism on their skin without succumbing to disease. We surveyed for the presence of B. dendrobatidis DNA among 211 lizards and 8 snakes at 8 sites at varying elevations in Panama where the syntopic amphibians were at pre-epizootic, epizootic or post-epizootic stages of chytridiomycosis. Detection of B. dendrobatidis DNA was done using qPCR analysis. Evidence of the amphibian pathogen was present at varying intensities in 29 of 79 examined Anolis humilis lizards (32%) and 9 of 101 A. lionotus lizards (9%), and in one individual each of the snakes Pliocercus euryzonus, Imantodes cenchoa, and Nothopsis rugosus. In general, B. dendrobatidis DNA prevalence among reptiles was positively correlated with the infection prevalence among co-occurring anuran amphibians at any particular site (r = 0.88, p = 0.004). These reptiles, therefore, may likely be vectors or reservoir hosts for B. dendrobatidis and could serve as disease transmission agents. Although there is no evidence of B. dendrobatidis disease-induced declines in reptiles, cases of coincidence of reptile and amphibian declines suggest this potentiality. Our study is the first to provide evidence of non-amphibian carriers for B. dendrobatidis in a natural Neotropical environment.     

Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide, associated with declines in amphibian populations. Diagnosis of chytridiomycosis to date has largely relied upon histological and immunohistochemical examination of toe clips. This technique is invasive and insensitive particularly at early stages of infection when treatment may be possible. We have developed a real-time PCR Taqman assay that can accurately detect and quantify one zoospore in a diagnostic sample. This assay will assist the early detection of B. dendrobatidis in both captive and wild populations, with a high degree of sensitivity and specificity, thus facilitating treatment and protection of endangered populations, monitoring of pristine environments and preventing further global spread via amphibian trade.     

Urinary corticosterone metabolites and chytridiomycosis disease prevalence in a free-living population of male Stony Creek frogs (Litoria wilcoxii)

The emerging amphibian disease chytridiomycosis, which is caused by the fungal pathogen (Batrachochytrium dendrobatidis, Bd), has caused mass mortalities of native amphibian populations globally. There have been no previous studies on the relationships between stress hormones in free-living amphibians and Bd infections. In this study, we measured urinary corticosterone metabolite concentrations and Bd infections within free-living populations of male Stony Creek frog (Litoria wilcoxii) in Queensland, Australia. Prevalence of Bd zoospores from frog skin swabs was quantified using a real-time quantitative PCR technique. A urinary corticosterone enzyme-immunoassay (EIA) was validated using adrenocorticotropic hormone (ACTH) challenge. Urinary corticosterone concentrations of male frogs increased within 1-2 days after ACTH challenge and returned to baseline levels within 3 days post-ACTH injection. None of the frogs showed any rise in urinary corticosterone after saline injections. Individual male frogs showed either low or high baseline corticosterone concentrations. Male frogs identified as positive for Bd infection had significantly higher baseline urinary corticosterone concentrations in comparison to Bd negative male frogs. Urinary corticosterone EIA provides a reliable indication of stress in this frog species and this non-invasive physiological tool can be used to further assess the dynamics of Bd infections and physiological stress responses in other native amphibians.     

Batrachochytrium dendrobatidis in amphibians confiscated from illegal wildlife trade and used in an ex situ breeding program in Brazil

This paper describes an outbreak of chytridiomycosis affecting a group of Dendrobates tinctorius, a Neotropical anuran species, confiscated from the illegal wildlife trade and housed in a private zoo in Brazil as part of an ex situ breeding program. We examined histological sections of the skin of 30 D. tinctorius and 20 Adelphobates galactonotus individuals. Twenty D. tinctorius (66.7%) and none of the A. galactonotus were positive for Batrachochytrium dendrobatidis (Bd). Multiple development stages of Bd infection were observed. The reasons for the inter-specific difference in the rate of infection could not be determined, and further studies are advised. Because the examined population consisted of confiscated frogs, detailed epidemiological aspects could not be investigated, and the source of the fungus remains uncertain. The existence of ex situ amphibian populations is important for protecting species at higher risk in the wild, and ex situ amphibian conservation and breeding programs in Brazil may be established using confiscated frogs as founders. However, this paper alerts these programs to the urgency of strict quarantine procedures to prevent the introduction of potential pathogens, particularly Bd, into ex situ conservation programs.     

First Detection of Batrachochytrium dendrobatidis in Wild Frogs from Bangladesh

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

Bd on the Beach: High Prevalence of Batrachochytrium dendrobatidis in the Lowland Forests of Gorgona Island (Colombia, South America)

The amphibian chytrid fungus, Batrachochytrium dendrobatidis, Bd, has been implicated in the decimation and extinction of many amphibian populations worldwide, especially at mid and high elevations. Recent studies have demonstrated the presence of the pathogen in the lowlands from Australia and Central America. We extend here its elevational range by demonstrating its presence at the sea level, in the lowland forests of Gorgona Island, off the Pacific coast of Colombia. We conducted two field surveys, separated by four?years, and diagnosed Bd by performing polymerase chain reactions on swab samples from the skin of five amphibian species. All species, including the Critically Endangered Atelopus elegans, tested positive for the pathogen, with prevalences between 3.9?% in A. elegans (in 2010) and 52?% in Pristimantis achatinus. Clinical signs of chytridiomycosis were not detected in any species. To our knowledge, this is the first report of B. dendrobatidis in tropical lowlands at sea level, where temperatures may exceed optimal growth temperatures of this pathogen. This finding highlights the need to understand the mechanisms allowing the interaction between frogs and pathogen in lowland ecosystems.     

Keratinocytes are cell targets of West Nile virus in vivo

West Nile virus (WNV) replicates in the skin; however, cell targets in the skin have not been identified. In the current studies, WNV infected the epidermis and adnexal glands of mouse skin, and the epidermal cells were identified as keratinocytes by double labeling for WNV antigen and keratin 10. Inoculation of mice with WNV replicon particles resulted in high levels of replication in the skin, suggesting that keratinocytes are an initial target of WNV. In addition, primary keratinocytes produced infectious virus in vitro. In conclusion, keratinocytes are cell targets of WNV in vivo and may play an important role in pathogenesis.