Transmission of Batrachochytrium dendrobatidis within and between amphibian life stages

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

The emerging amphibian pathogen Batrachochytrium dendrobatidis globally infects introduced populations of the North American bullfrog, Rana catesbeiana

Batrachochytrium dendrobatidis is the chytridiomycete fungus which has been implicated in global amphibian declines and numerous species extinctions. Here, we show that introduced North American bullfrogs (Rana catesbeiana) consistently carry this emerging pathogenic fungus. We detected infections by this fungus on introduced bullfrogs from seven of eight countries using both PCR and microscopic techniques. Only native bullfrogs from eastern Canada and introduced bullfrogs from Japan showed no sign of infection. The bullfrog is the most commonly farmed amphibian, and escapes and subsequent establishment of feral populations regularly occur. These factors taken together with our study suggest that the global threat of B. dendrobatidis disease transmission posed by bullfrogs is significant.     

Effect of temperature on host response to Batrachochytrium dendrobatidis infection in the mountain yellow-legged frog (Rana muscosa)

The pathogenic chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis, has been implicated in declines of amphibian populations throughout the world, including declines and extinctions of local populations of mountain yellow-legged frogs, Rana muscosa, in the California Sierra Nevada. Previous studies have shown B. dendrobatidis achieves its maximum growth rate in culture in the temperature range of 17-25 C, and exposure to very high temperatures can clear frogs of B. dendrobatidis infection. Here we present the results of a laboratory experiment in which experimentally infected R. muscosa tadpoles were followed through metamorphosis at temperatures of 17 and 22 C. All infected animals developed clinical disease within a similar time frame. However, frogs housed at 22 C exhibited a significantly lower mortality than those housed at 17 C. Within 35 days after metamorphosis, 50% of the frogs housed at 22 C died, while 95% of the frogs housed at 17 C died. Clinical signs subsided in the surviving frogs at 22 C, despite persistent infection. Because both temperatures are within the optimal thermal range for growth of B. dendrobatidis, we propose that the difference in outcome indicates the effect of temperature on the host's resistance to chytridiomycosis, rather than an effect on the fungus alone.     

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.     

Transition of Chytrid Fungus Infection from Mouthparts to Hind Limbs During Amphibian Metamorphosis

EcoHealth - The chytrid fungus, Batrachochytrium dendrobatidis (Bd), is implicated in worldwide amphibian declines. Bd has been shown to qualitatively transition from the mouthparts of tadpoles to...     

Transmission of Batrachochytrium dendrobatidis to wood frogs (Lithobates sylvaticus) via a bullfrog (L. catesbeianus) vector

Chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis, threatens anuran populations worldwide. Effects of B. dendrobatidis on frog species are variable. Some species typically develop nonlethal infections and may function as carriers; others typically develop lethal infections that can lead to population declines. Nonlethal infections in the bullfrog (Lithobates catesbeianus) are well-documented. In contrast, recently metamorphosed wood frogs (L. sylvaticus) can die from chytridiomycosis. We conducted an ex-situ experiment between May and July 2010 to determine whether B. dendrobatidis-infected bullfrogs could transmit the fungus to wood frog tadpoles when the two species shared a body of water. We tested for B. dendrobatidis infections with quantitative polymerase chain reactions (qPCR) in a subsample of the wood frog tadpoles and in all metamorphosed wood frogs and compared risk of death of froglets exposed and unexposed to infected bullfrogs. We detected B. dendrobatidis sporadically in subsampled treatment tadpoles (nine of 90, 10%) and frequently in treatment froglets (112 of 113, 99.1%). Pooled risk of froglet death was higher (P<0.001) in treatment enclosures than in control enclosures. Our results indicate that, at the low infection loads bullfrogs tend to carry, swabbing for PCR analyses may underestimate prevalence of B. dendrobatidis in this species. We highlight bullfrog disease screening as a management challenge, especially in light of exotic bullfrog colonies on multiple continents and large-scale global trade in this species. We document the importance of quantifying lethal and sublethal effects of bullfrog vectors on B. dendrobatidis-susceptible species.     

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.     

Antimicrobial peptide defenses of the Tarahumara frog,Rana tarahumarae

Populations of the Tarahumara frog Rana tarahumarae have decreased markedly in recent years in the northern part of their range. Infection by the chytrid fungus Batrachochytrium dendrobatidis has been implicated in these declines. To determine whether antimicrobial peptides in the skin provide protection against this pathogen, norepinephrine-stimulated skin secretions were tested for their ability to inhibit growth of B. dendrobatidis in vitro. After concentration, crude mixtures of skin peptides inhibited the growth of the chytrid in a concentration-dependent manner. Proteomic analysis led to the identification and characterization of three peptides belonging to the brevinin-1 family of antimicrobial peptides and three belonging to the ranatuerin-2 family. The two most abundant peptides, ranatuerin-2TRa (GIMDSIKGAAKEIAGHLLDNLKCKITGC) and brevinin-1TRa (FLPVIAGIAANVLPKLFCKLTKRC), were active against B. dendrobatidis (MIC of 50 microM for ranatuerin-2TRa and 12.5 microM for brevinin-1TRa against zoospores). These data clearly show that antimicrobial peptides in the skin secretions of the Tarahumara frog are active against B. dendrobatidis and should provide some protection against infection. Therefore, the observed susceptibility of these frogs to this pathogen in the wild may be due to the effects of additional environmental factors that impair this innate defense mechanism, leading to the observed population declines.     

Chytridiomycosis in native Arizona frogs

Twenty-seven adult/sub-adult lowland leopard frogs (Rana yavapaiensis), two larval lowland leopard frogs, two adult Chirichahua leopard frogs (Rana chiricahuensis), and two adult canyon tree frogs (Hyla arenicolor) collected from populations experiencing mortality events at eight sites were found to have characteristic lesions of chytrid fungus infection (Batrachochytrium dendrobatidis). The mortalities occurred during December 1992 and between October and February in 1997-98 and December and February in 1998-99. Gross lesions varied from none to diffuse reddening of the skin of the abdomen, pelvic area, and legs. Microscopic lesions were characteristic of those previously reported for the disease and included diffuse epidermal hyperplasia, hyperkeratosis, and colonization of the keratinized layers of the epidermis by sporangia of the chytrid. Bacterial cultures did not yield a primary pathogenic agent. Virus isolation from frog tissues was negative. Batrachochytrium dendrobatidis was isolated from the skin of two of 10 R. yavapaiensis and one of two H. arenicolor cultured following necropsy. An additional nine of 11 clinically affected or dead R. yavapaiensis from the same locations, but not necropsied, were culture positive for B. dendrobatidis.     

Amphibian chytrid fungus Batrachochytrium dendrobatidis in Cusuco National Park, Honduras

Amphibian population declines in Honduras have long been attributed to habitat degradation and pollution, but an increasing number of declines are now being observed from within the boundaries of national parks in pristine montane environments. The amphibian chytrid fungus Batrachochytrium dendrobatidis has been implicated in these declines and was recently documented in Honduras from samples collected in Pico Bonito National Park in 2003. This report now confirms Cusuco National Park, a protected cloud forest reserve with reported amphibian declines, to be the second known site of infection for Honduras. B. dendrobatidis infection was detected in 5 amphibian species: Craugastor rostralis, Duellmanohyla soralia, Lithobates maculata, Plectrohyla dasypus, and Ptychohyla hypomykter. D. soralia, P. dasypus, and P. hypomykter are listed as critically endangered in the IUCN Red List of Threatened Species and have severely fragmented or restricted distributions. Further investigations are necessary to determine whether observed infection levels indicate an active B. dendrobatidis epizootic with the potential to cause further population declines and extinction.     

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.     

Predicted disease susceptibility in a Panamanian amphibian assemblage based on skin peptide defenses

Chytridiomycosis is an emerging infectious disease of amphibians caused by a chytrid fungus, Batrachochytrium dendrobatidis. This panzootic does not equally affect all amphibian species within an assemblage; some populations decline, others persist. Little is known about the factors that affect disease resistance. Differences in behavior, life history, biogeography, or immune function may impact survival. We found that an innate immune defense, antimicrobial skin peptides, varied significantly among species within a rainforest stream amphibian assemblage that has not been exposed to B. dendrobatidis. If exposed, all amphibian species at this central Panamanian site are at risk of population declines. In vitro pathogen growth inhibition by peptides from Panamanian species compared with species with known resistance (Rana pipiens and Xenopus laevis) or susceptibility (Bufo boreas) suggests that of the nine species examined, two species (Centrolene prosoblepon and Phyllomedusa lemur) may demonstrate strong resistance, and the other species will have a higher risk of disease-associated population declines. We found little variation among geographically distinct B. dendrobatidis isolates in sensitivity to an amphibian skin peptide mixture. This supports the hypothesis that B. dendrobatidis is a generalist pathogen and that species possessing an innate immunologic defense at the time of disease emergence are more likely to survive.     

Surveillance for batrachochytrium dendrobatidis using Mixophyes (Anura: Myobatrachidae) larvae

Fourteen populations of anuran larvae (tadpoles), including three populations of the endangered Fleay's Barred Frog (Mixophyes fleayi) and 11 populations of the common Great Barred Frog (Mixophyes fasciolatus), in creek sites in the southeast region of Queensland were selected. Site selection was based on a history (within the district) of adult frog population declines and/or disappearances or records of infection of adult frogs or larvae by Batrachochytrium dendrobatidis. Larvae were collected once from each creek site between October 2002 and October 2004, and were between Gosner developmental stages 25 and 40. Total body length ranged from 18 mm to 100 mm. Mouthparts were examined under a dissecting microscope for grossly visible abnormalities, and then examined for histologic evidence of B. dendrobatidis. The most consistent mouthpart abnormalities found were multifocal depigmentation of the jaw sheaths and loss or shortening of the tooth rows. At the individual larva level, presence of mouthpart abnormalities was strongly associated with histologic diagnosis of B. dendrobatidis (93%). At least one larva with abnormal mouthparts was detected at 12 of the 14 sites and histologic evidence of B. dendrobatidis was detected at 13 of the 14 sites. These findings suggest that larvae of Mixophyes species are suitable for surveillance for B. dendrobatidis. We conclude that surveillance of B. dendrobatidis where individual larva prevalences of mouthpart abnormalities and histologic evidence of B. dendrobatidis are as high as those observed in this study (66% and 78%, respectively), relatively small numbers of larvae are required to detect these infections. Medium to large larvae (body length >30 mm) were much more likely to be affected than small larvae (body length < or =30 mm), suggesting that larger individuals should be targeted for surveillance.     

The chytrid fungus Batrachochytrium dendrobatidis is non-randomly distributed across amphibian breeding habitats

The chytrid fungus Batrachochytrium dendrobatidis has been implicated as the causative agent of mass mortalities, population declines, and the extinctions of stream-breeding amphibian species worldwide. While the factors that limit the distribution and abundance of B. dendrobatidis across large geographical regions are fairly well understood, little is known about the distribution of the fungus within localized areas such as individual catchments. The accurate identification of amphibian populations likely to be exposed to the fungus is urgently required for effective disease management. We conducted disease surveys of frogs representing five ecological guilds in south-east Queensland, Australia, and hypothesized that if B. dendrobatidis were responsible for the disappearance of stream-breeding amphibian populations, infection prevalence and intensity would be greatest in frogs breeding in permanent, flowing water. Overall, 30.3% of the 519 frogs we sampled were infected with B. dendrobatidis . However, infections were not evenly distributed across the ecological guilds, being almost completely restricted to frogs breeding at permanent waterbodies. Of these, stream breeders were significantly more likely to be infected than were pond breeders, though the intensity of frogs' infections did not differ significantly between the two guilds. Batrachochytrium dendrobatidis was detected on only one of the 117 frogs that were found at ephemeral ponds, ephemeral streams, or terrestrial sites. These findings provide strong support for the hypothesis that B. dendrobatidis was responsible for many of the unexplained disappearances of stream-breeding amphibian populations in recent decades, and will enable wildlife managers to more accurately focus conservation efforts on those species at highest risk of disease-related decline.     

Amphibian immune defenses against chytridiomycosis: impacts of changing environments

Eco-immunology is the field of study that attempts to understand the functions of the immune system in the context of the host's environment. Amphibians are currently suffering devastating declines and extinctions in nearly all parts of the world due to the emerging infectious disease chytridiomycosis caused by the chytrid fungus, Batrachochytrium dendrobatidis. Because chytridiomycosis is a skin infection and remains confined to the skin, immune defenses of the skin are critical for survival. Skin defenses include secreted antimicrobial peptides and immunoglobulins as well as antifungal metabolites produced by symbiotic skin bacteria. Low temperatures, toxic chemicals, and stress inhibit the immune system and may impair natural defenses against B. dendrobatidis. Tadpoles' mouth parts can be infected by B. dendrobatidis. Damage to the mouth parts can impair growth, and the affected tadpoles maintain the pathogen in the environment even when adults have dispersed. Newly metamorphosing frogs appear to be especially vulnerable to infection and to the lethal effects of this pathogen because the immune system undergoes a dramatic reorganization at metamorphosis, and postmetamorphic defenses are not yet mature. Here we review our current understanding of amphibian immune defenses against B. dendrobatidis and the ability of the pathogen to resist those defenses. We also briefly review what is known about the impacts of temperature, environmental chemicals, and stress on the host-pathogen interactions and suggest future directions for research.     

Quantifying the disease transmission function: effects of density on Batrachochytrium dendrobatidis transmission in the mountain yellow-legged frog Rana muscosa

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

Early-spring survey for Batrachochytrium dendrobatidis in wild Rana dybowskii in Heilongjiang Province, China

Batrachochytrium dendrobatidis has been investigated worldwide because of its importance in population declines in multiple species of amphibians; however, little is known regarding the disease status of all native amphibian species in China. The present study is the first survey of chytridiomycosis in free-ranging amphibian populations in China, and it examined the possible presence of B. dendrobatidis in Rana dybowskii in northeastern China (Heilongjiang Province). R. dybowskii is mainly distributed in the northeast part of China and is intensively hunted for human consumption, making populations vulnerable to extirpation in the event of additional stresses from disease epidemics. The survey was performed in early spring of 2008, using a PCR assay, histological examination of skin samples, and zoospore culture. In total, 191 frogs were examined; thus, a 95% confidence limit for prevalence of 1.57% was selected. Our results demonstrate that R. dybowskii is currently free from chytridiomycosis in Heilongjiang, even though the natural conditions of the sampling sites are suitable for the occurrence of B. dendrobatidis. Central and local governments should implement strict management measures to prevent the escape of non-native commercial amphibian species into this area, which might endanger local populations of native species.     

Disease associated with integumentary and cloacal parasites in tadpoles of northern red-legged frog Rana aurora aurora

A total of 6830 northern red-legged frog Rana aurora aurora tadpoles were examined under a dissecting microscope for oral disc, integumentary, and cloacal abnormalities in 13 ponds in and near Redwood National Park in northern California. Of these, 163 tadpoles were collected for histopathological investigation, including 115 randomly collected individuals, 38 collected with oral disc abnormalities, and 10 collected due to severe morbidity of unknown etiology. The tadpoles were infected with 8 parasites, including Batrachochytrium dendrobatidis (the amphibian chytrid), trematodes, leeches, and protozoa. Chytridiomycosis was detected at an overall prevalence of 6.4%, but prevalence was higher in tadpoles with oral disc lesions than in those with normal oral discs (43.5% versus 6.1%). Interestingly, infection was associated with some environmental and co-infection risk factors. Individual tadpoles possessed 0 to 5 species of parasites in varying intensities. Apiosoma sp. was the most prevalent (66%) and widespread. Tadpoles infected with B. dendrobatidis had a lower diversity of oral parasites than those uninfected. During the field portion of the study, a large number (approximately 500) of moribund and dead tadpoles was seen occurring at multiple locations within and surrounding Redwood National Park. Ten animals were collected for histological examination and a diverse protozoal infection was discovered, including some known pathogens of fish. This study is the first reporting parasitism and disease in natural populations of northern red-legged frogs.     

Tadpole erythrocytes: luminescent properties with dark field microscopy

To characterize and classify erythrocytes of ranid tadpoles, alcohol-fixed blood smears were studied with dark field illumination. All preclimax stage (limb bud-foot) Rana castesbeiana tadpoles from ponds in Massachusetts had red blood cells that were polymorphic. The majority of cells (88%) showed a bright, granular luminescence varying from white to blue-grey, whereas, cytoplasm of the other cells was smooth, black, and nonluminescent. On the other hand, tadpoles in similar stages from other species (Rana clamitans, Mass. and Rana pipens, Vermont) and from R. catesbeiana tadpoles from other locations (Wisconsin and North Carolina) had no observable cytoplasmic luminescence in any of their red blood cells. Moreover, as Mass. R. catesbeiana underwent metamorphic climax their luminescent cells disappeared and were replaced by small, round, dark, nonluminescent cells, precursors of the oval, nonluminescent erythrocytes characteristic of adult frogs. Cells with black nonluminescent cytoplasm generally contained nuclei which were luminescent. In conclusion, two main types of red blood cells-those with and those without cytoplasmic luminescence-are distinguishable with dark field microscopy. Luminescence of the cells varies with species, geographic location, and developmental stage of the tadpoles.