PLoS One. 2010; 5(7):
Tobler U, Schmidt BR

[This corrects the article on p. e10927 in vol. 5.].

Proc Natl Acad Sci U S A. 2010 Jul 19;
Crawford AJ, Lips KR, Bermingham E

Amphibian populations around the world are experiencing unprecedented declines attributed to a chytrid fungal pathogen, Batrachochytrium dendrobatidis. Despite the severity of the crisis, quantitative analyses of the effects of the epidemic on amphibian abundance and diversity have been unavailable as a result of the lack of equivalent data collected before and following disease outbreak. We present a community-level assessment combining long-term field surveys and DNA barcode data describing changes in abundance and evolutionary diversity within the amphibian community of El Copé, Panama, following a disease epidemic and mass-mortality event. The epidemic reduced taxonomic, lineage, and phylogenetic diversity similarly. We discovered that 30 species were lost, including five undescribed species, representing 41% of total amphibian lineage diversity in El Copé. These extirpations represented 33% of the evolutionary history of amphibians within the community, and variation in the degree of population loss and decline among species was random with respect to the community phylogeny. Our approach provides a fast, economical, and informative analysis of loss in a community whether measured by species or phylogenetic diversity.

Cell Tissue Res. 2010 Jul 17;
Conlon JM

Cationic peptides with the propensity to adopt an amphipathic alpha-helical conformation in a membrane-mimetic environment are synthesized in the skins of many species of anurans (frogs and toads). These peptides frequently display cytolytic activities against a range of pathogenic bacteria and fungi consistent with the idea that they play a role in the host's system of innate immunity. However, the importance of the peptides in the survival strategy of the animal is not clearly understood. It is a common misconception that antimicrobial peptides are synthesized in the skins of all anurans. In fact, the species distribution is sporadic suggesting that their production may confer some evolutionary advantage to the organism but is not necessary for survival. Although growth inhibitory activity against the chytrid fungus Batrachochytrium dendrobatidis, responsible for anuran population declines worldwide, has been demonstrated in vitro, the ability of frog skin antimicrobial peptides to protect the animal in the wild appears to be limited and there is no clear correlation between their production by a species and its resistance to fatal chytridiomycosis. The low potency of many frog skin antimicrobial peptides is consistent with the hypothesis that cutaneous symbiotic bacteria may provide the major system of defense against pathogenic microorganisms in the environment with antimicrobial peptides assuming a supplementary role in some species.

Ecohealth. 2010 Jun 29;
Longo AV, Burrowes PA

The chytrid fungus, Batrachochytrium dendrobatidis (Bd) has been linked to extinction and decline of numerous amphibians. We studied the population-level effects of Bd in two post-decline anuran species, Eleutherodactylus coqui and E. portoricensis, at El Yunque National Forest, Puerto Rico. Data on amphibian abundance was updated to report long-term population trends. Mark-recapture data was used to monitor Bd-infection status and estimate survival probabilities of infected versus uninfected adults. Prevalence of Bd (number of infected/total sampled) and individual infection level (number of zoospores) were compared among age classes at Palo Colorado Forest (661 m) and Elfin Forest (850 m). Results revealed that both species continued to decrease in Palo Colorado Forest, while in the Elfin Forest, E. portoricensis recuperated from drastic declines. Age class, season, and locality significantly predicted zoospore load. Age was also significantly associated with high zoospores loads among Bd-positive frogs, and the prevalence of Bd was higher in juveniles than adults in all populations studied. We suggest that early age represents a critical life stage in the survival of direct-developing frogs infected by this fungus. Survival probability was always higher for uninfected frogs, but recapture rates of infected versus uninfected adults were significantly different only in Palo Colorado, alerting that the negative effect of Bd infection under enzootic conditions is greater at mid-elevations. This work contributes to our understanding of how direct-developing amphibians persist with Bd, pointing to critical life stages and synergistic interactions that may induce fluctuations and/or declines in the wild.

Infect Immun. 2010 Jun 28;
Ramsey JP, Reinert LK, Harper LK, Woodhams DC, Rollins-Smith LA

Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. Here we show that the South African clawed frog, Xenopus laevis, is a suitable model to investigate immunity to this pathogen. Following an experimental exposure, a mild infection developed over 20-30 days and declined by 45 days post-exposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections.

J Parasitol. 2010 Jun; 96(3): 552-7
Venesky MD, Wassersug RJ, Parris MJ

Pathogens can alter host life-history traits by affecting host feeding activities. In anuran tadpoles, keratinized mouthparts (teeth and jaw sheaths) are essential for feeding. Batrachochytrium dendrobatidis ( Bd ) is a pathogenic fungus of amphibians that can infect these mouthparts and reduce tadpole survival. However, the precise way that Bd-induced changes in tadpole mouthparts impact tadpole feeding is unknown. We use high-speed (500 frames/sec) videography to study how Bd-induced mouthpart deformities affect the feeding kinematics of Fowler's toad (Anaxyrus [= Bufo] fowleri ) and grey tree frog (Hyla chrysoscelis) tadpoles. We tested for species-specific patterns of Bd-induced mouthpart deformities to assess how deformations to specific areas of tadpole mouthparts alter feeding kinematics. The teeth of tadpoles from the Bd-exposed treatment slipped off of surfaces on which tadpoles graze and were in contact with an algal-covered substratum for a shorter duration in each gape cycle compared to teeth of control tadpoles. We also found that the jaw sheaths had significantly more deformations than labial teeth; however, how this relates to feeding kinematics is unclear. Our data show explicitly how Bd infection reduces foraging efficiency of anuran tadpoles by altering feeding kinematics and elucidate a mechanistic link between the pathogen infection and reduced host fitness.

Environ Microbiol. 2010 Jun 1;
Van Wichelen J, van Gremberghe I, Vanormelingen P, Debeer AE, Leporcq B, Menzel D, Codd GA, Descy JP, Vyverman W

Summary Despite its importance for bloom toxicity, the factors determining the population structure of cyanobacterial blooms are poorly understood. Here, we report the results of a two-year field survey of the population dynamics of Microcystis blooms in a small hypertrophic urban pond. Microscopic enumeration of Microcystis and its predators and parasites was combined with pigment and microcystin analysis and denaturing gradient gel electrophoresis of the ITS rDNA region to assess population dynamics and structure. Two main Microcystis morpho- and ITS types were revealed, corresponding to M. aeruginosa and M. viridis. In both years, high population densities of naked amoebae grazing on Microcystis coincided with rapid decreases in Microcystis biomass. In one year, there was a shift from heavily infested M. aeruginosa to the less-infested M. viridis, allowing the bloom to rapidly recover. The preference of amoebae for M. aeruginosa was confirmed by grazing experiments, in which several amoeba strains were capable of grazing down a strain of M. aeruginosa, but not of M. viridis. Zooplankton and chytrid parasites appeared to be of minor importance for these strong and fast reductions in Microcystis biomass. These findings demonstrate a strong impact of small protozoan grazers on the biomass and genetic structure of Microcystis blooms.

PLoS One. 2010; 5(6): e10927
Tobler U, Schmidt BR

BACKGROUND: Chytridiomycosis is a fungal disease linked to local and global extinctions of amphibians. Susceptibility to chytridiomycosis varies greatly between amphibian species, but little is known about between- and within-population variability. However, this kind of variability is the basis for the evolution of tolerance and resistance evolution to disease. METHODOLOGY/PRINCIPAL FINDINGS: In a common garden experiment, we measured mortality after metamorphosis of Alytes obstetricans naturally infected with Batrachochytrium dendrobatidis. Mortality rates differed significantly among populations and ranged from 27 to 90%. Within populations, mortality strongly depended on mass at and time through metamorphosis. CONCLUSIONS/SIGNIFICANCE: Although we cannot rule out that the differences observed resulted from differences in skin microbiota, different pathogen strains or environmental effects experienced by the host or the pathogen prior to the start of the experiment, we argue that genetic differences between populations are a likely source of at least part of this variation. To our knowledge, this is the first study showing differences in survival between and within populations under constant laboratory conditions. Assuming that some of this intraspecific variation has a genetic basis, this may suggest that there is the potential for the evolution of resistance or tolerance, which might allow population persistence.

PLoS One. 2010; 5(6): e10957
Becker MH, Harris RN

Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is an infectious disease that causes population declines of many amphibians. Cutaneous bacteria isolated from redback salamanders, Plethodon cinereus, and mountain yellow-legged frogs, Rana muscosa, inhibit the growth of Bd in vitro. In this study, the bacterial community present on the skin of P. cinereus individuals was investigated to determine if it provides protection to salamanders from the lethal and sub-lethal effects of chytridiomycosis. When the cutaneous bacterial community was reduced prior to Bd exposure, salamanders experienced a significantly greater decrease in body mass, which is a symptom of the disease, when compared to infected individuals with a normal bacterial community. In addition, a greater proportion of infected individuals with a reduced bacterial community experienced limb-lifting, a behavior seen only in infected individuals. Overall, these results demonstrate that the cutaneous bacterial community of P. cinereus provides protection to the salamander from Bd and that alteration of this community can change disease resistance. Therefore, symbiotic microbes associated with this species appear to be an important component of its innate skin defenses.

Ecohealth. 2010 Jun 2;
Wollenberg KC, Jenkins RK, Randrianavelona R, Ralisata M, Rampilamanana R, Ramanandraibe A, Ravoahangimalala OR, Vences M

Chytridiomycosis (Bd) is contributing to amphibian extinctions worldwide but has so far not been detected in Madagascar. The high likelihood for Bd to spread to the island and efface this amphibian diversity and endemism hotspot requires respective conservation policies to be developed. Bd could be introduced by the large number of tourists that visit protected areas; therefore, increasing awareness among tourists and encouraging them to participate in safety measures should be a priority conservation action. However, concerns have been raised that tourists would not be able to distinguish between an amphibian disease harmless to humans and emerging diseases that would imply a danger for human health, invoking a negative image of Madagascar as an ecotourism destination. We evaluated whether informing tourists about this infectious animal disease would cause health scare and diminish trip satisfaction. Based on 659 respondents we found that most ecotourists favored to be informed about Bd and were proactive about participating in prevention measures, refuting previous concerns.

Proc Natl Acad Sci U S A. 2010 May 25; 107(21): 9695-700
Briggs CJ, Knapp RA, Vredenburg VT

Chytridiomycosis, the disease caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd), has contributed to amphibian population declines and extinctions worldwide. The impact of this pathogen, however, varies markedly among amphibian species and populations. Following invasion into some areas of California's Sierra Nevada, Bd leads to rapid declines and local extinctions of frog populations (Rana muscosa, R. sierrae). In other areas, infected populations of the same frog species have declined but persisted at low host densities for many years. We present results of a 5-year study showing that infected adult frogs in persistent populations have low fungal loads, are surviving between years, and frequently lose and regain the infection. Here we put forward the hypothesis that fungal load dynamics can explain the different population-level outcomes of Bd observed in different areas of the Sierra Nevada and possibly throughout the world. We develop a model that incorporates the biological details of the Bd-host interaction. Importantly, model results suggest that host persistence versus extinction does not require differences in host susceptibility, pathogen virulence, or environmental conditions, and may be just epidemic and endemic population dynamics of the same host-pathogen system. The different disease outcomes seen in natural populations may result solely from density-dependent host-pathogen dynamics. The model also shows that persistence of Bd is enhanced by the long-lived tadpole stage that characterize these two frog species, and by nonhost Bd reservoirs.

Proc Natl Acad Sci U S A. 2010 May 25; 107(21): 9689-94
Vredenburg VT, Knapp RA, Tunstall TS, Briggs CJ

Epidemiological theory generally suggests that pathogens will not cause host extinctions because the pathogen should fade out when the host population is driven below some threshold density. An emerging infectious disease, chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is directly linked to the recent extinction or serious decline of hundreds of amphibian species. Despite continued spread of this pathogen into uninfected areas, the dynamics of the host-pathogen interaction remain unknown. We use fine-scale spatiotemporal data to describe (i) the invasion and spread of Bd through three lake basins, each containing multiple populations of the mountain yellow-legged frog, and (ii) the accompanying host-pathogen dynamics. Despite intensive sampling, Bd was not detected on frogs in study basins until just before epidemics began. Following Bd arrival in a basin, the disease spread to neighboring populations at approximately 700 m/yr in a wave-like pattern until all populations were infected. Within a population, infection prevalence rapidly reached 100% and infection intensity on individual frogs increased in parallel. Frog mass mortality began only when infection intensity reached a critical threshold and repeatedly led to extinction of populations. Our results indicate that the high growth rate and virulence of Bd allow the near-simultaneous infection and buildup of high infection intensities in all host individuals; subsequent host population crashes therefore occur before Bd is limited by density-dependent factors. Preventing infection intensities in host populations from reaching this threshold could provide an effective strategy to avoid the extinction of susceptible amphibian species in the wild.

Conserv Biol. 2010 Apr 20;
Pilliod DS, Muths E, Scherer RD, Bartelt PE, Corn PS, Hossack BR, Lambert BA, McCaffery R, Gaughan C

Chytridiomycosis is linked to the worldwide decline of amphibians, yet little is known about the demographic effects of the disease. We collected capture-recapture data on three populations of boreal toads (Bufo boreas [Bufo = Anaxyrus]) in the Rocky Mountains (U.S.A.). Two of the populations were infected with chytridiomycosis and one was not. We examined the effect of the presence of amphibian chytrid fungus(Batrachochytrium dendrobatidis[Bd]; the agent of chytridiomycosis) on survival probability and population growth rate. Toads that were infected with Bd had lower average annual survival probability than uninfected individuals at sites where Bd was detected, which suggests chytridiomycosis may reduce survival by 31-42% in wild boreal toads. Toads that were negative for Bd at infected sites had survival probabilities comparable to toads at the uninfected site. Evidence that environmental covariates (particularly cold temperatures during the breeding season) influenced toad survival was weak. The number of individuals in diseased populations declined by 5-7%/year over the 6 years of the study, whereas the uninfected population had comparatively stable population growth. Our data suggest that the presence of Bd in these toad populations is not causing rapid population declines. Rather, chytridiomycosis appears to be functioning as a low-level, chronic disease whereby some infected individuals survive but the overall population effects are still negative. Our results show that some amphibian populations may be coexisting with Bd and highlight the importance of quantitative assessments of survival in diseased animal populations.

Conserv Biol. 2010 Jun; 24(3): 897-9; discussion 900-2
Schmidt BR

Proc Natl Acad Sci U S A. 2010 May 4; 107(18): 8269-74
Rohr JR, Raffel TR

The role of global climate change in the decline of biodiversity and the emergence of infectious diseases remains controversial, and the effect of climatic variability, in particular, has largely been ignored. For instance, it was recently revealed that the proposed link between climate change and widespread amphibian declines, putatively caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), was tenuous because it was based on a temporally confounded correlation. Here we provide temporally unconfounded evidence that global El Niño climatic events drive widespread amphibian losses in genus Atelopus via increased regional temperature variability, which can reduce amphibian defenses against pathogens. Of 26 climate variables tested, only factors associated with temperature variability could account for the spatiotemporal patterns of declines thought to be associated with Bd. Climatic predictors of declines became significant only after controlling for a pattern consistent with epidemic spread (by temporally detrending the data). This presumed spread accounted for 59% of the temporal variation in amphibian losses, whereas El Niño accounted for 59% of the remaining variation. Hence, we could account for 83% of the variation in declines with these two variables alone. Given that global climate change seems to increase temperature variability, extreme climatic events, and the strength of Central Pacific El Niño episodes, climate change might exacerbate worldwide enigmatic declines of amphibians, presumably by increasing susceptibility to disease. These results suggest that changes to temperature variability associated with climate change might be as significant to biodiversity losses and disease emergence as changes to mean temperature.

Ecohealth. 2010 Apr 6;
Bai C, Garner TW, Li Y

Although the chytrid fungus Batrachochytrium dendrobatidis (Bd), the etiological agent of amphibian chytridiomycosis, has been implicated in mass mortality and population declines on several continents around the world, there have been no reports on the presence of Bd infections in amphibians in China. We employed quantitative PCR and histological techniques to investigate the presence of Bd in introduced North American bullfrogs (Rana catesbeiana) (referred to hereafter as bullfrog) and native amphibians in bullfrog-invaded areas of the Yunnan Province, China. A total of 259 samples at five wild sites were collected between June and September in 2007 and 2008, including bullfrogs and four native amphibian species (Rana pleuraden, Rana chaochiaoensis, Odorrana andersonii, and Bombina maxima). In addition, 37 samples of adult bullfrogs were obtained from a food market. Bd infections were discovered in bullfrogs and three native amphibian species from all of the surveyed sites. Of the 39 Bd-positive samples, 35 were from wild-caught bullfrog tadpoles, postmetamorphic bullfrogs, R. pleuraden, R. chaochiaoensis, and O. andersonii, and four were from adult bullfrogs from the market. Our results provide the first evidence of the presence of Bd in Chinese amphibians, suggesting that native amphibian diversity in China is at risk from Bd. There is an urgent need to monitor the distribution of Bd in amphibians in China and understand the susceptibility of native amphibian species to chytridiomycosis. Strict regulations on the transportation of bullfrogs and the breeding of bullfrogs in markets and farms should be drafted in order to stop the spread of Bd by bullfrogs.

J Am Assoc Lab Anim Sci. 2010; 49(2): 215-20
Hill WA, Newman SJ, Craig L, Carter C, Czarra J, Brown JP

Here we describe diagnosis of concurrent infection with Aeromonas hydrophila, Mycobacterium spp., and Batrachochytrium dendrobatidis in a wild female Xenopus laevis captured in Chile and transported to the United States. After approximately 130 d in the laboratory, the frog was presented for dysecdysis and obtundation. After euthanasia, tissues were submitted for histopathologic evaluation and PCR analysis for B. dendrobatidis and Ranavirus. Clinically significant gross lesions included cutaneous ulcerations on the lip, right forelimb, and ventral chest. Microscopic findings included regionally extensive splenic necrosis, diffuse pneumonia, and fibrinous coelomitis all containing intralesional bacteria. PCR analysis yielded positive results for B. dendrobatidis only. Bacterial culture of the ulcerated skin and liver yielded A. hydrophila. Infection with Contracaecum spp. was diagnosed as an incidental finding. To our knowledge, this case is the first report of simultaneous infection with Aeromonas hydrophila, Mycobacterium spp., and Batrachochytrium dendrobatidis in a laboratory-maintained X. laevis captured from the wild.

Ecol Appl. 2010 Jan; 20(1): 289-302
Adams MJ, Chelgren ND, Reinitz D, Cole RA, Rachowicz LJ, Galvan S, McCreary B, Pearl CA, Bailey LL, Bettaso J, Bull EL, Leu M

Batrachochytrium dendrobatidis is a fungal pathogen that is receiving attention around the world for its role in amphibian declines. Study of its occurrence patterns is hampered by false negatives: the failure to detect the pathogen when it is present. Occupancy models are a useful but currently underutilized tool for analyzing detection data when the probability of detecting a species is <1. We use occupancy models to evaluate hypotheses concerning the occurrence and prevalence of B. dendrobatidis and discuss how this application differs from a conventional occupancy approach. We found that the probability of detecting the pathogen, conditional on presence of the pathogen in the anuran population, was related to amphibian development stage, day of the year, elevation, and human activities. Batrachochytrium dendrobatidis was found throughout our study area but was only estimated to occur in 53.4% of 78 populations of native amphibians and 66.4% of 40 populations of nonnative Rana catesbeiana tested. We found little evidence to support any spatial hypotheses concerning the probability that the pathogen occurs in a population, but did find evidence of some taxonomic variation. We discuss the interpretation of occupancy model parameters, when, unlike a conventional occupancy application, the number of potential samples or observations is finite.

Dis Aquat Organ. 2010 Jan 25; 88(2): 177-81
Stockwell MP, Clulow J, Mahony MJ

The amphibian chytrid fungus Batrachochytrium dendrobatidis is a recently described pathogen that has been implicated as a causal agent in the global decline in amphibians. Research into its biology and epidemiology has frequently involved in vitro experimentation. However, this research is currently limited by the inability to differentiate between viable and inviable zoospores. Stains are frequently used to determine cell viability, and this study tested a 2-colour fluorescence assay for the detection and quantification of viable B. dendrobatidis zoospores. The results show that the nucleic acid stains SYBR 14 and propidium iodide are effective in distinguishing live from dead zoospores, and a protocol has been optimized for their use. This viability assay provides an efficient and reliable tool that will have applications in B. dendrobatidis challenge and amphibian exposure experiments.

Ecohealth. 2009 Sep; 6(3): 358-72
Lötters S, Kielgast J, Bielby J, Schmidtlein S, Bosch J, Veith M, Walker SF, Fisher MC, Rödder D

Amphibians are globally declining and approximately one-third of all species are threatened with extinction. Some of the most severe declines have occurred suddenly and for unknown reasons in apparently pristine habitats. It has been hypothesized that these "rapid enigmatic declines" are the result of a panzootic of the disease chytridiomycosis caused by globally emerging amphibian chytrid fungus. In a Species Distribution Model, we identified the potential distribution of this pathogen. Areas and species from which rapid enigmatic decline are known significantly overlap with those of highest environmental suitability to the chytrid fungus. We confirm the plausibility of a link between rapid enigmatic decline in worldwide amphibian species and epizootic chytridiomycosis.