Survivorship in Wild Frogs Infected with Chytridiomycosis

Chytridiomycosis is an emerging infectious disease that has been implicated as the causative agent of many recent amphibian population declines and extinctions that have taken place in relatively pristine locations worldwide. While there exists a growing body of literature regarding the effect of the fungus on experimentally infected frogs, few studies have examined the effect of the fungus on apparently healthy wild frogs from nondeclining, infected populations. We examined the temporal pattern of chytrid infection in individually marked Stony Creek Frogs (Litoria wilcoxii) at a lowland site in southeast Queensland, Australia. We provide the first evidence that wild frogs are capable of both acquiring chytridiomycosis as adults, and also of clearing their infections entirely. Changes in disease status in individual frogs largely tracked changing climatic conditions, with infections tending to appear in cooler months and disappearing in warmer months. Though 27.2% of the adult frogs we sampled were infected at some point in the study, we found no evidence that chytridiomycosis was negatively affecting adult survivorship, suggesting either: (1) chytrid-induced mortality in this population is generally restricted to metamorphs and juveniles; (2) this population was not exposed to conditions which favored lethal disease outbreaks; or (3) this population has evolved sufficient resistance to the disease to persist relatively unaffected.     

Chytridiomycosis and Amphibian Population Declines Continue to Spread Eastward in Panama

Chytridiomycosis is a globally emerging disease of amphibians and the leading cause of population declines and extirpations at species-diverse montane sites in Central America. We continued long-term monitoring efforts for the presence of the fungal pathogen Batrachochytrium dendrobatidis (Bd) and for amphibian populations at two sites in western Panama, and we began monitoring at three new sites to the east. Population declines associated with chytridiomycosis emergence were detected at Altos de Campana National Park. We also detected Bd in three species east of the Panama Canal at Soberanía National Park, and prevalence data suggests that Bd may be enzootic in the lowlands of the park. However, no infected frogs were found further east at Tortí (prevalence <7.5% with 95% confidence). Our results suggest that Panama’s diverse and not fully described amphibian communities east of the canal are at risk. Precise predictions of future disease emergence events are not possible until factors underlying disease emergence, such as dispersal, are understood. However, if the fungal pathogen spreads in a pattern consistent with previous disease events in Panama, then detection of Bd at Tortí and other areas east of the Panama Canal is imminent. Therefore, development of new management strategies and increased precautions for tourism, recreation, and biology are urgently needed.     

<Emphasis Type="Italic">Batrachochytrium</Emphasis><Emphasis Type="Italic">dendrobatidis</Emphasis> and Chytridiomycosis in Anuran Amphibians of Colombia

In order to investigate the possible presence of the chytrid fungus Batrachochytrium dendrobatidis (Chytridiomycota: Chytridiales) in frogs (Amphibia: Anura) of Colombia, we made a retrospective examination of formalin-fixed specimens preserved in natural history collections. Using the staining technique of hematoxylin and eosin to identify B. dendrobatidis in histological slices, we found evidence of the fungus in 3 of the 53 frog species examined from a total of 672 specimens collected in 17 departments within Colombia between 1968 and 2006. The infected specimens were found dead or dying in recent years in high elevation sites, suggesting that chytridiomycosis (the disease caused by the fungus) may represent a significant threat to Colombian amphibians. We conclude that a more extensive search for B. dendrobatidis in museum specimens and wild-caught frogs should be undertaken as soon as possible, using both histological and molecular genetic techniques, in order to further characterize the geographic and taxonomic extent of infections of B. dendrobatidis.     

Chytridiomycosis in Wild Frogs from Pico Bonito National Park, Honduras

Almost half of the endemic species of Honduran amphibians have declining populations; some of which seem to be extinct since they have not been seen in several years in places where they were once common. Disappearances in pristine and protected habitats have occurred in several highland localities throughout the country. The highland amphibian fauna of Pico Bonito National Park declined sometime between 1989 and 1995. An amphibian chytrid fungus, Batrachochytrium dendrobatidis has been linked to similar declines in other neotropical regions. We checked 19 specimens for this disease, which were collected in the park in 2003. The only Rana maculata examined was found to be infected, as were three of the six Eleutherodactylus aurilegulus surveyed for the disease. Two of the infected E. aurilegulus were collected at 120 m elevation and showed strong infections. One of these was lethargic and did not react when it was collected in the field, although it was still alive. A complete necropsy could help determine if the B. dendrobatidis infection was responsible for these symptoms, and further research might show how susceptible E. aurilegulus is to this pathogen at low altitudes. More research should be focused on the distribution of this pathogen in Honduras, and on how this disease has affected the local amphibian fauna.     

Chytridiomycosis Survey in Wild and Captive Mexican Amphibians

Mexico, a rich country in terms of amphibian diversity, hosts about 375 described species. Population declines have been documented for several species where it is evident that their habitat is being destroyed or modified. However, other species which inhabit pristine areas are declining as well. It has been suggested that the chytrid fungus Batrachochytrium dendrobatidis (B.d.) may be one of the causes of the enigmatic declines in Mexico. We surveyed a total of 45 localities, in 12 states across Mexico, examining a total of 360 specimens representing 14 genera and 30 species. We also examined 91 specimens of Ambystoma mexicanum from a captive population in Mexico City as well as one Pachymedusa dacnicolor obtained in a pet shop. We used a two-tiered technique to detect the pathogen. For wild-caught specimens, we utilized light microscopy to identify presence of B.d. sporangia in amphibian skin. Then, to verify the infection, we used a quantitative real-time PCR assay on collected skin sections which is specific for B.d. For captive animals, we used a nonlethal version of the real-time PCR technique. We found evidence of B.d. infection in 111 animals comprising 14 species in 13 localities. A large percentage (84%) of Ambystoma mexicanum from the colony were infected with B.d. The two most highly infected individuals were the endangered Ambystoma mexicanum, from a captive colony, and Pachymedusa dacnicolor, purchased at a pet shop. The online version of this article (doi:) contains electronic supplementary material, which is available to authorized users.     

Evidence of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> Infection in Water Frogs of the <Emphasis Type="Italic">Rana esculenta</Emphasis> Complex in Central Italy

Batrachochytrium dendrobatidis (phylum Chytridiomycota, order Chytridiales) is the causative organism of chytridiomycosis in amphibians, a disease associated with their population decline worldwide. In this work, we report a cutaneous infection in water frogs of the Rana esculenta complex in agricultural areas of Umbria, central Italy. Histological, immunohistochemical, ultrastructural, and molecular analyses demonstrated for the first time the presence of the Batrachochytrium dendrobatidis in this complex; to date, no association between the presence of chytrid fungal infection and mortality has been found, to our knowledge. However, the presence of Batrachochytrium dendrobatidis infection in the water frogs of the Rana esculenta complex is of concern because the frogs could act as a reservoir species and contribute to the decline of less resistant species.     

The Amphibian Chytrid Batrachochytrium dendrobatidis Occurs on Freshwater Shrimp in Rain Forest Streams in Northern Queensland, Australia

Chytridiomycosis is a disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis. It can be highly virulent and is unusual in that it appears to drive many host species to local extinction during outbreaks. One mechanism that could facilitate this is the ability to grow saprophytically or on alternative hosts. This is common in other chytrids but has not been demonstrated for B. dendrobatidis in the field. B. dendrobatidis can grow on arthropod exoskeletons in the laboratory, and freshwater shrimp can be the most abundant animals in tropical rain forest streams. We therefore used diagnostic quantitative polymerase chain reaction to determine the infection status of freshwater shrimp from areas in which they are sympatric with frog species that have suffered declines in association with outbreaks of chytridiomycosis. We detected B. dendrobatidis on three individual shrimp belonging to two genera and collected from two widely separated streams. Two of the individuals had high levels of infection. This indicates that the presence of alternative hosts is likely to contribute to the extreme virulence of chytridiomycosis outbreaks in some systems. The presence of alternative hosts may allow B. dendrobatidis to remain in the environment after local extinctions of amphibian hosts, preventing the recovery of amphibian populations.     

Presence of the Chytrid Fungus Batrachochytrium dendrobatidis in Populations of the Critically Endangered Frog Mannophryne olmonae in Tobago, West Indies

The emerging infectious disease chytridiomycosis is prevalent in Central and South America, and has caused catastrophic declines of amphibian populations in the Neotropics. The responsible organism, Batrachochytrium dendrobatidis, has been recorded on three West Indian islands, but the whole of the Caribbean region is predicted to offer a suitable environment for the disease. Monitoring the spread of chytridiomycosis is thus a priority in this region, which has exceptionally high levels of amphibian endemism. PCR analysis of 124 amphibian skin swabs in Tobago (Republic of Trinidad and Tobago) demonstrated the presence of B. dendrobatidis in three widely separated populations of the frog Mannophryne olmonae, which is listed as Critically Endangered on the basis of recent population declines. Chytridiomycosis is presently endemic in this species, with a prevalence of about 20% and no associated clinical disease. Increased susceptibility to chytridiomycosis from climate change is unlikely in amphibian populations in Tobago, as this island does not have high montane environments, but remains a possibility in the sister island of Trinidad. Preventing the spread of chytridiomycosis within and between these and other Caribbean islands should be a major goal of practical conservation measures for amphibians in the region.     

Amphibian Chytrid Fungus Broadly Distributed in the Brazilian Atlantic Rain Forest

To investigate the occurrence of the chytrid fungus Batrachochytrium dendrobatidis in Brazil, we conducted histological screenings of 96 preserved specimens of anurans collected at 10 sites in the Atlantic rain forest. Data show this fungus to be widely distributed. Infected specimens included Colostethus olfersioides (Dendrobatidae), Bokermannohyla gouveai and Hypsiboas freicanecae (Hylidae), as well as Thoropa miliaris and Crossodactylus caramaschii (Leptodactylidae), extending the area of B. dendrobatidis occurrence in Brazil approximately 1,600 km N, 200 km S, and 270 km E. The altitudinal range of the chytrid is broad, spanning from less than 100 m (Estação Ecológica Juréia-Itatins, Reserva Biológica do Tinguá) to about 2,400 m (Parque Nacional do Itatiaia). An infection record dating to 1981 roughly coincides with the time of the first observations of amphibian declines in the country. Widespread occurrence of B. dendrobatidis in the Atlantic Forest adds to the challenge of conserving an already endangered biome given the potential risk of further local biodiversity loss. Further research is needed to understand how environmental and genetic factors relate to chytridiomycosis in leading to or preventing local die-offs. Protected sites at mid and high elevations may be particularly threatened, while lowland populations may be functioning as reservoirs. Conservation efforts should also involve monitoring studies and habitat protection.     

Experimental Infection and Repeat Survey Data Indicate the Amphibian Chytrid Batrachochytrium dendrobatidis May Not Occur on Freshwater Crustaceans in Northern Queensland, Australia

Chytridiomycosis is a fatal disease of amphibians, caused by the amphibian chytrid Batrachochytrium dendrobatidis. The disease is unusual in that it may drive many amphibian species to local extinction during outbreaks. These dramatic declines in host population numbers could be facilitated if the pathogen can grow as a saprobe or on alternative hosts, a feature common to other chytrid species. This is also supported by in vitro work that demonstrates B. dendrobatidis can grow and reproduce in the absence of amphibian cells. In a previous study, B. dendrobatidis was detected on freshwater shrimp from rain forest streams in northern Queensland, Australia, using diagnostic PCR. We set out to confirm and further investigate the presence of B. dendrobatidis on crustaceans by carrying out more extensive sampling of shrimp in the field, experimental B. dendrobatidis infection trials using shrimp and crayfish, and PCR verification of the presence of B. dendrobatidis from shrimp samples that previously tested positive. We could not confirm the presence of B. dendrobatidis on shrimp, and report that original positive tests in shrimp reported by Rowley et al. (2006) were likely false. Thus, we suggest that shrimp may not be an important reservoir host for B. dendrobatidis.     

Experimental Exposures of Boreal Toads (Bufo boreas) to a Pathogenic Chytrid Fungus (Batrachochytrium dendrobatidis)

One of the major causes of worldwide amphibian declines is a skin infection caused by a pathogenic chytrid fungus (Batrachochytrium dendrobatidis). This study documents the interactions between this pathogen and a susceptible amphibian host, the boreal toad (Bufo boreas). The amount of time following exposure until death is influenced by the dosage of infectious zoospores, duration of exposure, and body size of the toad. The significant relation between dosage and the number of days survived (dose-response curve) supports the hypothesis that the degree of infection must reach a particular threshold of about 10⁷–10⁸ zoosporangia before death results. Variation in air temperature between 12°C and 23°C had no significant effect on survival time. The infection can be transmitted from infected to healthy animals by contact with water containing zoospores; no physical contact between animals is required. These results are correlated with observations on the population biology of boreal toads in which mortalities associated with B. dendrobatidis have been identified.     

Acknowledgments

One of the major causes of worldwide amphibian declines is a skin infection caused by a pathogenic chytrid fungus (Batrachochytrium dendrobatidis). This study documents the interactions between this pathogen and a susceptible amphibian host, the boreal toad (Bufo boreas). The amount of time following exposure until death is influenced by the dosage of infectious zoospores, duration of exposure, and body size of the toad. The significant relation between dosage and the number of days survived (dose-response curve) supports the hypothesis that the degree of infection must reach a particular threshold of about 10⁷–10⁸ zoosporangia before death results. Variation in air temperature between 12°C and 23°C had no significant effect on survival time. The infection can be transmitted from infected to healthy animals by contact with water containing zoospores; no physical contact between animals is required. These results are correlated with observations on the population biology of boreal toads in which mortalities associated with B. dendrobatidis have been identified.     

Context-dependent symbioses and their potential roles in wildlife diseases

It is well known in ecology, evolution and medicine that both the nature (commensal, parasitic and mutualistic) and outcome (symbiont fitness, survival) of symbiotic interactions are often context-dependent. Less is known about the importance of context-dependence in symbioses involved in wildlife disease. We review variable symbioses, and use the amphibian disease chytridiomycosis to demonstrate how understanding context-dependence can improve the understanding and management of wildlife diseases. In chytridiomycosis, the host-pathogen interaction is context-dependent; it is strongly affected by environmental temperature. Skin bacteria can also modify the interaction; some bacteria reduce amphibians' susceptibility to chytridiomycosis. Augmentation of protective microbes is being considered as a possible management tool, but informed application of bioaugmentation requires understanding of how the interactions between host, beneficial bacteria and pathogen depend upon environmental context. The community-level response of the amphibian skin microbiota to environmental conditions may explain the relatively narrow range of environmental conditions in which past declines have occurred. Environmental context affects virulence and the protection provided by mutualists in other host-pathogen systems, including threatened bats and corals. Increased focus on context-dependence in interactions between wildlife and their symbionts is likely to be crucial to the future investigation and management of emerging diseases of wildlife.     

Population trends associated with skin peptide defenses against chytridiomycosis in Australian frogs

Many species of amphibians in the wet tropics of Australia have experienced population declines linked with the emergence of a skin-invasive chytrid fungus, Batrachochytrium dendrobatidis. An innate defense, antimicrobial peptides produced by granular glands in the skin, may protect some species from disease. Here we present evidence that supports this hypothesis. We tested ten synthesized peptides produced by Australian species, and natural peptide mixtures from five Queensland rainforest species. Natural mixtures and most peptides tested in isolation inhibited growth of B. dendrobatidis in vitro. The three most active peptides (caerin 1.9, maculatin 1.1, and caerin 1.1) were found in the secretions of non-declining species (Litoria chloris, L. caerulea, and L. genimaculata). Although the possession of a potent isolated antimicrobial peptide does not guarantee protection from infection, non-declining species (L. lesueuri and L. genimaculata) inhabiting the rainforest of Queensland possess mixtures of peptides that may be more protective than those of the species occurring in the same habitat that have recently experienced population declines associated with chytridiomycosis (L. nannotis, L. rheocola, and Nyctimystes dayi). This study demonstrates that in vitro effectiveness of skin peptides correlates with the degree of decline in the face of an emerging pathogen. Further research is needed to assess whether this non-specific immune defense may be useful in predicting disease susceptibility in other species.     

Successful elimination of a lethal wildlife infectious disease in nature

Methods to mitigate the impacts of emerging infectious diseases affecting wildlife are urgently needed to combat loss of biodiversity. However, the successful mitigation of wildlife pathogens in situ has rarely occurred. Indeed, most strategies for combating wildlife diseases remain theoretical, despite the wealth of information available for combating infections in livestock and crops. Here, we report the outcome of a 5-year effort to eliminate infection with Batrachochytrium dendrobatidis affecting an island system with a single amphibian host. Our initial efforts to eliminate infection in the larval reservoir using a direct application of an antifungal were successful ex situ but infection returned to previous levels when tadpoles with cleared infections were returned to their natal sites. We subsequently combined antifungal treatment of tadpoles with environmental chemical disinfection. Infection at four of the five pools where infection had previously been recorded was eradicated, and remained so for 2 years post-application.     

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.     

Discovery of a Novel Alveolate Pathogen Affecting Southern Leopard Frogs in Georgia: Description of the Disease and Host Effects

In April of 2006, we observed southern leopard frog (Rana sphenocephala) tadpoles in a pond in northeast Georgia that were dying from an unknown pathogen. Examination of affected specimens, as well as PCR characterization, revealed that all were infected with a novel alveolate pathogen closely related to freshwater and marine eukaryotic organisms and, to a lesser degree, to members of the genus Perkinsus. This pathogen has been documented in numerous mortality events in anuran tadpoles in the United States, although it has not yet been named nor clearly described. We subsequently conducted a systematic survey of this and four other ponds in the same area to document the extent of the pathogen and to describe the nature of infections in leopard frog tadpoles. Of 87 live tadpoles examined, 25% were infected with the alveolate pathogen, based on visual inspection of tadpole liver tissue. Affected tadpoles frequently had enlarged abdomens, swam erratically, and could be captured by hand. All organs of infected tadpoles were infiltrated but typically to a lesser extent than the liver and kidneys, which often had hundreds of thousands of the spherical, 6-μm organisms. Infected tadpoles tended to weigh more than noninfected ones, likely due to the massive organ swelling that coincided with infections. Infected tadpoles did not differ in developmental stage from noninfected tadpoles. Infection prevalence varied widely among ponds, and in one pond, we witnessed a rapid die-off of R. spenocephala tadpoles during our surveys, although we did find infected metamorphic frogs. The rapid mortality we observed as well as the vast number of organisms seen in specimens suggests that this pathogen has tremendous transmission potential, and therefore deserves further monitoring and study.     

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. Captive assurance colonies are important worldwide for threatened amphibian species and may be the only lifeline for those in critical threat of extinction. Maintaining disease free colonies is a priority of captive managers, yet safe and effective treatments for all species and across life stages have not been identified. The most widely used chemotherapeutic treatment is itraconazole, although the dosage commonly used can be harmful to some individuals and species. We performed a clinical treatment trial to assess whether a lower and safer but effective dose of itraconazole could be found to cure Bd infections. We found that by reducing the treatment concentration from 0.01-0.0025% and reducing the treatment duration from 11-6 days of 5 min baths, frogs could be cured of Bd infection with fewer side effects and less treatment-associated mortality.     

The Decline of the Sharp-Snouted Day Frog (Taudactylus acutirostris): The First Documented Case of Extinction by Infection in a Free-Ranging Wildlife Species?

Infectious diseases are increasingly recognized as the cause of mass mortality events, population declines, and the local extirpation of wildlife species. In a number of cases, it has been hypothesized that pathogens have caused species extinctions in wildlife. However, there is only one definitively proven case of extinction by infection, and this was in a remnant captive population of a Polynesian tree snail. In this article, we review the potential involvement of infectious disease in the recent extinction of the sharp-snouted day frog Taudactylus acutirostris. Our review of available evidence suggests that a virulent pathogen of amphibians, Batrachochytrium dendrobatidis, caused a rapid, catastrophic decline of this species, from which it did not recover. We propose that this is the first case of extinction by infection of a free-ranging wildlife species where disease acted as both the proximate and ultimate cause of extinction. This highlights a probable underreporting of infectious disease as a cause of biodiversity loss historically and currently.     

Integrating species traits with extrinsic threats: closing the gap between predicting and preventing species declines

In studies of extinction risk, it is often insufficient to conclude that species with narrow ranges or small clutch sizes require prioritized protection. To improve conservation outcomes, we also need to know which threats interact with these traits to endanger some species but not others. In this study, we integrated the spatial patterns of key threats to Australian amphibians with species' ecological/life-history traits to both predict declining species and identify their likely threats. In addition to confirming the importance of previously identified traits (e.g. narrow range size), we find that extrinsic threats (primarily the disease chytridiomycosis and invasive mosquitofish) are equally important and interact with intrinsic traits (primarily ecological group) to create guild-specific pathways to decline in our model system. Integrating the spatial patterns of extrinsic threats in extinction risk analyses will improve our ability to detect and manage endangered species in the future, particularly where data deficiency is a problem.