Short-term exposure to warm microhabitats could explain amphibian persistence with Batrachochytrium dendrobatidis

Environmental conditions can alter the outcomes of symbiotic interactions. Many amphibian species have declined due to chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), but many others persist despite high Bd infection prevalence. This indicates that Bd's virulence is lower, or it may even be a commensal, in some hosts. In the Australian Wet Tropics, chytridiomycosis extirpated Litoria nannotis from high-elevation rain forests in the early 1990 s. Although the species is recolonizing many sites, no population has fully recovered. Litoria lorica disappeared from all known sites in the early 1990 s and was thought globally extinct, but a new population was discovered in 2008, in an upland dry forest habitat it shares with L. nannotis. All frogs of both species observed during three population censuses were apparently healthy, but most carried Bd. Frogs perch on sun-warmed rocks in dry forest streams, possibly keeping Bd infections below the lethal threshold attained in cooler rain forests. We tested whether short-term elevated temperatures can hamper Bd growth in vitro over one generation (four days). Simulating the temperatures available to frogs on strongly and moderately warmed rocks in dry forests, by incubating cultures at 33°C for one hour daily, reduced Bd growth below that of Bd held at 15°C constantly (representing rain forest habitats). Even small decreases in the exponential growth rate of Bd on hosts may contribute to the survival of frogs in dry forests.     

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.     

Strain differences in the amphibian chytrid Batrachochytrium dendrobatidis and non-permanent, sub-lethal effects of infection

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is likely the cause of numerous recent amphibian population declines worldwide. While the fungus is generally highly pathogenic to amphibians, hosts express a wide range of responses to infection, probably due to variation among hosts and environmental conditions, but possibly also due to variation in Bd. We investigated variation in Bd by exposing standardized host groups to 2 Bd strains in a uniform environment. All exposed frogs became infected, but subsequent lethal and sub-lethal (weight loss) responses differed among groups. These results demonstrate variation in Bd and suggest variation occurs even at small geographical scales, likely explaining some of the variation in host responses. With lower than expected mortality among infected frogs, we continued our study opportunistically to determine whether or not frogs could recover from chytridiomycosis. Using heat, we cleared infection from half of the surviving frogs, leaving the other half infected, then continued to monitor mortality and weight. Mortality ceased among disinfected frogs but continued among infected frogs. Disinfected frogs gained weight significantly more than infected frogs, to the point of becoming indistinguishable from controls, demonstrating that at least some of the effects of sub-lethal chytridiomycosis on hosts can be non-permanent and reversible.     

Survey protocol for detecting chytridiomycosis in all Australian frog populations

Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused the decline and extinction of frogs, but the distribution of Bd is not completely known. This information is crucial to implementing appropriate quarantine strategies, preparing for outbreaks of chytridiomycosis due to introduction of Bd, and for directing conservation actions towards affected species. This survey protocol provides a simple and standard method for sampling all frog populations in Australia to maximise the chances of detecting Bd. In order to structure and prioritise the protocol, areas are divided by bioregion and frog species are allocated depending on the water bodies they utilize into 3 groups representing different levels of risk of exposure to Bd. Sixty individuals per population need to be tested to achieve 95% certainty of detecting 1 positive frog, based on the minimum apparent prevalence of > or =5% in infected Australian frog populations and using a quantitative real-time TaqMan PCR test. The appropriate season to sample varies among bioregions and will ideally incorporate temperatures favourable for chytridiomycosis (e.g. maximum air temperatures generally <27 degrees C). Opportunistic collection and testing of sick frogs and tadpoles with abnormal mouth-parts should also be done to increase the probability of detecting Bd. The survey priorities in order are (1) threatened species that may have been exposed to Bd, (2) bioregions surrounding infected bioregions/ecological groups, and (3) species of frogs of unknown infection status in infected bioregions. Within these priority groups, sampling should first target ecological groups and species likely to be exposed to Bd, such as those associated with permanent water, and areas within bioregions that have high risk for Bd as indicated by climatic modelling. This protocol can be adapted for use in other countries and a standard protocol will enable comparison among amphibian populations globally.     

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.     

Distribution and Pathogenicity of Batrachochytrium dendrobatidis in Boreal Toads from the Grand Teton Area of Western Wyoming

The pathogen Batrachochytrium dendrobatidis (Bd), which causes the skin disease chytridiomycosis, has been linked to amphibian population declines and extinctions worldwide. Bd has been implicated in recent declines of boreal toads, Bufo boreas boreas, in Colorado but populations of boreal toads in western Wyoming have high prevalence of Bd without suffering catastrophic mortality. In a field and laboratory study, we investigated the prevalence of Bd in boreal toads from the Grand Teton ecosystem (GRTE) in Wyoming and tested the pathogenicity of Bd to these toads in several environments. The pathogen was present in breeding adults at all 10 sites sampled, with a mean prevalence of 67%. In an experiment with juvenile toadlets housed individually in wet environments, 10⁶ zoospores of Bd isolated from GRTE caused lethal disease in all Wyoming and Colorado animals within 35 days. Survival time was longer in toadlets from Wyoming than Colorado and in toadlets spending more time in dry sites. In a second trial involving Colorado toadlets exposed to 35% fewer Bd zoospores, infection peaked and subsided over 68 days with no lethal chytridiomycosis in any treatment. However, compared with drier aquaria with dry refuges, Bd infection intensity was 41% higher in more humid aquaria and 81% higher without dry refuges available. Our findings suggest that although widely infected in nature, Wyoming toads may escape chytridiomycosis due to a slight advantage in innate resistance or because their native habitat hinders Bd growth or provides more opportunities to reduce pathogen loads behaviorally than in Colorado.     

Temperature, hydric environment, and prior pathogen exposure alter the experimental severity of chytridiomycosis in boreal toads

Prevalence of the pathogen Batrachochytrium dendrobatidis (Bd), implicated in amphibian population declines worldwide, is associated with habitat moisture and temperature, but few studies have varied these factors and measured the response to infection in amphibian hosts. We evaluated how varying humidity, contact with water, and temperature affected the manifestation of chytridiomycosis in boreal toads Anaxyrus (Bufo) boreas boreas and how prior exposure to Bd affects the likelihood of survival after re-exposure, such as may occur seasonally in long-lived species. Humidity did not affect survival or the degree of Bd infection, but a longer time in contact with water increased the likelihood of mortality. After exposure to approximately 10(6) Bd zoospores, all toads in continuous contact with water died within 30 d. Moreover, Bd-exposed toads that were disease-free after 64 d under dry conditions, developed lethal chytridiomycosis within 70 d of transfer to wet conditions. Toads in unheated aquaria (mean = 15 degrees C) survived less than 48 d, while those in moderately heated aquaria (mean = 18 degrees C) survived 115 d post-exposure and exhibited behavioral fever, selecting warmer sites across a temperature gradient. We also found benefits of prior Bd infection: previously exposed toads survived 3 times longer than Bd-na?ve toads after re-exposure to 106 zoospores (89 vs. 30 d), but only when dry microenvironments were available. This study illustrates how the outcome of Bd infection in boreal toads is environmentally dependent: when continuously wet, high reinfection rates may overwhelm defenses, but periodic drying, moderate warming, and previous infection may allow infected toads to extend their survival.     

Detection of Batrachochytrium dendrobatidis in Mexican Bolitoglossine Salamanders Using an Optimal Sampling Protocol

The role of the chytrid fungus Batrachochytrium dendrobatidis (Bd), which is the causal agent of chytridiomycosis, in the declines of Central American bolitoglossine salamanders is unknown. Here we establish a swabbing protocol to maximize the detection probability of Bd in salamanders. We then used this protocol to examine captive and wild Mexican bolitoglossine salamanders of 14 different species for the presence of Bd. Of the seven body parts sampled, the pelvic region, hindlimbs, forelimbs, and the ventral side of the tail had the most Bd per surface area and thus might provide the best sampling regions of salamanders to detect Bd infections. Sixteen out of 33 (48%) of the dead captive salamanders had Bd infections and epidermal hyperkeratosis, whereas none of the 28 clinically healthy captive animals were infected. Nine out of 17 (53%) of the wild salamanders carried low zoospore loads of Bd but had no clinical signs of disease. The high prevalence of Bd in dead captive salamanders, its absence in clinically healthy living ones and its presence in wild salamanders is consistent with Bd being involved in recent bolitoglossine population declines, but further studies would be required to draw a causal link.     

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.     

Seasonality of Batrachochytrium dendrobatidis infection in direct-developing frogs suggests a mechanism for persistence

Batrachochytrium dendrobatidis (Bd), a disease-causing amphibian-specific fungus, is widely distributed in Puerto Rico, but is restricted to elevations above 600 m. The effect of this pathogen in the wild was studied by monitoring Eleutherodactylus coqui and E. portoricensis in 2 upland forests at El Yunque, a site characterized by historic population declines in the presence of chytridiomycosis. We tested a potential synergistic interaction between climate and Bd by measuring prevalence of infection and level of infection per individual sampled (number of zoospores), across the dry and wet seasons for 2 yr (between 2005 and 2007). Infection levels in adult frogs were significantly higher during the dry season in both species studied, suggesting a cyclic pattern of dry/ cool-wet/warm climate-driven synergistic interaction. These results are consistent with ex situ experiments in which E. coqui infected with Bd were more susceptible to chytridiomycosis when subjected to limited water treatments resembling drought. Long-term data on the prevalence of Bd in the populations studied versus intensity of infection in individual frogs provided contradictory information. However, the conflicting nature of these data was essential to understand the status of Bd in the species and geographical area studied. We conclude that in Puerto Rico, Bd is enzootic, and vulnerability of eleutherodactylid frogs to this pathogen is related to seasonal climatic variables. Our data suggest a mechanism by which this disease can persist in tropical frog communities without decimation of its hosts, but that complex interactions during severe droughts may lead to population crashes.     

Batrachochytrium dendrobatidis: requirement for further isolate collection and archiving

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis, which is lethal to many species of amphibians worldwide. Many studies have investigated the epidemiology of chytridiomycosis in amphibian populations, but few have considered possible host-pathogen coevolution. More specifically, investigations focused on the evolution of Bd, and the link with Bd virulence, are needed. Such studies, which may be important for conservation management of amphibians, depend on access to Bd isolates. Here we provide a summary of known Bd isolates that have been collected and archived in various locations around the world. Of 257 Bd isolates, we found that 53% originate from ranids in the United States. In many cases, detailed information on isolate origin is unavailable, and it is unknown how many isolates are cryo-archived. We suggest the creation of a centralized database of isolate information, and we urge researchers and managers to isolate and archive Bd to facilitate future research on chytridiomycosis.     

High Prevalence of Batrachochytrium dendrobatidis in Wild Populations of Lowland Leopard Frogs Rana yavapaiensis in Arizona

Batrachochytrium dendrobatidis (Bd) is a fungus that can potentially lead to chytridiomycosis, an amphibian disease implicated in die-offs and population declines in many regions of the world. Winter field surveys in the last decade have documented die-offs in populations of the lowland leopard frog Rana yavapaiensis with chytridiomycosis. To test whether the fungus persists in host populations between episodes of observed host mortality, we quantified field-based Bd infection rates during nonwinter months. We used PCR to sample for the presence of Bd in live individuals from nine seemingly healthy populations of the lowland leopard frog as well as four of the American bullfrog R. catesbeiana (a putative vector for Bd) from Arizona. We found Bd in 10 of 13 sampled populations. The overall prevalence of Bd was 43% in lowland leopard frogs and 18% in American bullfrogs. Our results suggest that Bd is widespread in Arizona during nonwinter months and may become virulent only in winter in conjunction with other cofactors, or is now benign in these species. The absence of Bd from two populations associated with thermal springs (water >30°C), despite its presence in nearby ambient waters, suggests that these microhabitats represent refugia from Bd and chytridiomycosis.     

Surviving Chytridiomycosis: Differential Anti-Batrachochytrium dendrobatidis Activity in Bacterial Isolates from Three Lowland Species of Atelopus

In the Neotropics, almost every species of the stream-dwelling harlequin toads (genus Atelopus) have experienced catastrophic declines. The persistence of lowland species of Atelopus could be explained by the lower growth rate of Batrachochytrium dendrobatidis (Bd) at temperatures above 25°C. We tested the complementary hypothesis that the toads' skin bacterial microbiota acts as a protective barrier against the pathogen, perhaps delaying or impeding the symptomatic phase of chytridiomycosis. We isolated 148 cultivable bacterial strains from three lowland Atelopus species and quantified the anti-Bd activity through antagonism assays. Twenty-six percent (38 strains representing 12 species) of the bacteria inhibited Bd growth and just two of them were shared among the toad species sampled in different localities. Interestingly, the strongest anti-Bd activity was measured in bacteria isolated from A. elegans, the only species that tested positive for the pathogen. The cutaneous bacterial microbiota is thus likely a fitness-enhancing trait that may (adaptation) or not (exaptation) have appeared because of natural selection mediated by chytridiomycosis. Our findings reveal bacterial strains for development of local probiotic treatments against chytridiomycosis and also shed light on the mechanisms behind the frog-bacteria-pathogen interaction.     

Is Chytridiomycosis an Emerging Infectious Disease in Asia?

The disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has caused dramatic amphibian population declines and extinctions in Australia, Central and North America, and Europe. Bd is associated with >200 species extinctions of amphibians, but not all species that become infected are susceptible to the disease. Specifically, Bd has rapidly emerged in some areas of the world, such as in Australia, USA, and throughout Central and South America, causing population and species collapse. The mechanism behind the rapid global emergence of the disease is poorly understood, in part due to an incomplete picture of the global distribution of Bd. At present, there is a considerable amount of geographic bias in survey effort for Bd, with Asia being the most neglected continent. To date, Bd surveys have been published for few Asian countries, and infected amphibians have been reported only from Indonesia, South Korea, China and Japan. Thus far, there have been no substantiated reports of enigmatic or suspected disease-caused population declines of the kind that has been attributed to Bd in other areas. In order to gain a more detailed picture of the distribution of Bd in Asia, we undertook a widespread, opportunistic survey of over 3,000 amphibians for Bd throughout Asia and adjoining Papua New Guinea. Survey sites spanned 15 countries, approximately 36° latitude, 111° longitude, and over 2000 m in elevation. Bd prevalence was very low throughout our survey area (2.35% overall) and infected animals were not clumped as would be expected in epizootic events. This suggests that Bd is either newly emerging in Asia, endemic at low prevalence, or that some other ecological factor is preventing Bd from fully invading Asian amphibians. The current observed pattern in Asia differs from that in many other parts of the world.     

Field and laboratory studies of the susceptibility of the green treefrog (Hyla cinerea) to Batrachochytrium dendrobatidis infection

Amphibians worldwide are experiencing devastating declines, some of which are due to the amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd). Populations in the southeastern United States, however, have not been noticeably affected by the pathogen. The green treefrog (Hyla cinerea) is abundant and widespread in the southeastern United States, but has not been documented to harbor Bd infection. This study examined the susceptibility of H. cinerea to two strains of Bd in the lab and the prevalence of infection in wild populations of this species in southeastern Louisiana. Although we were able to infect H. cinerea with Bd in the lab, we did not observe any clinical signs of chytridiomycosis. Furthermore, infection by Bd does not appear to negatively affect body condition or growth rate of post-metamorphic individuals. We found no evidence of infection in surveys of wild H. cinerea. Our results suggest that H. cinerea is not susceptible to chytridiomycosis post-metamorphosis and probably is not an important carrier of the fungal pathogen Bd in the southeastern United States, although susceptibility at the larval stage remains unknown.     

Seasonal pattern of Batrachochytrium dendrobatidis infection and mortality in Lithobates areolatus: affirmation of Vredenburg's "10,000 zoospore rule"

To fully comprehend chytridiomycosis, the amphibian disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), it is essential to understand how Bd affects amphibians throughout their remarkable range of life histories. Crawfish Frogs (Lithobates areolatus) are a typical North American pond-breeding species that forms explosive spring breeding aggregations in seasonal and semipermanent wetlands. But unlike most species, when not breeding Crawfish Frogs usually live singly--in nearly total isolation from conspecifics--and obligately in burrows dug by crayfish. Crayfish burrows penetrate the water table, and therefore offer Crawfish Frogs a second, permanent aquatic habitat when not breeding. Over the course of two years we sampled for the presence of Bd in Crawfish Frog adults. Sampling was conducted seasonally, as animals moved from post-winter emergence through breeding migrations, then back into upland burrow habitats. During our study, 53% of Crawfish Frog breeding adults tested positive for Bd in at least one sample; 27% entered breeding wetlands Bd positive; 46% exited wetlands Bd positive. Five emigrating Crawfish Frogs (12%) developed chytridiomycosis and died. In contrast, all 25 adult frogs sampled while occupying upland crayfish burrows during the summer tested Bd negative. One percent of postmetamorphic juveniles sampled were Bd positive. Zoospore equivalents/swab ranged from 0.8 to 24,436; five out of eight frogs with zoospore equivalents near or >10,000 are known to have died. In summary, Bd infection rates in Crawfish Frog populations ratchet up from near zero during the summer to over 25% following overwintering; rates then nearly double again during and just after breeding--when mortality occurs--before the infection wanes during the summer. Bd-negative postmetamorphic juveniles may not be exposed again to this pathogen until they take up residence in crayfish burrows, or until their first breeding, some years later.     

Infection intensity and sampling locality affect Batrachochytrium dendrobatidis distribution among body regions on green-eyed tree frogs Litoria genimaculata

Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, which has caused devastating amphibian population declines. Little is known about the biology of Bd on hosts, and techniques for diagnosing it on living and preserved animals are still evolving. We investigated the spatial distribution of Bd on the integument of naturally infected Australian hylid frogs Litoria genimaculata at 4 rain forest localities in northern Queensland, Australia. We collected 555 samples by swabbing 111 individuals on 5 regions of the body (back, abdomen, legs, forefeet and hindfeet). Numbers of zoospore equivalents on each body region were quantified using a real-time TaqMan PCR assay. The intensity of infection differed significantly among body regions and this pattern of differences differed among sampling localities. The lightest infections were usually centered on the abdomen, while heavier infections were concentrated on the legs and feet. The back was always either lightly infected or uninfected. Many frogs with light infections had positive PCR results only for the abdomen or the legs. We compared swabs taken from the legs and abdomen and found that they provided similar sensitivity to detect infections, but using both regions together led to greater sensitivity than either region alone. Because swabbing may transfer zoospores from infected to uninfected regions within individuals, we suggest that the best procedure for all species is to employ separate swabs for each body region. If that cannot be done, swabbing patterns that minimize potential harm should be determined for each species, and possibly each class of individuals (e.g. males, females, juveniles) within species, by examining the distribution of infection among body parts in naturally infected individuals.     

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.     

High Prevalence of the Amphibian Chytrid Pathogen in Gabon

Amphibian chytridiomycosis is an infectious disease caused by the fungus Batrachochytrium dendrobatidis (Bd) that is implicated in the worldwide decline and extinction of amphibians. Africa has been proposed as a potential source for the global expansion of Bd, yet the distribution of Bd across the continent remains largely unexplored. Using quantitative polymerase chain reaction (qPCR), we screened for the presence of Bd in 166 adult anurans from two national parks in Gabon (Monts de Cristal and Ivindo). Bd was detected in 20 of the 42 species and was present at all three sites surveyed (two in Monts de Cristal, and one in Ivindo) with high prevalence (19.6%-36.0%). Both national parks were Bd-positive at all elevations and across habitat types, though no dead or dying frogs were encountered. To our knowledge, this study presents the first evidence of Bd in Gabon and the first record of infection for 19 of the 20 species that were Bd-positive. Documenting the distribution and virulence of Bd across Africa will be essential for understanding the dynamics of amphibian chytridiomycosis across the globe.     

Prevalence of Batrachochytrium dendrobatidis in three species of wild frogs on Prince Edward Island, Canada

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has resulted in the decline or extinction of approximately 200 frog species worldwide. It has been reported throughout much of North America, but its presence on Prince Edward Island (PEI), on the eastern coast of Canada, was unknown. To determine the presence and prevalence of Bd on PEI, skin swabs were collected from 115 frogs from 18 separate sites across the province during the summer of 2009. The swabs were tested through single round end-point PCR for the presence of Bd DNA. Thirty-one frogs were positive, including 25/93 (27%) green frogs Lithobates (Rana) clamitans, 5/20 (25%) northern leopard frogs L. (R.) pipiens, and 1/2 (50%) wood frogs L. sylvaticus (formerly R. sylvatica); 12 of the 18 (67%) sites had at least 1 positive frog. The overall prevalence of Bd infection was estimated at 26.9% (7.2-46.7%, 95% CI). Prevalence amongst green frogs and leopard frogs was similar, but green frogs had a stronger PCR signal when compared to leopard frogs, regardless of age (p < 0.001) and body length (p = 0.476). Amongst green frogs, juveniles were more frequently positive than adults (p = 0.001). Green frogs may be the most reliable species to sample when looking for Bd in eastern North America. The 1 wood frog positive for Bd was found dead from chytridiomycosis; none of the other frogs that were positive for Bd by PCR showed any obvious signs of illness. Further monitoring will be required to determine what effect Bd infection has on amphibian population health on PEI.