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.     

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.     

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.     

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.     

Towards a Better Understanding of the Use of Probiotics for Preventing Chytridiomycosis in Panamanian Golden Frogs

Populations of native Panamanian golden frogs (Atelopus zeteki) have collapsed due to a recent chytridiomycosis epidemic. Reintroduction efforts from captive assurance colonies are unlikely to be successful without the development of methods to control chytridiomycosis in the wild. In an effort to develop a protective treatment regimen, we treated golden frogs with Janthinobacterium lividum, a skin bacterium that has been used to experimentally prevent chytridiomycosis in North American amphibians. Although J. lividum appeared to colonize A. zeteki skin temporarily, it did not prevent or delay mortality in A. zeteki exposed to Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis. After introduction of J. lividum, average bacterial cell counts reached a peak of 1.7 × 10(6) cells per frog ~2 weeks after treatment but declined steadily after that. When J. lividum numbers declined to ~2.8 × 10(5) cells per frog, B. dendrobatidis infection intensity increased to greater than 13,000 zoospore equivalents per frog. At this point, frogs began to die of chytridiomycosis. Future research will concentrate on isolating and testing antifungal bacterial species from Panama that may be more compatible with Atelopus skin.     

Behaviour of Australian rainforest stream frogs may affect the transmission of chytridiomycosis

The amphibian disease chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis, has been implicated in mass mortalities, population declines and extinctions of amphibians around the world. In almost all cases, amphibian species that have disappeared or declined due to chytridiomycosis coexist with non-declining species. One reason why some species decline from chytridiomycosis and others do not may be interspecific differences in behaviour. Host behaviour could either facilitate or hinder pathogen transmission, and transmission rates in the field are likely to vary among species according the frequency of factors such as physical contact between frogs, contact with infected water and contact with environmental substrates containing B. dendrobatidis. We tracked 117 frogs (28 Litoria nannotis, 27 L. genimaculata and 62 L. lesueuri) at 5 sites where B. dendrobatidis is endemic in the rainforest of tropical northern Queensland and recorded the frequency of frog-to-frog contact and the frequency of contact with stream water and environmental substrates. Frequency of contact with other frogs and with water were highest in L. nannotis, intermediate in L. genimaculata and lowest in L. lesueueri. Environmental substrate use also differed among species. These species-specific opportunities for disease transmission were correlated with conservation status: L. nannotis is the species most susceptible to chytridiomycosis-related declines and L. lesueuri is the least susceptible. Interspecific variation in transmission probability may, therefore, play a large role in determining why chytridiomycosis drives some populations to extinction and not others.     

MS-222 (tricaine methane sulfonate) does not kill the amphibian chytrid fungus Batrachochytrium dendrobatidis

MS-222 (tricaine methane sulfonate) is an agent commonly used to anaesthetise or euthanize amphibians used in experiments. It is administered by immersing the animal to allow absorption through the skin. Chytridiomycosis is an important disease of amphibians and research involves experiments with live animals. Batrachochytrium dendrobatidis, the fungus which causes chytridiomycosis, is located in the skin and therefore the organism should come into contact with MS-222 when it is used. B. dendrobatidis is a sensitive organism which could possibly be killed by MS-222. Hence, results of chytridiomycosis studies in which MS-222 is used could be unreliable. A concentration of 2 g l(-1) and an exposure duration of 1 h is at the high end of the range at which MS-222 would be most commonly used. Exposure to 2 g l(-1) MS-222 for 1 h does not kill B. dendrobatidis cultures, suggesting that MS-222 is safe to use in chytridiomycosis studies.     

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.     

Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis

An overview of the morphology and life cycle of Batrachochytrium dendrobatidis, the cause of chytridiomycosis of amphibians, is presented. We used a range of methods to examine stages of the life cycle in culture and in frog skin, and to assess ultrastructural pathology in the skin of 2 frogs. Methods included light microscopy, transmission electron microscopy with conventional methods as well as high pressure freezing and freeze substitution, and scanning electron microscopy with critical point drying as well as examination of bulk-frozen and freeze-fractured material. Although chytridiomycosis is an emerging disease, B. dendrobatidis has adaptations that suggest it has long been evolved to live within cells in the dynamic tissue of the stratified epidermis. Sporangia developed at a rate that coincided with the maturation of the cell, and fungal discharge tubes usually opened onto the distal surface of epidermal cells of the stratum corneum. A zone of condensed, fibrillar, host cytoplasm surrounded some sporangia. Hyperkeratosis may be due to (1) a hyperplastic response that leads to an increased turnover of epidermal cells, and (2) premature keratinization and death of infected cells.     

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.     

Climate change and outbreaks of amphibian chytridiomycosis in a montane area of Central Spain; is there a link?

Amphibian species are declining at an alarming rate on a global scale in large part owing to an infectious disease caused by the chytridiomycete fungus, Batrachochytrium dendrobatidis. This disease of amphibians has recently emerged within Europe, but knowledge of its effects on amphibian assemblages remains poor. Importantly, little is known about the environmental envelope that is associated with chytridiomycosis in Europe and the potential for climate change to drive future disease dynamics. Here, we use long-term observations on amphibian population dynamics in the Pe?alara Natural Park, Spain, to investigate the link between climate change and chytridiomycosis. Our analysis shows a significant association between change in local climatic variables and the occurrence of chytridiomycosis within this region. Specifically, we show that rising temperature is linked to the occurrence of chytrid-related disease, consistent with the chytrid-thermal-optimum hypothesis. We show that these local variables are driven by general circulation patterns, principally the North Atlantic Oscillation. Given that B. dendrobatidis is known to be broadly distributed across Europe, there is now an urgent need to assess the generality of our finding and determine whether climate-driven epidemics may be expected to impact on amphibian species across the wider region.     

Co-localisation of Batrachochytrium dendrobatidis and keratin for enhanced diagnosis of chytridiomycosis in frogs

Chytridiomycosis is a disease of post-metamorphic frogs caused by the fungus Batrachochytrium dendrobatidis and is associated with large declines in frog populations on a global scale. B. dendrobatidis is found only in the keratinised tissues, which include the mouthparts of healthy tadpoles. The epidermis of infected post-metamorphic frogs is thickened (hyperkeratosis) and the superficial layer can sometimes slough. Diagnosis is most commonly performed on stained sections of toe clips or ventral skin. Accurate interpretation can be difficult and requires a high level of expertise, particularly in infected animals exhibiting hyperkeratosis with sloughing. Misdiagnosis can occur when zoosporangia of B. dendrobatidis are shed with the superficial keratin layers. We have developed a staining protocol based on previously described methods to detect both B. dendrobatidis and keratin, to improve the sensitivity and specificity of diagnosis of chytridiomycosis by inexperienced diagnosticians.     

Persistence of the emerging pathogen Batrachochytrium dendrobatidis outside the amphibian host greatly increases the probability of host extinction

Pathogens do not normally drive their hosts to extinction; however, Batrachochytrium dendrobatidis, which causes amphibian chytridiomycosis, has been able to do so. Theory predicts that extinction can be caused by long-lived or saprobic free-living stages. The hypothesis that such a stage occurs in B. dendrobatidis is supported by the recent discovery of an apparently encysted form of the pathogen. To investigate the effect of a free-living stage of B. dendrobatidis on host population dynamics, a mathematical model was developed to describe the introduction of chytridiomycosis into a breeding population of Bufo bufo, parametrized from laboratory infection and transmission experiments. The model predicted that the longer that B. dendrobatidis was able to persist in water, either due to an increased zoospore lifespan or saprobic reproduction, the more likely it was that it could cause local B. bufo extinction (defined as decrease below a threshold level). Establishment of endemic B. dendrobatidis infection in B. bufo, with severe host population depression, was also possible, in agreement with field observations. Although this model is able to predict clear trends, more precise predictions will only be possible when the life history of B. dendrobatidis, including free-living stages of the life cycle, is better understood.     

Experimental transmission of cutaneous chytridiomycosis in dendrobatid frogs

In a series of three experiments during March-October, 1998, two species of captive-bred poison dart frogs (Dendrobates tinctorius and D. auratus) were exposed to Batrachochytrium dendrobatidis, a recently-described chytridiomycete fungus (chytrid) that was originally isolated from a blue poison dart frog (D. azureus). All frogs exposed to the chytrids developed a fatal skin disease, whereas none of the control frogs developed skin lesions. The most consistent clinical sign in chytrid-exposed frogs was excessive shedding of skin. Gross lesions were subtle, usually affected the legs and ventrum, and consisted of mild skin thickening and discoloration. Microscopic examination of shed skin pieces and/or skin imprints demonstrated the presence of chytrids and was used for ante mortem and post mortem confirmation of chytrid infection. Histologically, there was epidermal hyperkeratosis, hyperplasia, and hypertrophy associated with low to moderate numbers of chytrids in the keratinized layers. These experiments demonstrated that Batrachochytrium dendrobatidis can be a fatal pathogen in poison dart frogs. The experimentally-induced disease in these frogs resembled cases of cutaneous chytridiomycosis that have recently been described in several other species of captive and wild amphibians.     

Populations of a susceptible amphibian species can grow despite the presence of a pathogenic chytrid fungus

Disease can be an important driver of host population dynamics and epizootics can cause severe host population declines. Batrachochytrium dendrobatidis (Bd), the pathogen causing amphibian chytridiomycosis, may occur epizootically or enzootically and can harm amphibian populations in many ways. While effects of Bd epizootics are well documented, the effects of enzootic Bd have rarely been described. We used a state-space model that accounts for observation error to test whether population trends of a species highly susceptible to Bd, the midwife toad Alytes obstetricans, are negatively affected by the enzootic presence of the pathogen. Unexpectedly, Bd had no negative effect on population growth rates from 2002-2008. This suggests that negative effects of disease on individuals do not necessarily translate into negative effects at the population level. Populations of amphibian species that are susceptible to the emerging disease chytridiomycosis can persist despite the enzootic presence of the pathogen under current environmental conditions.     

Examining the evidence for chytridiomycosis in threatened amphibian species

Extinction risks are increasing for amphibians due to rising threats and minimal conservation efforts. Nearly one quarter of all threatened/extinct amphibians in the IUCN Red List is purportedly at risk from the disease chytridiomycosis. However, a closer look at the data reveals that Batrachochytrium dendrobatidis (the causal agent) has been identified and confirmed to cause clinical disease in only 14% of these species. Primary literature surveys confirm these findings; ruling out major discrepancies between Red List assessments and real-time science. Despite widespread interest in chytridiomycosis, little progress has been made between assessment years to acquire evidence for the role of chytridiomycosis in species-specific amphibian declines. Instead, assessment teams invoke the precautionary principle when listing chytridiomycosis as a threat. Precaution is valuable when dealing with the world's most threatened taxa, however scientific research is needed to distinguish between real and predicted threats in order to better prioritize conservation efforts. Fast paced, cost effective, in situ research to confirm or rule out chytridiomycosis in species currently hypothesized to be threatened by the disease would be a step in the right direction. Ultimately, determining the manner in which amphibian conservation resources are utilized is a conversation for the greater conservation community that we hope to stimulate here.     

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.     

Retreat sites of rain forest stream frogs are not a reservoir for Batrachochytrium dendrobatidis in northern Queensland, Australia

Chytridiomycosis is a potentially fatal disease of amphibians caused by Batrachochytrium dendrobatidis, and is implicated in declines and extinctions of amphibian populations and species around the world. To cause local host extinction, a disease organism must persist at low host densities. One mechanism that could facilitate this is the ability to persist in the environment. In the laboratory, B. dendrobatidis spreads by both frog-to-frog and environment-to-frog transmission, and can persist on a number of biotic substrates. In the field, B. dendrobatidis has been detected on environmental samples taken during an epidemic, but it is not known if it persists in the environment when endemic. Retreat sites of 2 species of Australian rain forest stream frogs Litoria lesueuri and L. nannotis were sampled 0 to 3 d after occupation during the wet and dry seasons in northern Queensland, Australia, where chytridiomycosis has been endemic for at least 10 yr. The intensity and prevalence of infection in frogs during sampling were comparatively low compared with epidemics. Diagnostic quantitative polymerase chain reaction did not detect B. dendrobatidis in any retreat site samples. It thus appears that retreat sites are not a major environmental source of infection when B. dendrobatidis occurs at low prevalence and intensity on frogs. This suggests that control efforts may not need to eliminate the organism from the environment, at least when prevalence and intensity of infection are low in frogs. Simply treating hosts may be effective at controlling the disease in the wild.     

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.