Amphibian chytridiomycosis in Japan: distribution,haplotypes and possible route of entry into Japan

A serious disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis was first found in Japan in December 2006 in imported pet frogs. This was the first report of chytridiomycosis in Asia. To assess the risk of pandemic chytridiomycosis to Japanese frogs, we surveyed the distribution of the fungus among captive and wild frog populations. We established a nested PCR assay that uses two pairs of PCR primers to amplify the internal transcribed spacer (ITS) region of a ribosomal RNA cassette to detect mild fungal infections from as little as 0.001 pg (1 fg) of B. dendrobatidis DNA. We collected swab samples from 265 amphibians sold at pet shops, 294 bred at institutes and 2103 collected at field sites from northern to southwestern Japan. We detected infections in native and exotic species, both in captivity and in the field. Sequencing of PCR products revealed 26 haplotypes of the B. dendrobatidis ITS region. Phylogenetic analysis showed that three of these haplotypes were specific to the Japanese giant salamander (Andrias japonicus) and appeared to have established a commensal relationship with this native amphibian. Many other haplotypes were carried by alien amphibians. The highest genetic diversity of B. dendrobatidis was found in the American bullfrog (Rana catesbeiana). Some strains of B. dendrobatidis appeared to be endemic to Japanese native amphibians, but many alien strains are being introduced into Japan via imported amphibians. To improve chytridiomycosis risk management, we must consider the risk of B. dendrobatidis changing hosts as a result of anthropogenic disturbance of the host‐specific distribution of the fungus.     

High Occupancy of Stream Salamanders Despite High Ranavirus Prevalence in a Southern Appalachians Watershed

The interactive effects of environmental stressors and emerging infectious disease pose potential threats to stream salamander communities and their headwater stream ecosystems. To begin assessing these threats, we conducted occupancy surveys and pathogen screening of stream salamanders (Family Plethodontidae) in a protected southern Appalachians watershed in Georgia and North Carolina, USA. Of the 101 salamanders screened for both chytrid fungus (Batrachochytrium dendrobatidis) and Ranavirus, only two exhibited low-level chytrid infections. Prevalence of Ranavirus was much higher (30.4% among five species of Desmognathus). Despite the ubiquity of ranaviral infections, we found high probabilities of site occupancy (≥0.60) for all stream salamander species.     

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.     

Distribution models for the amphibian chytrid Batrachochytrium dendrobatidis in Costa Rica: proposing climatic refuges as a conservation tool

Aim We use novel data on the occurrence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in Costa Rica to model its potential distribution in that country. Location Lowland and montane areas of Costa Rica. Methods We use published and new data on the presence of B. dendrobatidis on 647 amphibians (35 species). Screening was performed through histological techniques by which 156 sites were surveyed. Of these, 21 were found to have the amphibian chytrid. Maxent, a presence‐only distribution modelling technique, was used to create 100 predictions of B. dendrobatidis occurrence, of which the most accurate 10 (based on area under the receiver‐operating characteristic curve) were chosen to create a composite distribution model. This approach increased confidence in model predictions, distinguishing areas of high probability of occurrence and low variability across model runs (higher confidence) from those with high probability but high variability (lower confidence). Results Predicted distribution patterns were not uniform along Costa Rica's mountains, where most amphibian declines have occurred. The pathogen was predicted to occur with greater probability on the Caribbean slopes than on the Pacific slopes. While high temperature seems to constrain the distribution of the pathogen, areas that also have small amounts of rainfall during the driest period of the year were predicted to have low probability of B. dendrobatidis occurrence. Main conclusions The model predicts that the Santa Elena Peninsula and the Central Valley have low probabilities of B. dendrobatidis occurrence, suggesting that they could function as refuges for amphibians. In such refugial areas, one could expect B. dendrobatidis to be absent, or to be present in low abundance (rendering an epidemic outbreak of chytridiomycosis unlikely). Craugastor ranoides, which belongs to a group of frogs particularly sensitive to chytridiomycosis outbreaks, persists in the hot and seasonally dry Santa Elena Peninsula but disappeared in the nearby colder and more humid Guanacaste Volcanic Chain. This information suggests that climatic refuges, where environmental conditions prevent disease outbreaks, could be an important component in amphibian conservation.     

Seasonal Ecology and Behavior of an Endangered Rainforest Frog (Litoria rheocola) Threatened by Disease

One of the most devastating wildlife diseases ever recorded is chytridiomycosis, a recently emerged amphibian disease that is caused by the chytrid fungus Batrachochytrium dendrobatidis. Understanding, predicting, and managing the impacts of chytridiomycosis on any amphibian species will require detailed information on its ecology and behavior because this pathogen is transmitted by contact with water or other individuals, and pathogen growth rates are thermally sensitive. The common mistfrog (Litoria rheocola) is an endangered tropical rainforest frog that has declined due to chytridiomycosis. We tracked L. rheocola during the winter (cool/dry) and summer (warm/wet) seasons at a low- and high-elevation site. We found that seasonal differences in environmental temperatures and frog behavior should render this species most vulnerable to B. dendrobatidis during cooler months and at higher elevations, which matches observed patterns of infection prevalence in this species. During winter, frogs moved shorter distances than during summer, and they spent less time in vegetation and more time in the stream, which should increase exposure to aquatic B. dendrobatidis zoospores. At a low-elevation site (40 m ASL), estimated body temperatures were within the optimal range for B. dendrobatidis growth (15-25°C) most of the time during winter, but they reached temperatures above this threshold frequently in summer. At a higher elevation (750 m ASL), estimated body temperatures were within the range most favorable for B. dendrobatidis year-round, and did not exceed 25°C, even during summer. Our study provides the first detailed information on the ecology and behavior of L. rheocola and suggests ecological mechanisms for infection dynamics that have been observed in this endangered species.     

Retrospective Survey of Museum Specimens Reveals Historically Widespread Presence of Batrachochytrium dendrobatidis in China

Chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has been implicated in amphibian population declines worldwide. However, no amphibian declines or extinctions associated with Bd have been reported in Asia. To investigate the history of this pathogen in China, we examined 1,007 museum-preserved amphibian specimens of 80 species collected between 1933 and 2009. Bd was detected in 60 individuals (6.0%), with the earliest case of Bd infection occurring in one specimen of Bufo gargarizans and two Fejervarya limnocharis, all collected in 1933 from Chongqing, southwest China. Although mainly detected in non-threatened native amphibians, Bd was also found in four endangered species. We report the first evidence of Bd for Taiwan and the first detection of Bd in the critically endangered Chinese giant salamander (Andrias davidianus). Bd appears to have been present at a low rate of infection since at least the 1930s in China, and no significant differences in prevalence were detected between decades or provinces, suggesting that a historical steady endemic relationship between Bd and Chinese amphibians has occurred. Our results add new insights on the global emergence of Bd and suggest that this pathogen has been more widely distributed in the last century than previously believed.     

Chytridiomycosis Widespread in Anurans of Northeastern United States

ABSTRACT An emerging disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis has been associated with morbidity, mortality, and extinction of species. Typically, researchers have detected B. dendrobatidis only when examining amphibians for causes of mortalities; few data exist on infection rates where mortalities are lacking. During May-September 2000–2002 we obtained amphibian specimens killed by vehicles and others collected at remote off-road sites throughout Maine, USA, and from federal lands in 5 states in the Northeast. We detected infected specimens, mostly green frogs (Rana clamitans), at 5 of 7 national wildlife refuges, a federal waterfowl production area, and Acadia National Park. Seven of 9 species, including all Ranidae species, were infected throughout Maine; rates ranged from 14.6% in American toads (Bufo americanus) to 25.7% in northern leopard frogs (Rana pipiens). We did not detect any infections in 50 eastern gray tree frogs (Hyla versicolor) or 21 spring peepers (Pseudacris crucifer). Species that hibernate in terrestrial habitats seem to have lower rates of infection than species that hibernate in aquatic habitats. Infections peaked in spring and autumn and were associated with air temperatures optimal for B. dendrobatidis growth. The relatively high infection rates among species without documented die-offs suggest that either losses have occurred undetected, that the fungus is endemic and species have attained a level of resistance to infections becoming lethal, or that climatic conditions of the Northeast have a role in preventing infections from being lethal. Data on prevalence and distribution of this chytrid fungus in the Northeast may be useful in modeling its origins and predicting long-term ecosystem effects involving anurans.     

Predicting the Distribution of the Amphibian Pathogen Batrachochytrium dendrobatidis in the New World1

One application of ecological niche modeling is predicting suitable areas for the establishment of invasive species. Herein, I model the fundamental niche of the chytrid fungus Batrachochytrium dendrobatidis, a pathogen linked to amphibian declines on several continents. Niche models were generated with the Genetic Algorithm of Rule‐Set Prediction using point distribution data of the pathogen and digital maps of environmental variables integrated in a GIS environment. The distribution of regions suitable for B. dendrobatidis in the New World is extensive and includes significant portions of: (1) Sierra Madre Occidental pine‐oak forest; (2) Sonoran and Sinaloan dry forest; (3) Veracruz moist forest; (4) Central America east from the Isthmus of Tehuantepec; (5) Caribbean Islands; (6) temperate forest in Chile and western Argentina south of latitude 30°S; (7) Andes above 1000 m of altitude in Venezuela, Colombia, and Ecuador; (8) eastern slopes of the Andes in Peru and Bolivia; (9) Brazilian Atlantic forest; (10) Uruguay, Paraguay, and northeastern Argentina; (11) southwestern and Madeira‐Tapajós Amazonian tropical rainforests. The regions with the highest suitability for B. dendrobatidis include habitats that contain the world's most diverse amphibian faunas. Models were built with New World localities, but also showed strong predictability for B. dendrobatidis localities in the Old World. Out of a total of 59 reported Old World localities for B. dendrobatidis, 56 occurred within regions with high predicted suitability. I also present analyses of the environmental envelope of B. dendrobatidis and discuss the implications of the results for the conservation of amphibians in the neotropics.     

The Last South American Redoubt? Tested Surinamese Anurans Still Chytrid Free

Batrachochytrium dendrobatidis, a chytrid fungus infecting amphibians’ cutaneous layer, is responsible for the greatest contemporary loss of amphibian biodiversity. In South America, Suriname is one of the only three countries where B. dendrobatidis infections of anurans (frogs and toads) have not been documented. To further examine this apparent gap in pathogen occurrence, frogs were sampled for B. dendrobatidis spores at eight disparate geographic locations in Suriname, including locations with high and low levels of anthropogenic activities, and near Suriname’s border with Brazil and French Guiana, countries where B. dendrobatidis infections have been documented. None of the 347 frogs sampled, representing 37 species from eight families, tested positive for B. dendrobatidis. Our results provide the baseline data for future comparative testing and one of the last opportunities for a country in South America to proactively plan mitigation measures to protect amphibians from B. dendrobatidis’ presumed eventual incursion into Suriname.

     

Ubiquity of the Pathogenic Chytrid Fungus, <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis>, in Anuran Communities in Panamá

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis, has been implicated as the main driver of many enigmatic amphibian declines in neotropical sites at high elevation. Batrachochytrium dendrobatidis is thought to be a waterborne pathogen limited by temperature, and the extent to which it persists and causes disease in amphibians at lower elevations in the neotropics is not known. It also is unclear by what mechanism(s) B. dendrobatidis has emerged as a pathogenic organism. To test whether B. dendrobatidis is limited by elevation in Panamá, we sought to determine the prevalence and intensity of B. dendrobatidis in relation to anuran abundance and diversity using quantitative PCR (qPCR) analyses. Sites were situated at varying elevations, from 45 to 1215 m, and were at varying stages of epizootic amphibian decline, including pre-epizootic, mid-epizootic, 2 years post-epizootic, and 10 years post-epizootic. Batrachochytrium dendrobatidis was found in all sites regardless of elevation or stage of epizootic decline. Levels of prevalence and infection intensity were comparable across all sites except at the mid-epizootic site, where both prevalence and intensity were significantly higher than at other sites. Symptoms of chytridiomycosis and corresponding declines in amphibian populations were variably seen at all elevations along a post-epizootic gradient. Because it is inherently difficult to prove a negative proposition, it can neither be proven that B. dendrobatidis is truly not present where it is not detected nor proven that it is only recently arrived where it is detected. Thus, there will always be doubts about whether B. dendrobatidis is enzootic or invasive. In any case, our results, coupled with current knowledge, suggest most clearly that the disease, chytridiomycosis, may be novel and invasive, and that the pathogen, B. dendrobatidis either is, or is becoming, globally ubiquitous.     

How Does Chytrid Infection Vary Among Habitats? The Case of Litoria wilcoxii (Anura, Hylidae) in SE Queensland, Australia

Most analyses dealing with the geographical distribution of the chytrid fungus (Batrachochytrium dendrobatidis) have been performed on large geographical scales and data on more localized distribution of the chytrid within catchments are scarce. In this study, we compare the prevalence and intensity of infection of chytrid within and outside rainforest habitats at five independent catchments in southeast Queensland. In each catchment, we sampled adult Litoria wilcoxii along two transects on the same stream: one in forested areas, and the other in open nearby farmland. We analyzed swabs using quantitative PCR techniques. Male frogs were in higher densities in open habitats compared with the nearby forested areas. Infected male frogs were found in all catchments surveyed; however, prevalence of B. dendrobatidis in adult males was higher in the forested habitats than in the open habitats in four of the catchments. There was no significant difference in intensity of infection between forested and open habitats. For adult females and juveniles, sample sizes were not high enough for comparisons. Our results suggest that habitat influences chytrid prevalence and open areas may provide refuge from chytrid-induced population declines.     

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.     

Amphibian Pathogen Batrachochytrium dendrobatidis Is Inhibited by the Cutaneous Bacteria of Amphibian Species

Population declines of amphibian species in many parts of the world are associated with a lethal fungal pathogen, Batrachochytrium dendrobatidis. Using laboratory challenge assays, we describe the inhibition of B. dendrobatidis by members of eight genera of bacteria isolated from the skin of two amphibian species that exhibit parental care behavior (Plethodon cinereus and Hemidactylium scutatum). We found that members of three genera of bacteria isolated from the skins of the salamander P. cinereus and members of seven genera isolated from the salamander H. scutatum inhibited the growth of B. dendrobatidis. Understanding how B. dendrobatidis interacts with an ecological community of cutaneous flora may be important in explaining and preventing amphibian population declines.     

<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.     

An interaction between climate change and infectious disease drove widespread amphibian declines

Climate change might drive species declines by altering species interactions, such as host–parasite interactions. However, few studies have combined experiments, field data, and historical climate records to provide evidence that an interaction between climate change and disease caused any host declines. A recently proposed hypothesis, the thermal mismatch hypothesis, could identify host species that are vulnerable to disease under climate change because it predicts that cool‐ and warm‐adapted hosts should be vulnerable to disease at unusually warm and cool temperatures, respectively. Here, we conduct experiments on Atelopus zeteki, a critically endangered, captively bred frog that prefers relatively cool temperatures, and show that frogs have high pathogen loads and high mortality rates only when exposed to a combination of the pathogenic chytrid fungus (Batrachochytrium dendrobatidis) and high temperatures, as predicted by the thermal mismatch hypothesis. Further, we tested various hypotheses to explain recent declines experienced by species in the amphibian genus Atelopus that are thought to be associated with B. dendrobatidis and reveal that these declines are best explained by the thermal mismatch hypothesis. As in our experiments, only the combination of rapid increases in temperature and infectious disease could account for the patterns of declines, especially in species adapted to relatively cool environments. After combining experiments on declining hosts with spatiotemporal patterns in the field, our findings are consistent with the hypothesis that widespread species declines, including possible extinctions, have been driven by an interaction between increasing temperatures and infectious disease. Moreover, our findings suggest that hosts adapted to relatively cool conditions will be most vulnerable to the combination of increases in mean temperature and emerging infectious diseases.     

Thermoregulatory behaviour affects prevalence of chytrid fungal infection in a wild population of Panamanian golden frogs

Predicting how climate change will affect disease dynamics requires an understanding of how the environment affects host–pathogen interactions. For amphibians, global declines and extinctions have been linked to a pathogenic chytrid fungus, Batrachochytrium dendrobatidis. Using a combination of body temperature measurements and disease assays conducted before and after the arrival of B. dendrobatidis, this study tested the hypothesis that body temperature affects the prevalence of infection in a wild population of Panamanian golden frogs (Atelopus zeteki). The timing of first detection of the fungus was consistent with that of a wave of epidemic infections spreading south and eastward through Central America. During the epidemic, many golden frogs modified their thermoregulatory behaviour, raising body temperatures above their normal set point. Odds of infection decreased with increasing body temperature, demonstrating that even slight environmental or behavioural changes have the potential to affect an individual''s vulnerability to infection. The thermal dependency of the relationship between B. dendrobatidis and its amphibian hosts demonstrates how the progression of an epidemic can be influenced by complex interactions between host and pathogen phenotypes and the environments in which they are found.     

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.     

Confronting inconsistencies in the amphibian‐chytridiomycosis system: implications for disease management

Chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is one of the largest threats to wildlife and is putatively linked to the extirpation of numerous amphibians. Despite over a decade of research on Bd, conflicting results from a number of studies make it difficult to forecast where future epizootics will occur and how to manage this pathogen effectively. Here, we emphasize how resolving these conflicts will advance Bd management and amphibian conservation efforts. We synthesize current knowledge on whether Bd is novel or endemic, whether amphibians exhibit acquired resistance to Bd, the importance of host resistance versus tolerance to Bd, and how biotic (e.g. species richness) and abiotic factors (e.g. climate change) affect Bd abundance. Advances in our knowledge of amphibian–chytrid interactions might inform the management of fungal pathogens in general, which are becoming more common and problematic globally.