Elevated temperature as a treatment for Batrachochytrium dendrobatidis infection in captive frogs

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines worldwide. In vitro laboratory studies and those done on wild populations indicate that Bd grows best at cool temperatures between 17 and 25 degrees C. In the present study, we tested whether moderately elevating the ambient temperature to 30 degrees C could be an effective treatment for frogs infected with Bd. We acquired 35 bullfrogs Rana catesbeiana from breeding facilities and 36 northern cricket frogs Acris crepitans from the wild and acclimated them to either 23 or 26 degrees C for 1 mo. Following the acclimation period, frogs were tested for the presence of Bd using qPCR TaqMan assays. The 12 R. catesbeiana and 16 A. crepitans that tested positive for Bd were subjected to 30 degrees C for 10 consecutive days before returning frogs to their starting temperatures. Post-treatment testing revealed that 27 of the 28 frogs that had tested positive were no longer infected with Bd; only a single A. crepitans remained infected following treatment. This result indicates that elevating ambient temperature to a moderate 30 degrees C can be effective as a treatment for Bd infection in captive amphibians, and suggests that heat may be a superior alternative to antifungal drugs.     

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.     

New records of Batrachochytrium dendrobatidis in Chilean frogs

We used molecular techniques to examine 11 species of frogs in 6 localities in southern Chile to ascertain the incidence of the chytrid fungus Batrachochytrium dendrobatidis (Bd). We detected the fungus in 2 localities (Co?aripe and Raúl Marín Balmaceda) in 3 species: Batrachyla leptopus, Pleurodema thaul and Rhinoderma darwinii. Our findings expand the list of Bd hosts to include B. leptopus and P. thaul and extend the spatial distribution in Chile to include the southernmost Bd record at Raúl Marín Balmaceda.     

Altitudinal distribution of chytrid (Batrachochytrium dendrobatidis) infection in subtropical Australian frogs

The disappearance of amphibian populations from seemingly pristine upland areas worldwide has become a major focus of conservation efforts in the last two decades, and a parasitic chytrid fungus, Batrachochytrium dendrobatidis, is thought to be the causative agent of the population declines. We examined the altitudinal distribution of chytrid infections in three stream‐dwelling frog species (Litoria wilcoxii, L. pearsoniana and L. chloris) in southeast Queensland, Australia, and hypothesized that if B. dendrobatidis were responsible for the disappearance of high‐altitude frog populations, infection prevalence and intensity would be greatest at higher altitudes. Overall, 37.7% of the 798 adult frogs we sampled were infected with B. dendrobatidis, and infections were found in all the populations we examined. Contrary to our initial hypothesis, we found no consistent evidence that high‐altitude frogs were more likely to be infected than were lowland frogs. Further, the intensity of fungal infections (number of zoospores) on high‐altitude frogs did not differ significantly from that of lowland frogs. Batrachochytrium dendrobatidis appears to be capable of infecting frogs at all altitudes in the subtropics, suggesting that all populations are at risk of decline when conditions favour disease outbreaks. We did find evidence, however, that chytrid infections persist longer into summer in upland as compared with lowland areas, suggesting that montane amphibian populations remain susceptible to disease outbreaks for longer periods than do lowland populations. Further, we found that at high altitudes, temperatures optimal for chytrid growth and reproduction coincide with frog metamorphosis, the life‐stage at which frogs are most susceptible to chytrid infections. While these altitudinal differences may account for the differential population‐level responses to the presence of B. dendrobatidis, the reason why many of southeast Queensland's montane frog populations declined precipitously while lowland populations remained stable has yet to be resolved.     

Widespread historical presence of Batrachochytrium dendrobatidis in African pipid frogs

Aim Amphibian chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), is associated with global amphibian population declines and species extinctions. Current evidence indicates that the pathogen has recently spread globally from an enzootic focus, with Xenopus spp. (family Pipidae) in South Africa having been identified as a likely source. The aim of this study was to investigate further the likelihood of African Xenopus spp. as the original source of Bd. Location We examined 665 museum specimens of 20 species of African and South American pipid frogs collected between 1844 and 1994 and held in the collection of the Natural History Museum, London. Methods Skin brushings taken from adult amphibians and brushings from the mouthparts, lips and developing hind limbs of larval pipid frogs were examined for the presence of Bd using real‐time PCR. Results We found six cases of Bd infection in three Xenopus spp. (from Africa), but none of the South American pipids was positive, although only 45 South American frogs were available for examination. The earliest case of Bd infection was in a specimen of Xenopus fraseri collected from Cameroon in 1933. A consistently low prevalence of infection over time indicates that a historical equilibrium existed between Xenopus spp. and Bd infection in Africa. Main conclusions Our results suggest that Bd infection was present in Xenopus spp. across sub‐Saharan Africa by the 1930s, providing additional support for the ‘out of Africa’ hypothesis. If this hypothesis is correct, it strengthens the argument for stringent control of human‐assisted movements of amphibians and other wildlife world‐wide to minimize the likelihood of pathogen introduction and disease emergence that can threaten species globally. Our findings help inform species selection for conservation in the face of the current Bd pandemic and also guide future research directions for selecting Bd isolates for sequencing and virulence testing.     

Co-localisation of Batrachochytrium dendrobatidis and keratin for enhanced diagnosis of chytridiomycosis in frogs

Chytridiomycosis is a disease of post-metamorphic frogs caused by the fungus Batrachochytrium dendrobatidis and is associated with large declines in frog populations on a global scale. B. dendrobatidis is found only in the keratinised tissues, which include the mouthparts of healthy tadpoles. The epidermis of infected post-metamorphic frogs is thickened (hyperkeratosis) and the superficial layer can sometimes slough. Diagnosis is most commonly performed on stained sections of toe clips or ventral skin. Accurate interpretation can be difficult and requires a high level of expertise, particularly in infected animals exhibiting hyperkeratosis with sloughing. Misdiagnosis can occur when zoosporangia of B. dendrobatidis are shed with the superficial keratin layers. We have developed a staining protocol based on previously described methods to detect both B. dendrobatidis and keratin, to improve the sensitivity and specificity of diagnosis of chytridiomycosis by inexperienced diagnosticians.     

Predators reduce Batrachochytrium dendrobatidis infection loads in their prey

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

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

Retreat sites of rain forest stream frogs are not a reservoir for Batrachochytrium dendrobatidis in northern Queensland, Australia

Chytridiomycosis is a potentially fatal disease of amphibians caused by Batrachochytrium dendrobatidis, and is implicated in declines and extinctions of amphibian populations and species around the world. To cause local host extinction, a disease organism must persist at low host densities. One mechanism that could facilitate this is the ability to persist in the environment. In the laboratory, B. dendrobatidis spreads by both frog-to-frog and environment-to-frog transmission, and can persist on a number of biotic substrates. In the field, B. dendrobatidis has been detected on environmental samples taken during an epidemic, but it is not known if it persists in the environment when endemic. Retreat sites of 2 species of Australian rain forest stream frogs Litoria lesueuri and L. nannotis were sampled 0 to 3 d after occupation during the wet and dry seasons in northern Queensland, Australia, where chytridiomycosis has been endemic for at least 10 yr. The intensity and prevalence of infection in frogs during sampling were comparatively low compared with epidemics. Diagnostic quantitative polymerase chain reaction did not detect B. dendrobatidis in any retreat site samples. It thus appears that retreat sites are not a major environmental source of infection when B. dendrobatidis occurs at low prevalence and intensity on frogs. This suggests that control efforts may not need to eliminate the organism from the environment, at least when prevalence and intensity of infection are low in frogs. Simply treating hosts may be effective at controlling the disease in the wild.     

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

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

Fluorescent microscopy of viable Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis ( Bd ), a chytrid fungus, is a causative agent of chytridiomycosis and amphibian population declines worldwide. The sequenced genome of Bd provides information necessary for studying the fungus and its molecular biology. Fluorescent microscopy is a technique used to image targeted molecules in live or fixed organisms to understand cellular trafficking and localization, but the use of fluorescent microscopy with Bd has not yet been demonstrated. Two fluorescent stains were tested for their use in live-cell imaging of Bd , i.e., the cell wall-specific fluorophore Solophenyl Flavine 7GFE and the DNA-specific fluorophore DRAQ5. These specific staining patterns were observed in live cultures of Bd when visualized with laser-scanning confocal microscopy.     

Environmental detection of Batrachochytrium dendrobatidis in a temperate climate

The aetiological agent of amphibian chytridiomycosis Batrachochytrium dendrobatidis is a primary cause of amphibian population declines. Current surveillance is based on the detection of B. dendrobatidis in its host but in vitro work suggests infective stages may survive in the abiotic environment for at least 3 mo. We describe here a surveillance system using filtration and quantitative PCR that can detect B. dendrobatidis in small (< 1 l) volumes of water. After assessing the analytical sensitivity of the protocol for both water and sediment samples in the laboratory, we analyzed environmental samples from the Sierra de Guadarrama mountain range in Spain at locations associated with chytrid-related die-offs and at other sites across Spain. B. dendrobatidis was detected in samples from 64% of the ponds in the Sierra de Guadarrama and at 2 sites outside this region, showing that levels of amphibian exposure to B. dendrobatidis are spatially heterogeneous. In experimental microcosms, we detected B. dendrobatidis for up to 12 wk, though we found no evidence for an overall increase in biomass. Our results emphasise the need to further investigate the life cycle of B. dendrobatidis to more completely understand the epidemiology of this emerging pathogen.     

Historical dynamics of Batrachochytrium dendrobatidis in Amazonia

The Amazon forest is known for its astonishing amphibian diversity, yet the potential distribution and underlying impacts of the most important amphibian pathogen is unknown for most of Amazonia. In this retrospective survey of preserved Leptodactylus frogs, collected over a 119 yr period, we used quantitative PCR to detect the fungal pathogen Batrachochytrium dendrobatidis (Bd) and performed spatial scan analyses to identify spatiotemporal clusters of Bd. We also quantified the potential effect of environmental factors on the likelihood of Bd occurrence and generated an updated suitability map for Bd in the Amazon that included our retrospective sampling. We detected Bd in lowland Amazon as early as 1935, in the state of Pará, Brazil, and we found low prevalence (～ 3.8%) over time. We identified two statistically significant spatiotemporal clusters of Bd: a recent and narrow cluster in the Amazon River delta and a spatiotemporally broad cluster in the southern edge of Amazon and Brazilian savanna. Furthermore, we found an increase in Bd‐positive samples in the southwestern Amazon after the 1990s, coinciding with reported amphibian declines in neighboring high elevation sites on Andean slopes of Peru. Spatial regressions indicated that higher human interference, higher precipitation, and lower temperatures were significant predictors of Bd occurrence. Environmental niche modeling predicted some narrow areas of suitable climates along the Amazon's periphery and generally low climatic suitability for Bd in the central Amazon; although, we found clusters of Bd‐positive samples with unexpectedly high infection loads in areas of predicted low suitability. Our study indicates that accelerated human development may put Amazonian amphibians at risk from Bd introductions, and it highlights the potential need to monitor Bd dynamics near Amazonian port cities.     

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.     

Cryo-archiving of Batrachochytrium dendrobatidis and other chytridiomycetes

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide. It has been associated with catastrophic declines of frog populations including those in pristine habitats in Queensland, Australia. To facilitate genetic and disease studies of this fungus and related species, it is essential to have a reliable long-term storage method to maintain genetic integrity of isolates. We have adapted well-established techniques used for the long-term storage of tissue-culture cell lines to the preservation of B. dendrobatidis and other chytridiomycetes. This simple method has allowed us to recover these fungi from storage at -80 degrees C and in liquid nitrogen over an extended period. With this technique it is now possible to preserve saprobic and parasitic isolates from a variety of environmental and disease situations for comparative genetic and biological studies.     

Relationships among size, development, and Batrachochytrium dendrobatidis infection in African tadpoles

The fungal pathogen Batrachochytrium dendrobatidis contributes to the global decline of amphibians. Although mortality from B. dendrobatidis infections occurs primarily in postmetamorphic individuals, infected tadpoles may suffer reduced growth and developmental rates as a result of oral chytridiomycosis, possibly affecting adult fitness. We conducted a field study in which we examined South African tadpoles for oral chytridiomycosis and compared the body sizes of infected and uninfected individuals of 2 species, Heleophryne natalensis and Strongylopus hymenopus. Presence of B. dendrobatidis was determined by microscopic inspection of mouthparts. Infection prevalence was high in both species, 62.5 and 38.6%, respectively, and infected individuals were significantly larger in both species. The inclusion of developmental stage in the analysis of S. hymenopus body size eliminated the relationship between body size and infection status, suggesting that differences in body size were not due to differences in growth, but to differences in developmental stage of infected larvae. These results suggest that larvae at more advanced developmental stages are more likely to be infected with B. dendrobatidis and that infection in larval amphibians may be dependent on time or developmental status of larvae. Contrary to the results of past studies, there was no evidence that oral chytridiomycosis resulted in decreased growth of tadpoles, despite the occurrence of oral abnormalities in infected individuals of 1 species. Because tadpole performance can subsequently affect the health of anuran populations and because tadpoles can act as reservoirs of infection, the study of B. dendrobatidis in larval amphibians is important to understanding the effects of this emerging disease.     

Landscape epidemiology of Batrachochytrium dendrobatidis in central California

Amphibian chytridiomycosis (caused by Batrachochytrium dendrobatis; Bd) was first identified in 1998 and has since been implicated in numerous amphibian declines worldwide. Most researchers have since investigated broad‐scale geographic and taxonomic occurrences of the pathogen in tropical lotic or cool montane systems. In this study, we analyzed how environmental factors, land use practices, and landscape structure may affect the dynamics of the pathogen's distribution in a landscape dominated by lentic systems within a region of Mediterranean climate. We quantified the occurrence of Bd testing the six resident amphibian species that occur in 54 isolated perennial and ephemeral ponds in central California between May and June annually from 2004 to 2007. The geographic distribution of Bd within the landscape varied markedly between years. Inter‐annual variation in climate affected the pond landscape structure indicating that climate conditions indirectly influence the distribution of the pathogen. Fourteen ponds, 12 perennial and 2 ephemeral, were positive for Bd≥3 yr of the study and were treated as Bd hotpots for comparative purposes. Occurrences of Bd within the landscape were spatially autocorrelated and ponds within ~1000–1500 m of Bd hotspots were more likely to test positive. Local land use, (presence/absence of grazing or recreational activity and developed lands), did not influence Bd status of a pond, indicating that the most likely means of Bd transmission between ponds may be waterfowl and/or amphibians.     

Evaluation of tadpole mouthpart depigmentation as a diagnostic test for infection by Batrachochytrium dendrobatidis for four California anurans

The objective of this study was to evaluate the utility of gross morphologic examination of larval mouthpart defects as a diagnostic screening test to detect Batrachochytrium dendrobatidis infection in four California, USA, anuran species. We examined mouthparts of 2,034 tadpoles of Bufo boreas, Pseudacris regilla, and Rana catesbeiana collected in 2003 and 2004 and Bufo canorus collected in 2004. Data were recorded for three morphologic features: upper toothrows, lower toothrows, and combined jaw sheaths. Mouthpart defects were observed in all four species (n=757), but only two species were infected with B. dendrobatidis (n=84). Sensitivity and specificity of the mouthparts test were 76% and 58%, respectively. Forty-two percent of B. dendrobatidis-negative animals would have been designated positive based on mouthpart defects. Observed prevalence was 43%, and true prevalence was 3.0%. Tests of the null hypothesis using logistic regression analysis showed that anuran larval mouthpart defects were not associated with B. dendrobatidis infection whether mouthparts scores were tested by individual morphologic feature or in combination (P=0.37). We conclude that B. dendrobatidis infection and anuran larval mouthpart defects are two separate processes that may occur concurrently and that evaluation of tadpole oral morphology is neither an accurate nor a reliable diagnostic test for B. dendrobatidis infection for the four species tested.     

Quantitative measurement of Batrachochytrium dendrobatidis in amphibian skin

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.