High Prevalence of Batrachochytrium dendrobatidis in Wild Populations of Lowland Leopard Frogs Rana yavapaiensis in Arizona

Batrachochytrium dendrobatidis (Bd) is a fungus that can potentially lead to chytridiomycosis, an amphibian disease implicated in die-offs and population declines in many regions of the world. Winter field surveys in the last decade have documented die-offs in populations of the lowland leopard frog Rana yavapaiensis with chytridiomycosis. To test whether the fungus persists in host populations between episodes of observed host mortality, we quantified field-based Bd infection rates during nonwinter months. We used PCR to sample for the presence of Bd in live individuals from nine seemingly healthy populations of the lowland leopard frog as well as four of the American bullfrog R. catesbeiana (a putative vector for Bd) from Arizona. We found Bd in 10 of 13 sampled populations. The overall prevalence of Bd was 43% in lowland leopard frogs and 18% in American bullfrogs. Our results suggest that Bd is widespread in Arizona during nonwinter months and may become virulent only in winter in conjunction with other cofactors, or is now benign in these species. The absence of Bd from two populations associated with thermal springs (water >30°C), despite its presence in nearby ambient waters, suggests that these microhabitats represent refugia from Bd and chytridiomycosis.     

Urinary corticosterone metabolites and chytridiomycosis disease prevalence in a free-living population of male Stony Creek frogs (Litoria wilcoxii)

The emerging amphibian disease chytridiomycosis, which is caused by the fungal pathogen (Batrachochytrium dendrobatidis, Bd), has caused mass mortalities of native amphibian populations globally. There have been no previous studies on the relationships between stress hormones in free-living amphibians and Bd infections. In this study, we measured urinary corticosterone metabolite concentrations and Bd infections within free-living populations of male Stony Creek frog (Litoria wilcoxii) in Queensland, Australia. Prevalence of Bd zoospores from frog skin swabs was quantified using a real-time quantitative PCR technique. A urinary corticosterone enzyme-immunoassay (EIA) was validated using adrenocorticotropic hormone (ACTH) challenge. Urinary corticosterone concentrations of male frogs increased within 1-2 days after ACTH challenge and returned to baseline levels within 3 days post-ACTH injection. None of the frogs showed any rise in urinary corticosterone after saline injections. Individual male frogs showed either low or high baseline corticosterone concentrations. Male frogs identified as positive for Bd infection had significantly higher baseline urinary corticosterone concentrations in comparison to Bd negative male frogs. Urinary corticosterone EIA provides a reliable indication of stress in this frog species and this non-invasive physiological tool can be used to further assess the dynamics of Bd infections and physiological stress responses in other native amphibians.     

Amphibian chytrid fungus and ranaviruses in the Northwest Territories, Canada

Pathogens can cause serious declines in host species, and knowing where pathogens associated with host declines occur facilitates understanding host-pathogen ecology. Suspected drivers of global amphibian declines include infectious diseases, with 2 pathogens in particular, Batrachochytrium dendrobatidis (Bd) and ranaviruses, causing concern. We explored the host range and geographic distribution of Bd and ranaviruses in the Taiga Plains ecoregion of the Northwest Territories, Canada, in 2007 and 2008. Both pathogens were detected, greatly extending their known geographic distributions. Ranaviruses were widespread geographically, but found only in wood frogs. In contrast, Bd was found at a single site, but was detected in all 3 species of amphibians in the survey area (wood frogs, boreal chorus frogs, western toads). The presence of Bd in the Northwest Territories is not congruent with predicted distributions based on niche models, even though findings from other studies at northern latitudes are consistent with those same models. Unexpectedly, we also found evidence that swabs routinely used to collect samples for Bd screening detected fewer infections than toe clips. Our use and handling of the swabs was consistent with other studies, and the cause of the apparent lack of integrity of swabs is unknown. The ranaviruses detected in our study were confirmed to be Frog Virus 3 by sequence analysis of a diagnostic 500 bp region of the major capsid protein gene. It is unknown whether Bd or ranaviruses are recent arrivals to the Canadian north. However, the genetic analyses required to answer that question can inform larger debates about the origin of Bd in North America as well as the potential effects of climate change and industrial development on the distributions of these important amphibian pathogens.     

Storage of samples at high temperatures reduces the amount of amphibian chytrid fungus Batrachochytrium dendrobatidis DNA detectable by PCR assay

Chytridiomycosis, caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), is an emerging infectious disease responsible for amphibian declines on several continents. In laboratory conditions, optimal temperatures for Bd growth and survivorship are between 17 and 25 degrees C. We investigated the effect of different storage temperatures, both in field and laboratory conditions, on detection of Bd from swabs stored for 7 d. We sampled 52 wild Litoria wilcoxii males for Bd by simultaneously running 2 cotton swabs along the skin of the frog. One group of swabs was stored in a freezer within 2 h of sampling and the other was kept in a car in an exposed environment for 7 d before being stored in the freezer. In the laboratory experiment, swabs were inoculated with zoospores of Bd and underwent one of 4 treatments: immediate DNA extraction, or storage at 27, 38 or 45 degrees C for 7 d prior to DNA extraction. Swabs from all treatments were analyzed by quantitative (real-time) PCR test. Though prevalence of Bd did not differ significantly between swabs that were frozen and those that remained in a car for 7 d (19.2 vs. 17.3%, respectively), the number of Bd zoospores detected on car swabs taken from infected frogs was, on average, 67% less than that detected on the corresponding frozen swab. In the laboratory experiment, the number of zoospore equivalents varied significantly with treatment (F(3,35) = 4.769, p = 0.007), indicating that there was reduced recovery of Bd DNA from swabs stored at higher temperatures compared with those stored at lower temperatures or processed immediately. We conclude that failure to store swabs in cool conditions can result in a significant reduction in the amount of Bd DNA detected using the PCR assay. Our results have important implications for researchers conducting field sampling of amphibians for Bd.     

Nothing a hot bath won't cure: infection rates of amphibian chytrid fungus correlate negatively with water temperature under natural field settings

Dramatic declines and extinctions of amphibian populations throughout the world have been associated with chytridiomycosis, an infectious disease caused by the pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd). Previous studies indicated that Bd prevalence correlates with cooler temperatures in the field, and laboratory experiments have demonstrated that Bd ceases growth at temperatures above 28°C. Here we investigate how small-scale variations in water temperature correlate with Bd prevalence in the wild. We sampled 221 amphibians, including 201 lowland leopard frogs (Rana [Lithobates] yavapaiensis), from 12 sites in Arizona, USA, and tested them for Bd. Amphibians were encountered in microhabitats that exhibited a wide range of water temperatures (10-50°C), including several geothermal water sources. There was a strong inverse correlation between the water temperature in which lowland leopard frogs were captured and Bd prevalence, even after taking into account the influence of year, season, and host size. In locations where Bd was known to be present, the prevalence of Bd infections dropped from 75-100% in water <15°C, to less than 10% in water >30°C. A strong inverse correlation between Bd infection status and water temperature was also observed within sites. Our findings suggest that microhabitats where water temperatures exceed 30°C provide lowland leopard frogs with significant protection from Bd, which could have important implications for disease dynamics, as well as management applications.There must be quite a few things a hot bath won't cure, but I don't know many of them--Sylvia Plath, "The Bell Jar" (1963).     

Survey protocol for detecting chytridiomycosis in all Australian frog populations

Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused the decline and extinction of frogs, but the distribution of Bd is not completely known. This information is crucial to implementing appropriate quarantine strategies, preparing for outbreaks of chytridiomycosis due to introduction of Bd, and for directing conservation actions towards affected species. This survey protocol provides a simple and standard method for sampling all frog populations in Australia to maximise the chances of detecting Bd. In order to structure and prioritise the protocol, areas are divided by bioregion and frog species are allocated depending on the water bodies they utilize into 3 groups representing different levels of risk of exposure to Bd. Sixty individuals per population need to be tested to achieve 95% certainty of detecting 1 positive frog, based on the minimum apparent prevalence of > or =5% in infected Australian frog populations and using a quantitative real-time TaqMan PCR test. The appropriate season to sample varies among bioregions and will ideally incorporate temperatures favourable for chytridiomycosis (e.g. maximum air temperatures generally <27 degrees C). Opportunistic collection and testing of sick frogs and tadpoles with abnormal mouth-parts should also be done to increase the probability of detecting Bd. The survey priorities in order are (1) threatened species that may have been exposed to Bd, (2) bioregions surrounding infected bioregions/ecological groups, and (3) species of frogs of unknown infection status in infected bioregions. Within these priority groups, sampling should first target ecological groups and species likely to be exposed to Bd, such as those associated with permanent water, and areas within bioregions that have high risk for Bd as indicated by climatic modelling. This protocol can be adapted for use in other countries and a standard protocol will enable comparison among amphibian populations globally.     

A non-lethal technique for detecting the chytrid fungus Batrachochytrium dendrobatidis on tadpoles

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.     

The disappearing northern leopard frog (Lithobates pipiens): conservation genetics and implications for remnant populations in western Nevada

Global amphibian declines suggest a major shift in the amount and quality of habitat for these sensitive taxa. Many species that were once widespread are now experiencing declines either in part of or across their historic range. The northern leopard frog (Rana [Lithobates] pipiens] has undergone significant declines particularly in the western United States and Canada. Leopard frog population losses in Nevada are largely due to habitat fragmentation and the introduction of nonnative fish, amphibian, and plant species. Only two populations remain in the Truckee and Carson River watersheds of western Nevada which represents the western boundary of this species range. We used sequence data for an 812 base pair fragment of the mitochondrial NADH dehydrogenase 1 (ND1) gene to support a native origin for western Nevada populations. All frogs had a single haplotype (W07) from the distinct western North America ND1 haplotype clade. Data from seven polymorphic microsatellite loci show that Truckee and Carson River populations are highly differentiated from each other and from leopard frogs collected from eastern Nevada sites. Lack of gene flow among and distinct color morphs among the western Nevada populations likely predates the current geographical isolation. Comparisons with other peripheral L. pipiens populations show western Nevada populations have similar levels of gene diversity despite their contemporary isolation (H(E) 0.411, 0.482). Restoration of leopard frog populations in these watersheds will be challenging given well-entrenched nonnative bullfrog populations and major changes to the riparian zone over the past century. Declines of once common amphibian species has become a major conservation concern. Contemporary isolation of populations on a species range periphery such as the leopard frog populations in the Truckee and Carson rivers further exacerbate extirpation risk as these populations are likely to have fewer genetic resources to adaptively respond to rapidly changing biotic and abiotic environments.     

Batrachochytrium dendrobatidis infection dynamics in the Columbia spotted frog Rana luteiventris in north Idaho, USA

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) is contributing to amphibian declines worldwide. Temperature plays an important role in both pathogen growth and host immune function, but little is known about seasonal dynamics of Bd infection in north temperate regions. Our objective was to increase understanding of Bd disease ecology by investigating patterns of Bd infection of Columbia spotted frogs Rana luteiventris across seasons, age classes, and sexes in north Idaho, USA. We collected skin swabs from 223 R. luteiventris in spring, summer, and fall 2009 at 7 ponds in the Palouse region and quantified Bd zoospores for each sample using quantitative PCR. Across seasons, Bd prevalence of adults was higher in summer than in spring or fall, suggesting that individuals may be clearing low-level infections over the summer. Among age classes, all but one late stage tadpole (Gosner stage 43-45) tested negative for Bd. Conversely, 100% of metamorphs tested positive for Bd and had the highest Bd loads of all age classes, suggesting they may be the most vulnerable age class. Adult R. luteiventris had high infection prevalence (> 60%) in all seasons, indicating that Bd infection is maintained within populations and that adults likely serve as disease reservoirs across seasons. Among adults, we also found weak evidence for females having higher infection prevalence than males. Further laboratory and field studies are needed to determine whether there are individual and population impacts from Bd on R. luteiventris and other amphibians in north Idaho.     

Assessment of rates of deformity in wild frog populations using in situ cages: a case study of Leopard Frogs (Rana pipiens) in Ontario, Canada

High rates of deformity in wild amphibian populations from north-eastern North America have been increasingly reported since 1995. In the St Lawrence River basin (Canada) elevated frequencies of limb and eye deformities in mudpuppies (Necturus maculosus) and leopard frogs (Rana pipiens) were recorded in the early 1990s. A caging study was conducted during 1998 to verify the rates recorded in leopard frogs and pursue the potential causes of deformities seen in juveniles and adults. Week-old leopard frog tadpoles were collected from a reference wetland and maintained through to metamorphosis in cages in previously identified high risk wetlands. Deformity frequencies were measured and compared with frequencies measured in wild populations of leopard frogs inhabiting the same wetlands. The results of caging studies and sampling of wild populations were also compared with corresponding data collected from a reference wetland. No deformities were observed in caged or wild reference animals. Very low deformity frequencies (up to 2.2%) were observed in frogs caged in high risk wetlands, but greater frequencies (3.4-10%) were observed in wild young-of-the-year frogs captured at the same sites. The types of deformities were similar among groups; they included fused, missing or extra digits and disproportionate hindlimb length or eye pupil size. In addition, mortality rates were elevated in two cages in high risk wetlands. In general, the caging procedure was effective in establishing the potential for production of deformities in the waters of a given wetland, but tended to underestimate the rates calculated for samples of wild populations. The ramifications of the first-year findings for similar assessments of amphibian deformity rates and establishment of cause-effect linkages are discussed.     

Pond canopy cover: a resource gradient for anuran larvae

1. The gradient in pond canopy cover strongly influences freshwater species distributions. This study tested the effects of canopy cover on the performance of two species of larval anurans, a canopy cover generalist (Rana sylvatica, the wood frog) and an open‐canopy specialist (R. pipiens, the leopard frog), and tested which factors co‐varying with canopy cover mediate these effects. 2. A field transplant experiment demonstrated that canopy cover had negative performance effects on both species. However, leopard frogs, which grow faster than wood frogs in open‐canopy ponds, were more strongly affected by closed‐canopy pond conditions. 3. Closed‐canopy ponds had lower temperature, dissolved oxygen (DO), and food nutritional quality as indicated by carbon‐to‐nitrogen ratio (C : N) analysis of field‐sampled food types, and of gut contents of transplanted larvae. 4. Laboratory experiments demonstrated that higher temperature and food quality but not DO substantially increased larval growth. However, only food quality increased growth rates of leopard frogs more than wood frogs. 5. The strong correlation of growth rates to gut content C : N in the field, and the similarity of growth curves as a function of resource quality in the field and laboratory, strongly suggest that resources are of primary importance in mediating intraspecific, and especially interspecific differences in performance across the canopy cover gradient.     

Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens.

The skins of frogs of the genus Rana synthesize a complex array of antimicrobial peptides that may be grouped into eight families on the basis of structural similarity. A total of 24 peptides with differential growth-inhibitory activity towards the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli and the yeast Candida albicans were isolated from extracts of the skins of three closely related North American frogs, Rana luteiventris (spotted frog), Rana berlandieri (Rio Grande leopard frog) and Rana pipiens (Northern leopard frog). Structural characterization of the antimicrobial peptides demonstrated that they belonged to four of the known families: the brevinin-1 family, first identified in skin of the Asian frog Rana porosa brevipoda; the esculentin-2 family, first identified in the European frog Rana esculenta; the ranatuerin-2 family, first identified in the North American bullfrog Rana catesbeiana; and the temporin family, first identified in the European frog Rana temporaria. Peptides belonging to the brevinin-2, ranalexin, esculentin-1 and ranatuerin-1 families were not identified in the extracts. Despite the close phylogenetic relationship between the various species of Ranid frogs, the distribution and amino-acid sequences of the antimicrobial peptides produced by each species are highly variable and species-specific, suggesting that they may be valuable in taxonomic classification and molecular phylogenetic analysis.     

Small frogs get their worms first: the role of nonodonate arthropods in the recruitment of Haematoloechus coloradensis and Haematoloechus complexus in newly metamorphosed northern leopard frogs, Rana pipiens, and woodhouse's toads, Bufo woodhousii

Studies on the life cycles and epizootiology of North American frog lung flukes indicate that most species utilize odonates as second intermediate hosts; adult frogs become infected by ingesting odonate intermediate hosts. Newly metamorphosed frogs are rarely infected with these parasites, predominantly because they are gape-limited predators that cannot feed on large intermediate hosts such as dragonflies. We examined the role of the frog diet and potential intermediate hosts in the recruitment of the frog lung fluke, Haematoloechus coloradensis, to metamorphosed northern leopard frogs (Rana pipiens), Woodhouse's toads (Bufo woodhousii), and bullfrogs (Rana catesbeiana) from western Nebraska. Because of the uncertain validity of H. coloradensis as a distinct species from Haematoloechus complexus, morphological characters of both species were reevaluated and the life cycles of both species were completed in the laboratory. The morphological data on H. coloradensis and H. coimplexus indicate that they differ in their oral sucker to pharynx ratio, uterine loop distribution, and placement of vitelline follicles. However, in terms of their life cycles, both species are quite similar in their use of physid snails as first intermediate hosts, a wide range of nonodonate and odonate arthropods as second intermediate hosts, and leopard frogs and toads as definitive hosts. These results indicate that H. coloradensis and H. complexus are generalists at the second intermediate host level and might be able to infect newly metamorphosed leopard frogs and toads by using small nonodonate arthropods more commonly than other frog lung fluke species. Comparisons of population structure of adult flukes in newly metamorphosed leopard frogs indicate that the generalist nature of H. coloradensis metacercariae enables it to colonize young of the year leopard frogs more commonly than other Haematoloechus spp. that only use odonates as second intermediate hosts. In this respect, the generalist nature of H. coloradensis and H. complexus at the second intermediate host level is an avenue for the colonization of young of year frogs.     

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and North American Rana frogs share the same families of skin antimicrobial peptides

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and the North American pickerel frog (Rana palustris) occupy different ecological niches on two different continents with no overlap in geographical distribution. R. palustris skin secretions contain a formidable array of antimicrobial peptides including homologs of brevinin-1, esculentin-1, esculentin-2, ranatuerin-2, a temporin and a family of peptides considered of unique structural attributes when isolated, palustrins 1-3. Here we describe the structures of mature peptides and precursors of eight putative antimicrobial peptides from the skin secretion of the Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis). Each peptide represents a structural homolog of respective peptide families isolated from R. palustris, including two peptides identical in primary structure to palustrin 1c and palustrin 3b. Additionally, two peptides were found to be structural homologs of ranatuerin 2B and ranatuerin 2P from the closely-related North American species, Rana berlandieri (the Rio Grande leopard frog) and Rana pipiens (the Northern leopard frog), respectively. Both palustrins and ranatuerins have hitherto been considered unique to North American ranid frogs. The use of primary structures of amphibian skin antimicrobial peptides is thus questionable as a taxonomic device or alternatively, the micro-evolution and/or ancestry of ranid frogs is more highly complex than previously thought.     

Glycation of wood frog (Rana sylvatica) hemoglobin and blood proteins: In vivo and in vitro studies

The effects of in vivo freezing and glucose cryoprotectant on protein glycation were investigated in the wood frog, Rana sylvatica. Our studies revealed no difference in the fructoselysine content of blood plasma sampled from control, 27 h frozen and 18 h thawed wood frogs. Glycated hemoglobin (GHb) decreased slightly with 48 h freezing exposure and was below control levels after 7 d recovery, while glycated serum albumin was unchanged by 48 h freezing but did increase after 7 d of recovery. In vitro exposure of blood lysates to glucose revealed that the GHb production in wood frogs was similar to that of the rat but was lower than in leopard frogs. We conclude that wood frog hemoglobin was glycated in vitro; however, GHb production was not apparent during freezing and recovery when in vivo glucose is highly elevated. It is possible that wood frog blood proteins have different in vivo susceptibilities to glycation.     

Ichthyophonus-like infection in wild amphibians from Québec, Canada

Myositis associated with infection by Ichthyophonus-like organisms was diagnosed in 35 of 260 (13%) wild amphibians collected in Quebec, Canada, from 1959 to 1964 (n = 30), and 1992 to 1999 (n = 230). Infection was diagnosed in 17 green frogs Rana clamitans, 9 wood frogs R. sylvatica, 4 red-spotted newts Notophthalmus viridescens, 3 bullfrogs R. catesbeiana, 1 spring peeper Pseudacris crucifer, and 1 pickerel frog R. palustris. The spring peeper and one of the bullfrogs were collected in 1964 from the Mont Saint-Hilaire Biosphere Reserve, indicating long-term presence of the organism. Spores of the organisms invaded striated muscle fibers and were associated with variable degrees of granulomatous and eosinophilic inflammation. Infection was considered fatal in 2 green frogs, 1 wood frog, and 1 red-spotted newt. It was considered potentially significant in 3 additional green frogs in which up to 100% of the fibers of some muscles were replaced by spores associated with a severe granulomatous reaction. Ultrastructural features of Ichthyophonus-like spores included a thick trilaminated wall, a paramural cytoplasm, multiple nuclei, oval mitochondria with short tubulo-vesicular cristae and numerous ribosomes. This report represents 4 new host records and shows that ichthyophonosis is enzootic in amphibians from Quebec.     

Seasonality of Batrachochytrium dendrobatidis infection in direct-developing frogs suggests a mechanism for persistence

Batrachochytrium dendrobatidis (Bd), a disease-causing amphibian-specific fungus, is widely distributed in Puerto Rico, but is restricted to elevations above 600 m. The effect of this pathogen in the wild was studied by monitoring Eleutherodactylus coqui and E. portoricensis in 2 upland forests at El Yunque, a site characterized by historic population declines in the presence of chytridiomycosis. We tested a potential synergistic interaction between climate and Bd by measuring prevalence of infection and level of infection per individual sampled (number of zoospores), across the dry and wet seasons for 2 yr (between 2005 and 2007). Infection levels in adult frogs were significantly higher during the dry season in both species studied, suggesting a cyclic pattern of dry/ cool-wet/warm climate-driven synergistic interaction. These results are consistent with ex situ experiments in which E. coqui infected with Bd were more susceptible to chytridiomycosis when subjected to limited water treatments resembling drought. Long-term data on the prevalence of Bd in the populations studied versus intensity of infection in individual frogs provided contradictory information. However, the conflicting nature of these data was essential to understand the status of Bd in the species and geographical area studied. We conclude that in Puerto Rico, Bd is enzootic, and vulnerability of eleutherodactylid frogs to this pathogen is related to seasonal climatic variables. Our data suggest a mechanism by which this disease can persist in tropical frog communities without decimation of its hosts, but that complex interactions during severe droughts may lead to population crashes.     

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

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

Presence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in threatened corroboree frog populations in the Australian Alps

Since the early 1980s, the southern corroboree frog Pseudophryne corroboree and northern corroboree frog P. pengilleyi have been in a state of decline from their sub-alpine and high montane bog environments on the southern tablelands of New South Wales, Australia. To date, there has been no adequate explanation as to what is causing the decline of these species. We investigated the possibility that a pathogen associated with other recent frog declines in Australia, the amphibian chytrid fungus Batrachochytrium dendrobatidis, may have been implicated in the decline of the corroboree frogs. We used histology of toe material and real-time PCR of skin swabs to investigate the presence and infection rates with B. dendrobatidis in historic and extant populations of both corroboree frog species. Using histology, we did not detect any B. dendrobatidis infections in corroboree frog populations prior to their decline. However, using the same technique, high rates of infection were observed in populations of both species after the onset of substantial population declines. The real-time PCR screening of skin swabs identified high overall infection rates in extant populations of P. corroboree (between 44 and 59%), while significantly lower rates of infection were observed in low-altitude P. pengilleyi populations (14%). These results suggest that the initial and continued decline of the corroboree frogs may well be attributed to the emergence of B. dendrobatidis in populations of these species.     

Infection intensity and sampling locality affect Batrachochytrium dendrobatidis distribution among body regions on green-eyed tree frogs Litoria genimaculata

Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, which has caused devastating amphibian population declines. Little is known about the biology of Bd on hosts, and techniques for diagnosing it on living and preserved animals are still evolving. We investigated the spatial distribution of Bd on the integument of naturally infected Australian hylid frogs Litoria genimaculata at 4 rain forest localities in northern Queensland, Australia. We collected 555 samples by swabbing 111 individuals on 5 regions of the body (back, abdomen, legs, forefeet and hindfeet). Numbers of zoospore equivalents on each body region were quantified using a real-time TaqMan PCR assay. The intensity of infection differed significantly among body regions and this pattern of differences differed among sampling localities. The lightest infections were usually centered on the abdomen, while heavier infections were concentrated on the legs and feet. The back was always either lightly infected or uninfected. Many frogs with light infections had positive PCR results only for the abdomen or the legs. We compared swabs taken from the legs and abdomen and found that they provided similar sensitivity to detect infections, but using both regions together led to greater sensitivity than either region alone. Because swabbing may transfer zoospores from infected to uninfected regions within individuals, we suggest that the best procedure for all species is to employ separate swabs for each body region. If that cannot be done, swabbing patterns that minimize potential harm should be determined for each species, and possibly each class of individuals (e.g. males, females, juveniles) within species, by examining the distribution of infection among body parts in naturally infected individuals.