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.     

Species-level correlates of susceptibility to the pathogenic amphibian fungus Batrachochytrium dendrobatidis in the United States

Disease is often implicated as a factor in population declines of wildlife and plants. Understanding the characteristics that may predispose a species to infection by a particular pathogen can help direct conservation efforts. Recent declines in amphibian populations world-wide are a major conservation issue and may be caused in part by a fungal pathogen, Batrachochytrium dendrobatidis (Bd). We used Random Forest, a machine learning approach, to identify species-level characteristics that may be related to susceptibility to Bd. Our results suggest that body size at maturity, aspects of egg laying behavior, taxonomic order and family, and reliance on water are good predictors of documented infection for species in the continental United States. These results suggest that, whereas local-scale environmental variables are important to the spread of Bd, species-level characteristics may also influence susceptibility to Bd. The relationships identified in this study suggest future experimental tests, and may target species for conservation efforts.     

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.     

Prevalence of infection by Batrachochytrium dendrobatidis and Ranavirus in eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) in eastern Tennessee

Hellbenders (n=97) were collected from the Little and Hiwassee Rivers in eastern Tennessee, USA, during 2009 and 2010. Location and morphometrics for each animal were recorded, and nonlethal tissue samples were collected to estimate the prevalence of infection with Batrachochytrium dendrobatidis (Bd) and Ranavirus in each watershed and year. Real-time polymerase chain reaction was performed on skin swabs for Bd and on tail clips for ranaviruses. Overall prevalences of DNA of Bd, Ranavirus, and coinfections (i.e., detectable DNA of both pathogens in the same individual) were 26%, 19%, and 5%, respectively. Differences in infection prevalence were detected between watersheds and years. Gross lesions were observed in 31 animals (32%), but the types of lesions were not consistent with chytridiomycosis or ranaviral disease. This is the first report of infection of eastern hellbenders with Bd and Ranavirus. Despite infection by both pathogens, it is unclear whether chytridiomycosis or ranaviral disease develops in wild populations of hellbenders. More research is needed to determine the susceptibility of hellbenders to Bd and ranaviruses and their role in the epidemiology of these pathogens.     

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.     

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.     

A reservoir species for the emerging Amphibian pathogen Batrachochytrium dendrobatidis thrives in a landscape decimated by disease

Chytridiomycosis, a disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is driving amphibian declines and extinctions in protected areas globally. The introduction of invasive reservoir species has been implicated in the spread of Bd but does not explain the appearance of the pathogen in remote protected areas. In the high elevation (>1500 m) Sierra Nevada of California, the native Pacific chorus frog, Pseudacris regilla, appears unaffected by chytridiomycosis while sympatric species experience catastrophic declines. We investigated whether P. regilla is a reservoir of Bd by comparing habitat occupancy before and after a major Bd outbreak and measuring infection in P. regilla in the field, monitoring susceptibility of P. regilla to Bd in the laboratory, examining tissues with histology to determine patterns of infection, and using an innovative soak technique to determine individual output of Bd zoospores in water. Pseudacris regilla persists at 100% of sites where a sympatric species has been extirpated from 72% in synchrony with a wave of Bd. In the laboratory, P. regilla carried loads of Bd as much as an order of magnitude higher than loads found lethal to sympatric species. Histology shows heavy Bd infection in patchy areas next to normal skin, a possible mechanism for tolerance. The soak technique was 77.8% effective at detecting Bd in water and showed an average output of 68 zoospores per minute per individual. The results of this study suggest P. regilla should act as a Bd reservoir and provide evidence of a tolerance mechanism in a reservoir species.     

Prevalence of Batrachochytrium dendrobatidis in Agalychnis moreletii (Hylidae) of El Salvador and association with larval jaw sheath depigmentation

Amphibian populations around the world have been declining at an alarming rate due to factors such as habitat destruction, pollution, and infectious diseases. Between May and July 2008, we investigated a fungal pathogen in the critically endangered Morelet's treefrog (Agalychnis moreletii) at sites in El Salvador. Larvae were screened with a hand lens for indications of infection with Batrachochytrium dendrobatidis (Bd), a fungus that can cause lethal chytridiomycosis in amphibians. Subsets of inspected tadpoles were preserved for analysis by polymerase chain reaction to determine the effectiveness of hand lens screening for presence of Bd and to estimate infection prevalence at various sites. Because individuals with signs of infection were preferentially included, we used a novel method to generate unbiased estimates of infection prevalence from these biased samples. External mouthpart deformities, identified with a hand lens, successfully predicted Bd infection across a large spatial scale. Two of 13 sites sampled had high (≥ 89%) estimated prevalence, whereas little or no Bd was detected at the remaining sites. Although it appears that A. moreletii populations in this region are not suffering rapid declines due to Bd, further monitoring is required to determine the extent to which these populations are stably coexisting with the pathogen.     

Experimental infection of self-cured Leiopelma archeyi with the amphibian chytrid Batrachochytrium dendrobatidis

The susceptibility of Archey's frog Leiopelma archeyi to Batrachochytrium dendrobatidis (Bd) is unknown, although one large population is thought to have declined sharply due to chytridiomycosis. As primary infection experiments were not permitted in this endangered New Zealand species, 6 wild-caught L. archeyi that naturally cleared infections with Bd while in captivity were exposed again to Bd to assess their immunity. These frogs were from an infected population at Whareorino, which has no known declines. All 6 L. archeyi became reinfected at low intensities, but rapidly self cured, most by 2 wk. Six Litoria ewingii were used as positive controls and developed heavier infections and clinical signs by 3 wk, demonstrating that the zoospore inoculum was virulent. Six negative controls of each species remained uninfected and healthy. Our results show that L. archeyi that have self cured have resistance to chytridiomycosis when exposed. The pattern is consistent with innate or acquired immunity to Bd, and immunological studies are needed to confirm this.     

Qualitative risk analysis of introducing Batrachochytrium dendrobatidis to the UK through the importation of live amphibians

The international amphibian trade is implicated in the emergence and spread of the amphibian fungal disease chytridiomycosis, which has resulted in amphibian declines and extinctions globally. The establishment of the causal pathogen, Batrachochytrium dendrobatidis (Bd), in the UK could negatively affect the survival of native amphibian populations. In recognition of the ongoing threat that it poses to amphibians, Bd was recently included in the World Organisation for Animal Health Aquatic Animal Health Code, and therefore is in the list of international notifiable diseases. Using standardised risk analysis guidelines, we investigated the likelihood that Bd would be introduced to and become established in wild amphibians in the UK through the importation of live amphibians. We obtained data on the volume and origin of the amphibian trade entering the UK and detected Bd infection in amphibians being imported for the pet and private collection trade and also in amphibians already held in captive pet, laboratory and zoological collections. We found that current systems for recording amphibian trade into the UK underestimate the volume of non-European Union trade by almost 10-fold. We identified high likelihoods of entry, establishment and spread of Bd in the UK and the resulting major overall impact. Despite uncertainties, we determined that the overall risk estimation for the introduction of Bd to the UK through the importation of live amphibians is high and that risk management measures are required, whilst ensuring that negative effects on legal trade are minimised.     

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.     

Temporal Variation in Infection Prevalence by the Amphibian Chytrid Fungus in Three Species of Frogs at La Selva,Costa Rica

The emerging infectious disease chytridiomycosis, caused by the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is implicated in widespread population declines, extirpations, and extinctions of amphibians throughout the world. In the Neotropics, most amphibian declines have occurred in cool mid‐ to high‐elevation sites (> 400 m asl), and it is hypothesized that high temperatures limit the growth of Bd in lowland tropical sites, despite few data available on the distribution of Bd in lowland forests. Here, we report the results of a 12‐mo pathogen surveillance program for three common species of frogs at a warm lowland site in northeastern Costa Rica. We combine standard non‐invasive skin swabbing techniques with a quantitative polymerase chain reaction assay to analyze the infection prevalence and Bd load across a 1‐yr period. Our data indicate an overall Bd infection rate of 6.1 percent, but prevalence varies from < 5 percent in warmer months to a peak of 34.7 percent in the coolest months of the year. Despite very little seasonal variation in temperature (< 4°C), our data indicate strong seasonal variation in the prevalence of Bd, with highest prevalence of infection in months with coolest air temperatures. While it has been suggested that Bd is primarily a riparian fungus, we find no difference in prevalence of infection among our species despite considerable differences in affiliation of these species with water. Our study provides further evidence that infection by Bd is regulated by temperature and shows that warm temperatures in lowland forests may restrict, but not prevent, infection by Bd.     

Prevalence of Batrachochytrium dendrobatidis infection is extremely low in direct-developing Australian microhylids

The emerging infectious disease chytridiomycosis has been implicated in declines and disappearances of amphibian populations around the world. However, susceptibility to infection and the extent of pathological effects of infection vary among hosts, and species with life histories that include parental care of direct-developing terrestrial eggs may tend to be less susceptible. We examined samples from a total of 595 individuals of 9 species of direct-developing Australian frogs in the family Microhylidae for the presence of infection by Batrachochytrium dendrobatidis (Bd). Between 1995 and 2004, 336 samples were collected; 102 of these were analysed histologically and 234 were tissues stored in alcohol, which were examined using diagnostic quantitative PCR (qPCR). Swab samples were collected from 259 frogs from 2005 to 2008 and were examined using qPCR. None of the 595 samples showed evidence of infection by Bd. If these data are regarded as a single sample representative of Australian microhylids, the upper 95% binomial confidence limit for the prevalence of infection in frogs of this family is 0.0062 (<1%). Even if only the data from the more powerful diagnostic qPCR tests are used, the upper 95% confidence limit for prevalence is 0.0075 (<1%). Our data suggest that Australian microhylids have a very low prevalence of infection by Bd in nature, and thus are either not susceptible, or are only slightly susceptible, to chytridiomycosis. This could be due solely to, or in combination with, low rates of transmission and to factors that promote resistance to infection, including ecological or behavioural characteristics, innate immune functions such as antimicrobial skin peptides, or antimicrobial symbionts in skin flora.     

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.     

Using amphibians in laboratory studies: precautions against the emerging infectious disease chytridiomycosis

The African clawed frog Xenopus laevis is by far the most widely used amphibian species in laboratories. In the wild, X. laevis is an asymptomatic carrier of an emerging infectious disease called chytridiomycosis. The vector is the chytrid fungus Batrachochytrium dendrobatidis (Bd), which has devastating effects on wild amphibian populations around the world. The impact of Bd on the metabolism of X. laevis has not been comprehended yet. However, even if asymptomatic, an infection is likely to affect the individual's physiology, immunology, development, reproduction and overall response to stress from a purely medical point of view, which will introduce noise and therefore increase variance within experimental groups of X. laevis. This could have implications on the scientific results from studies using this species. Here, we review the current knowledge on treatments of infected amphibians and propose a hygiene protocol adapted to laboratory populations and amphibian husbandry. Following the presented sanitation guidelines could further prevent the spread of Bd and probably of other amphibian pathogens. The sanitation guidelines will help to reduce the impact of amphibian husbandry on natural populations and must be considered a crucial contribution to amphibian conservation, as today 32% of all amphibians are considered threatened.     

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.     

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.     

Underestimated ranges and overlooked refuges from amphibian chytridiomycosis

Aim

Accurately documenting and predicting declines and shifts in species’ distributions is fundamental for implementing effective conservation strategies and directing future research; species distribution models (SDM) have become a powerful tool for such work. Nevertheless, much of the data used to create these models are opportunistic and often violate some of their basic assumptions. We use amphibian declines and extinctions linked to the fungus Batrachochytrium dendrobatidis (Bd) to examine how sampling biases in data collection can affect what we know of this disease and its effect on amphibians in the wild.

Location

Queensland, Australia.

Methods

We developed a distribution model for Bd incorporating known locality records for Bd and a subset of climatic variables that should correctly characterize its distribution. We tested this (original) model with additional surveys, recorded new Bd observations in novel environments and reran the distribution model. We then investigated the difference between the original and new models, and used frog abundance and infection status data from two of these new localities to look at the susceptibility of the torrent frog Litoria nannotis to chytridiomycosis.

Results

While largely correct, the original SDM underestimated the distribution of Bd; sampling in ‘unsuitable’ drier environments discovered abundant populations of susceptible frogs with pathogen prevalences of up to 100%. The validation surveys further uncovered a new population of the frog Litoria lorica coexisting with the pathogen; this species was previously believed to be an extinct rain forest endemic.

Main conclusion

Our results indicate that SDMs constructed using opportunistically collected data can be biased if species are not at equilibrium with their environment or because environmental gradients have not been adequately sampled. For disease ecology, the better estimations of pathogen distribution may lead to the discovery of new populations persisting at the edge of their range, which has important implications for the conservation of species threatened by chytridiomycosis.

     

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.     

Quantitative PCR detection of Batrachochytrium dendrobatidis DNA from sediments and water

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease implicated in amphibian declines on 5 continents. Polymerase chain reaction (PCR) primer sets exist with which amphibians can be tested for this disease, and advances in sampling techniques allow non-invasive testing of animals. We developed filtering and PCR based quantitative methods by modifying existing PCR assays to detect Bd DNA in water and sediments, without the need for testing amphibians; we tested the methods at 4 field sites. The SYBR based assay using Boyle primers (SYBR/Boyle assay) and the Taqman based assay using Wood primers performed similarly with samples generated in the laboratory (Bd spiked filters), but the SYBR/Boyle assay detected Bd DNA in more field samples. We detected Bd DNA in water from 3 of 4 sites tested, including one pond historically negative for chytridiomycosis. Zoospore equivalents in sampled water ranged from 19 to 454 1(-1) (nominal detection limit is 10 DNA copies, or about 0.06 zoospore). We did not detect DNA of Bd from sediments collected at any sites. Our filtering and amplification methods provide a new tool to investigate critical aspects of Bd in the environment.