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Amphibian declines caused by chytridiomycosis have been severe, but some susceptible populations have persisted or even recovered. Resistance to the causal agent Batrachochytrium dendrobatidis (Bd) could result from alleles of the adaptive immune system. During metamorphosis, however, immune systems may not be fully functional, implying that an effective immune response to Bd may be life-stage dependent. We evaluated the susceptibility of the relict leopard frog (Rana onca) sourced from two areas where Bd was present or absent, and where the populations appeared to show differences in pathogen resistance. We evaluated whether population-level resistance manifested across life stages using challenge experiments with late-stage tadpoles (Gosner stage 31-38), metamorphs (stage 45-46), and juvenile frogs. We used three different Bd isolates including one from wild R. onca to challenge juvenile frogs and focused on the isolate from R. onca to challenge tadpoles and resulting metamorphs. We found that juveniles from the Bd exposed population were 5.5 times more likely to survive Bd infection and 10 times more likely to clear infections than those from the area without Bd. In contrast, and regardless of the source area, we observed 98% survivorship of tadpoles, but only 19% survivorship of resulting metamorphs following re-exposure. Given the low survivorship of exposed metamorphs in the laboratory, we speculate on how resistance characteristics, whether adaptive or innate, that do not manifest at each life stage could develop in the wild. We suggest that seasonal high temperatures during times when metamorphosis appears common may modulate the effects of the pathogen during this most susceptible life stage.

Ranaviruses and the fungus Batrachochytrium dendrobatidis are globally important agents of emerging infectious amphibian diseases. Amphibians on Oahu, the Hawaiian Island with the greatest potential for disease introduction through the movement of goods and people, have never been surveyed for ranaviruses or B. dendrobatidis . We surveyed all five species of frogs on Oahu, Hawaii, US for these pathogens. Of 325 individuals sampled from six sites, none were positive for ranavirus. However, we found B. dendrobatidis in a total of four individuals of three species, the cane toad ( Bufo marinus ), the American bullfrog ( Rana catesbeiana ), and the greenhouse frog ( Eleutherodactylus planirostris ), but not in the green and black poison dart frog ( Dendrobates auratus ) or the Japanese wrinkled frog ( Rana rugosa ). The apparent lack of ranavirus and low prevalence of B. dendrobatidis are noteworthy given how widespread these pathogens are in terms of both global distribution and host range. Surveillance should continue to document any changes in B. dendrobatidis prevalence or the arrival of ranaviruses in Hawaii.

Infection experiments are critical for understanding wildlife disease dynamics. Although infection experiments are typically designed to reduce complexity, disease outcomes still result from complex interactions between host, pathogen, and environmental factors. Cryptic variation across factors can lead to decreased repeatability of infection experiments within and between research groups and hinder research progress. Furthermore, studies with unexpected results are often relegated to the "file drawer" and potential insights gained from these experimental outcomes are lost. Here, we report unexpected results from an infection experiment studying the response of two differentially-susceptible but related frogs (American Bullfrog Rana catesbeiana and the Mountain yellow-legged frog Rana muscosa) to the amphibian-killing chytrid fungus (Batrachochytrium dendrobatidis, Bd). Despite well-documented differences in susceptibility between species, we found no evidence for antibody-mediated immune response and no Bd-related mortality in either species. Additionally, during the study, the sham-inoculated R. catesbeiana control group became unexpectedly Bd-positive. We used a custom genotyping assay to demonstrate that the aberrantly-infected R. catesbeiana carried a Bd genotype distinct from the inoculation genotype. Thus R. catesbeiana individuals were acquired with low-intensity infections that could not be detected with qPCR. In the Bd-inoculated R. catesbeiana treatment group, the inoculated genotype appeared to out-compete the cryptic infection. Thus, our results provide insight into Bd coinfection dynamics, a phenomenon that is increasingly relevant as different pathogen strains are moved around the globe. Our experiment highlights how unexpected experimental outcomes can serve as both cautionary tales and opportunities to explore unanswered research questions. We use our results as a case study to highlight common sources of anomalous results for infection experiments. We argue that understanding these factors will aid researchers in the design, execution, and interpretation of experiments to understand wildlife disease processes.

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) has been associated with global amphibian declines, but it is often difficult to discern the relative importance of Bd as a causal agent in declines that have already occurred. Retrospective analyses of museum specimens have allowed researchers to associate the timing of Bd arrival with the timing of past amphibian declines. Cascades frogs (Rana cascadae) have experienced dramatic declines in northern California, but it is not clear whether the onset of these declines corresponds to the arrival of Bd. We used quantitative real-time PCR assays of samples collected from museum specimens to determine historical Bd prevalence in the northern California range of Cascades frogs. We detected Bd in 13 of 364 (3.5%) Cascades frog specimens collected between 1907 and 2003, with the first positive result from 1978. A Bayesian analysis suggested that Bd arrived in the region between 1973 and 1978, which corresponds well with the first observations of declines in the 1980s.

Infections by the fungal pathogen Batrachochytrium dendrobatidis (Bd) and members of the genus Ranavirus (Rv) are increasingly reported as significant determinants of amphibian population die-offs. The complexity associated with their transmission and spatial distribution leads to an increase in demand for comprehensive reporting systems and global mapping of their distribution. Here, we document the distribution of these 2 pathogens in a remote northern temperate lowland where environmental sensitivity is high, providing important insight into the pathogens' natural history and infection patterns. Wood frog Lithobates sylvaticus tissues were collected from the James Bay area in northeastern Canada and were screened for the presence of Bd and Rv using conventional and real-time PCR. Both pathogens were present in the study area, which is the northernmost record in eastern North America. Interestingly, different patterns of distribution were observed between the eastern and western coasts of James Bay, suggesting differences in the spatial and transmission dynamics for each pathogen. Anthropogenic introduction may still influence the distribution patterns observed, even at these latitudes. The presence of infections in this remote area also raises further questions on the risk these pathogens pose to northern amphibian communities. We encourage further research in remote locations for a better understanding of these pathogens, their transmission dynamics, and especially their respective impacts on amphibian populations worldwide.

The amphibian skin fungus Batrachochytrium dendrobatidis (Bd) occurs widely in Puerto Rico and is thought to be responsible for the apparent extinction of 3 species of endemic frogs in the genus Eleutherodactylus, known as coquis. To examine immune defenses which may protect surviving species, we induced secretion of skin peptides from adult common coqui frogs E. coqui collected from upland forests at El Yunque. By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we were unable to detect peptide signals suggestive of antimicrobial peptides, and enriched peptides showed no capacity to inhibit growth of Bd. Thus, it appears that E. coqui depend on other skin defenses to survive in the presence of this deadly fungus.

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) has been associated with amphibian declines worldwide but has not been well-studied among Critically Endangered amphibian species in Bolivia. We sampled free-living marbled water frogs Telmatobius marmoratus (Anura: Leptodactylidae) from Isla del Sol, Bolivia, for Bd using skin swabs and quantitative polymerase chain reactions. We detected Bd on 44% of T. marmoratus sampled. This is the first record of Bd in amphibians from waters associated with Lake Titicaca, Bolivia. These results further confirm the presence of Bd in Bolivia and substantiate the potential threat of this pathogen to the Critically Endangered, sympatric Titicaca water frog T. culeus and other Andean amphibians.

Amphibian populations are globally threatened by emerging infectious diseases, and 2 pathogens in particular are recognized as major threats: the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) and ranaviruses. Here, we evaluated the prevalence of infection by Bd and ranavirus in an assemblage of frogs from a lowland wet forest in Costa Rica. We found an overall prevalence of 21.3% for Bd and 16.6% for ranavirus, and detected both pathogens widely among our 20 sampled species. We found a positive association between ranavirus and Bd infection in one of our 4 most commonly sampled species. We also found a positive but non-significant association between infection by ranavirus and infection by Bd among species overall. Our study is among the first detailed evaluations of ranavirus prevalence in the American tropics, and to our knowledge is the first to detect a positive association between Bd and ranavirus in any species. Considerable research attention has focused on the ecology of Bd in tropical regions, yet we argue that greater research focus is necessary to understand the ecology and conservation impact of ranaviruses on amphibian populations already decimated by the emergence of Bd.

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.

The pathogen Batrachochytrium dendrobatidis (Bd) has been associated with amphibian declines in multiple continents, including western North America. We investigated Bd prevalence in Oregon spotted frog (Rana pretiosa), a species that has declined across its range in the Pacific Northwest. Polymerase chain reaction analysis of skin swabs indicated that Bd was prevalent within populations (420 of 617 juvenile and adults) and widespread among populations (36 of 36 sites) where we sampled R. pretiosa in Oregon and Washington. We rarely detected Bd in R. pretiosa larvae (2 of 72). Prevalence of Bd in postmetamorphic R. pretiosa was inversely related to frog size. We found support for an interactive effect of elevation and sampling date on Bd: prevalence of Bd generally increased with date, but this effect was more pronounced at lower elevations. We also found evidence that the body condition of juvenile R. pretiosa with Bd decreased after their first winter. Our data indicate that some Oregon spotted frog populations are currently persisting with relatively high Bd prevalence, but the risk posed by Bd is unknown.

The recent emergence of amphibian chytridiomycosis has precipitated competing hypotheses regarding the endemic versus novel nature of the causative agent, Batrachochytrium dendrobatidis (Bd). We conducted a retrospective survey of the California Academy of Sciences' (San Francisco, California, USA) amphibian collection, testing for presence of Bd in 4 amphibian species collected from central California between 1897 and 2005. The earliest detection of Bd was found in 2 Rana catesbeiana in 1961, and the data support the hypothesis that Bd was a novel pathogen introduced into central California prior to 1961 that spread out geographically and taxonomically from at least one central location and is now endemic throughout most of central California. The taxonomic pattern of infection prevalence and the ecological constraints of the 4 species we tested suggest that, although Bd was initially detected in R. catesbeiana, the more efficient and most likely local vector for Bd in central California is actually Pseudacris regilla.

The introduction of a new group of dendrobatid frogs to an established captive amphibian collection was followed by several acute mortalities in both resident and introduced frog populations. Chytridiomycosis, caused by Batrachochytrium dendrobatidis, was diagnosed by histology in two of the dead frogs. Following the diagnosis, all amphibians were moved to a specially made quarantine room with strict handling protocols and treated with itraconazole. Frogs, being terrestrial amphibians, were treated with itraconazole (Sporanox, 10 mg/ml) at 0.01% in 0.6% saline in a 5-min bath for 11 consecutive days. Axolotls (Ambystoma mexicanum) and Kaup's caecilians (Potymotyphlus kaupii), being aquatic amphibians, were treated with itraconazole administered directly in their primary tank water to achieve a concentration of 0.01% for 30 min every 5 days for four treatments. Itraconazole was removed from the tank water after 30 min by high-rate-of-flow activated charcoal filters. The treatment and quarantine procedures were successful in eradicating the disease. The few amphibian mortalities that occurred in the 18 mo after the start of the treatment have been histologically negative for the presence of chytrid fungi. The collection is now considered free of chytridiomycosis.

Chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis, is responsible for many amphibian declines and has been identified in wild amphibian populations on all continents where they exist, except for Asia. In order to assess whether B. dendrobatidis is present on the native amphibians of Hong Kong, we sampled wild populations of Amolops hongkongensis, Paa exilispinosa, P. spinosa and Rana chloronota during 2005-2006. Amphibians infected with B. dendrobatidis have been found in the international trade, so we also examined the extent and nature of the amphibian trade in Hong Kong during 2005-2006, and assessed whether B. dendrobatidis was present in imported amphibians. All 274 individuals of 4 native amphibian species sampled tested negative for B. dendrobatidis, giving an upper 95% confidence limit for prevalence of 1.3%. Approximately 4.3 million amphibians of 45 species from 11 countries were imported into Hong Kong via air over 12 mo; we did not detect B. dendrobatidis on any of 137 imported amphibians sampled. As B. dendrobatidis generally occurs at greater than 5% prevalence in infected populations during favorable environmental conditions, native amphibians in Hong Kong appear free of B. dendrobatidis, and may be at severe risk of impact if it is introduced. Until it is established that the pathogen is present in Hong Kong, management strategies should focus on preventing it from being imported and decreasing the risk of it escaping into the wild amphibian populations if imported. Further research is needed to determine the status of B. dendrobatidis in Hong Kong with greater certainty.

Chytridiomycosis is a recently emerged, infectious skin disease of amphibians that has been linked directly to mass mortalities, population declines, and species extinctions worldwide. An understanding of the factors that limit the distribution and abundance of Batrachochytrium dendrobatidis (the etiological agent of chytridiomycosis) is urgently required. We conducted disease surveys at 31 lowland sites distributed north-south along 2315 km of the Australian east coast that encompassed 20.8 degrees of latitude. A total of 863 adult male stony creek frogs (Litoria lesueuri complex) were sampled, and the overall prevalence of B. dendrobatidis infection was 26%. B. dendrobatidis was detected at 77% of the sites, including sites at the northern and southern limits of the latitudinal transect. Frogs from temperate regions, however, had significantly more intense infections than did their tropical counterparts, often carrying an order of magnitude more B. dendrobatidis zoospores, suggesting that at low elevations, temperate frogs are at higher risk of chytridiomycosis-induced mortality than are tropical frogs. The prevalence and intensity of B. dendrobatidis infections were significantly greater at sites with high rainfall (>33 mm in the 30 days prior to sampling) and cool temperatures (stream temperature 1 h after sunset < 23 degrees C). Although climatic variables explained much of the variation in the prevalence and intensity of B. dendrobatidis infections between infected and uninfected sites, frog snout-vent length was consistently the best predictor of infection levels across infected sites. Small frogs were more likely to be infected and carried more intense infections than larger frogs, suggesting either that frogs can outgrow their chytrid infections or that the disease induces developmental stress that limits growth. Our results will directly assist amphibian disease researchers and wildlife managers, whose conservation efforts should focus on those amphibian populations living within the B. dendrobatidis climatic envelope that we have described.

1. Chytridiomycosis is an emerging infectious disease of amphibians, caused by the fungal pathogen Batrachochytrium dendrobatidis, which has been implicated recently in population declines and possible extinctions throughout the world. 2. The transmission rate of this pathogen was quantified in the mountain yellow-legged frog Rana muscosa through laboratory and field experiments, and a maximum likelihood approach was used to determine the form of the transmission function that was best supported by the experimental data. 3. The proportion of R. muscosa tadpole hosts that became infected increased with the number of previously infected R. muscosa tadpoles to which they were exposed, as would be expected in an infectious disease. 4. The laboratory experiment revealed some support for a transmission function in which the transmission rate levels off as the density of infected individuals increases. However, there was not enough power to distinguish between a frequency-dependent form and several other asymptotic forms of the transmission function. 5. The impacts of crowding and temperature on transmission were also investigated; however, neither of these factors significantly affected the transmission rate.

The ability to quantify infections provides a tool with which to perform comparative pathological research. The need exists for a simplistic standard method to compare infection levels of Batrachochytrium dendrobatidis, a major cause of global amphibian declines. Through examination of skin sloughs of the Cape river frog Afrana fuscigula, we present an accessible method that not only provides quantitative measurements of B. dendrobatidis, but also provides information that increases the confidence of detection through histological surveys. The method relies on the availability of live animals that are actively shedding skin. By employing a direct microscopic count of sporangia, it is possible to express infection in terms of density. Micro-spatial infection in the skin of A. fuscigula is characterised by significant differences in sporangium density among the different components of the foot, and by similar differences in site infection frequency. Notably, toe tips and tubercles contain higher infection densities and are more often infected than webbing or the base of the foot. This pattern of infection might facilitate disease transmission due to the increased exposure of these components to abrasion. Density data can be used with the Poisson frequency function to approximate binomial probabilities of detecting B. dendrobatidis through histology. The probability matrix produced for A. fuscigula indicated that foot-site selection for histology markedly influenced the number of sections required to detect B. dendrobatidis at a specific level of probability. Thus, examination of a test sample of skin tissue with direct-count quantification can help in planning the sampling of tissues for histological surveys.

Batrachochytrium dendrobatidis (Bd) infection on post-metamorphic frogs and salamanders is commonly diagnosed using polymerase chain reaction (PCR) of skin scrapings taken with mildly abrasive swabs. The technique is sensitive, non-lethal, and repeatable for live animals. Tadpoles are generally not sampled by swabbing but are usually killed and their mouthparts excised to test for the pathogen. We evaluated a technique for non-lethal Bd diagnosis using quantitative PCR (qPCR) on swabs scraped over the mouthparts of live tadpoles. The sensitivity of non-lethal (swabbing) and lethal (removal of mouthparts) sampling was assessed using 150 Bd-infected Rana subaquavocalis tadpoles. Swabbing was consistently less sensitive than lethal sampling, but still detected Bd. Experimental Bd prevalence was 41.1% when estimated by destructively sampling mouthparts and 4.7 to 36.6% (mean = 21.4%) when estimated with swabs. Detection rates from swabbing varied with investigator and time since infection. The likelihood of detecting Bd-infected tadpoles was similar regardless of size and developmental stage. Swabbing mouthparts of live tadpoles is a feasible and effective survey technique for Bd, but, because it is less sensitive, more tadpoles must be sampled to estimate prevalence at a confidence level comparable to destructive sampling.

Batrachochytrium dendrobatidis is a fungal pathogen that has been implicated in amphibian declines worldwide. Histopathologic techniques have been used to diagnose the disease, but their sensitivity has not been determined. It is also unclear whether the probability of detection varies between skin samples derived from different body parts. We examined 24 Fitzinger's rainfrogs (Eleutherodactylus fitzingeri) with chytridiomycosis. This is a common frog species with a broad range and high abundance throughout most of Costa Rica. We sampled 12 different body parts from each animal, and alternated the staining between a routinely used stain (hematoxylin and eosin [H&E]), and a more fungus-specific stain (periodic acid-Schiff [PAS]). The pelvic patch and the innermost finger of the hand were consistently the best places to detect the disease, although significant differences were found only with the gular area, the abdomen, and toes four and five. We found more positive samples using PAS than using H and E in all body parts, although significant differences were detected only in samples derived from the pelvic patch. Using the best combination of factors (stain and body part) and animals with the lightest infections (to test the sensitivity of the technique), we calculated that at least 17 sections are needed in order to reach 95% confidence that a frog is or is not infected. We conclude that the choice of stain and body part can significantly alter estimates of prevalence of B. dendrobatidis.

Chytridiomycosis is an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis, which has been implicated in amphibian declines worldwide. The mountain yellow-legged frog Rana muscosa is a declining amphibian species that can be infected by B. dendrobatidis; however, transmission between conspecifics has not been documented. Here, we present experimental evidence that R. muscosa tadpoles can be infected by fungal zoospores and that they can transmit infection to each other and to postmetamorphic animals. We compared several techniques for detecting B. dendrobatidis transmission and found that histology with serial sectioning was able to detect infection before cytology or visual inspections. We also show that R. muscosa tadpoles appear healthy with B. dendrobatidis infection, while postmetamorphic animals experience mortality. In addition, we provide guidelines for visually detecting B. dendrobatidis in R. muscosa tadpoles, which may be useful in other affected species. Field surveys of infected and uninfected populations verify this identification technique.

The 1st recorded histological evidence of chytridiomycete fungal infection in a free-ranging ranid amphibian in South Africa is presented. Literature on causes of a worldwide decline in amphibian populations is briefly reviewed.