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1.  Germ Tube Mediated Invasion of Batrachochytrium dendrobatidis in Amphibian Skin Is Host Dependent 
PLoS ONE  2012;7(7):e41481.
Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.
We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis, Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro, comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea.
Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus.
In X. laevis, interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.
doi:10.1371/journal.pone.0041481
PMCID: PMC3401113  PMID: 22911798
2.  Seasonal Ecology and Behavior of an Endangered Rainforest Frog (Litoria rheocola) Threatened by Disease 
PLoS ONE  2015;10(5):e0127851.
One of the most devastating wildlife diseases ever recorded is chytridiomycosis, a recently emerged amphibian disease that is caused by the chytrid fungus Batrachochytrium dendrobatidis. Understanding, predicting, and managing the impacts of chytridiomycosis on any amphibian species will require detailed information on its ecology and behavior because this pathogen is transmitted by contact with water or other individuals, and pathogen growth rates are thermally sensitive. The common mistfrog (Litoria rheocola) is an endangered tropical rainforest frog that has declined due to chytridiomycosis. We tracked L. rheocola during the winter (cool/dry) and summer (warm/wet) seasons at a low- and high-elevation site. We found that seasonal differences in environmental temperatures and frog behavior should render this species most vulnerable to B. dendrobatidis during cooler months and at higher elevations, which matches observed patterns of infection prevalence in this species. During winter, frogs moved shorter distances than during summer, and they spent less time in vegetation and more time in the stream, which should increase exposure to aquatic B. dendrobatidis zoospores. At a low-elevation site (40 m ASL), estimated body temperatures were within the optimal range for B. dendrobatidis growth (15-25°C) most of the time during winter, but they reached temperatures above this threshold frequently in summer. At a higher elevation (750 m ASL), estimated body temperatures were within the range most favorable for B. dendrobatidis year-round, and did not exceed 25°C, even during summer. Our study provides the first detailed information on the ecology and behavior of L. rheocola and suggests ecological mechanisms for infection dynamics that have been observed in this endangered species.
doi:10.1371/journal.pone.0127851
PMCID: PMC4437910  PMID: 25993520
3.  Immune Defenses against Batrachochytrium dendrobatidis, a Fungus Linked to Global Amphibian Declines, in the South African Clawed Frog, Xenopus laevis▿  
Infection and Immunity  2010;78(9):3981-3992.
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. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. 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.
doi:10.1128/IAI.00402-10
PMCID: PMC2937463  PMID: 20584973
4.  Successful treatment of Batrachochytrium salamandrivorans infections in salamanders requires synergy between voriconazole, polymyxin E and temperature 
Scientific Reports  2015;5:11788.
Chytridiomycosis caused by the chytrid fungus Batrachochytrium salamandrivorans (Bsal) poses a serious threat to urodelan diversity worldwide. Antimycotic treatment of this disease using protocols developed for the related fungus Batrachochytrium dendrobatidis (Bd), results in therapeutic failure. Here, we reveal that this therapeutic failure is partly due to different minimum inhibitory concentrations (MICs) of antimycotics against Bsal and Bd. In vitro growth inhibition of Bsal occurs after exposure to voriconazole, polymyxin E, itraconazole and terbinafine but not to florfenicol. Synergistic effects between polymyxin E and voriconazole or itraconazole significantly decreased the combined MICs necessary to inhibit Bsal growth. Topical treatment of infected fire salamanders (Salamandra salamandra), with voriconazole or itraconazole alone (12.5 μg/ml and 0.6 μg/ml respectively) or in combination with polymyxin E (2000 IU/ml) at an ambient temperature of 15 °C during 10 days decreased fungal loads but did not clear Bsal infections. However, topical treatment of Bsal infected animals with a combination of polymyxin E (2000 IU/ml) and voriconazole (12.5 μg/ml) at an ambient temperature of 20 °C resulted in clearance of Bsal infections. This treatment protocol was validated in 12 fire salamanders infected with Bsal during a field outbreak and resulted in clearance of infection in all animals.
doi:10.1038/srep11788
PMCID: PMC4485233  PMID: 26123899
5.  Fungal Infection Intensity and Zoospore Output of Atelopus zeteki, a Potential Acute Chytrid Supershedder 
PLoS ONE  2014;9(3):e93356.
Amphibians vary in their response to infection by the amphibian-killing chytrid fungus, Batrachochytrium dendrobatidis (Bd). Highly susceptible species are the first to decline and/or disappear once Bd arrives at a site. These competent hosts likely facilitate Bd proliferation because of ineffective innate and/or acquired immune defenses. We show that Atelopus zeteki, a highly susceptible species that has undergone substantial population declines throughout its range, rapidly and exponentially increases skin Bd infection intensity, achieving intensities that are several orders of magnitude greater than most other species reported. We experimentally infected individuals that were never exposed to Bd (n = 5) or previously exposed to an attenuated Bd strain (JEL427-P39; n = 3). Within seven days post-inoculation, the average Bd infection intensity was 18,213 zoospores (SE: 9,010; range: 0 to 66,928). Both average Bd infection intensity and zoospore output (i.e., the number of zoospores released per minute by an infected individual) increased exponentially until time of death (t50 = 7.018, p<0.001, t46 = 3.164, p = 0.001, respectively). Mean Bd infection intensity and zoospore output at death were 4,334,422 zoospores (SE: 1,236,431) and 23.55 zoospores per minute (SE: 22.78), respectively, with as many as 9,584,158 zoospores on a single individual. The daily percent increases in Bd infection intensity and zoospore output were 35.4% (SE: 0.05) and 13.1% (SE: 0.04), respectively. We also found that Bd infection intensity and zoospore output were positively correlated (t43 = 3.926, p<0.001). All animals died between 22 and 33 days post-inoculation (mean: 28.88; SE: 1.58). Prior Bd infection had no effect on survival, Bd infection intensity, or zoospore output. We conclude that A. zeteki, a highly susceptible amphibian species, may be an acute supershedder. Our results can inform epidemiological models to estimate Bd outbreak probability, especially as they relate to reintroduction programs.
doi:10.1371/journal.pone.0093356
PMCID: PMC3968150  PMID: 24675899
6.  The Bacterially Produced Metabolite Violacein Is Associated with Survival of Amphibians Infected with a Lethal Fungus ▿  
Applied and Environmental Microbiology  2009;75(21):6635-6638.
The disease chytridiomycosis, which is caused by the chytrid fungus Batrachochytrium dendrobatidis, is associated with recent declines in amphibian populations. Susceptibility to this disease varies among amphibian populations and species, and resistance appears to be attributable in part to the presence of antifungal microbial species associated with the skin of amphibians. The betaproteobacterium Janthinobacterium lividum has been isolated from the skins of several amphibian species and produces the antifungal metabolite violacein, which inhibits B. dendrobatidis. In this study, we added J. lividum to red-backed salamanders (Plethodon cinereus) to obtain an increased range of violacein concentrations on the skin. Adding J. lividum to the skin of the salamander increased the concentration of violacein on the skin, which was strongly associated with survival after experimental exposure to B. dendrobatidis. As expected from previous work, some individuals that did not receive J. lividum and were exposed to B. dendrobatidis survived. These individuals had concentrations of bacterially produced violacein on their skins that were predicted to kill B. dendrobatidis. Our study suggests that a threshold violacein concentration of about 18 μM on a salamander's skin prevents mortality and morbidity caused by B. dendrobatidis. In addition, we show that over one-half of individuals in nature support antifungal bacteria that produce violacein, which suggests that there is a mutualism between violacein-producing bacteria and P. cinereus and that adding J. lividum is effective for protecting individuals that lack violacein-producing skin bacteria.
doi:10.1128/AEM.01294-09
PMCID: PMC2772424  PMID: 19717627
7.  Experimental evolution alters the rate and temporal pattern of population growth in Batrachochytrium dendrobatidis, a lethal fungal pathogen of amphibians 
Ecology and Evolution  2014;4(18):3633-3641.
Virulence of infectious pathogens can be unstable and evolve rapidly depending on the evolutionary dynamics of the organism. Experimental evolution can be used to characterize pathogen evolution, often with the underlying objective of understanding evolution of virulence. We used experimental evolution techniques (serial transfer experiments) to investigate differential growth and virulence of Batrachochytrium dendrobatidis (Bd), a fungal pathogen that causes amphibian chytridiomycosis. We tested two lineages of Bd that were derived from a single cryo-archived isolate; one lineage (P10) was passaged 10 times, whereas the second lineage (P50) was passaged 50 times. We quantified time to zoospore release, maximum zoospore densities, and timing of zoospore activity and then modeled population growth rates. We also conducted exposure experiments with a susceptible amphibian species, the common green tree frog (Litoria caerulea) to test the differential pathogenicity. We found that the P50 lineage had shorter time to zoospore production (Tmin), faster rate of sporangia death (ds), and an overall greater intrinsic population growth rate (λ). These patterns of population growth in vitro corresponded with higher prevalence and intensities of infection in exposed Litoria caerulea, although the differences were not significant. Our results corroborate studies that suggest that Bd may be able to evolve relatively rapidly. Our findings also challenge the general assumption that pathogens will always attenuate in culture because shifts in Bd virulence may depend on laboratory culturing practices. These findings have practical implications for the laboratory maintenance of Bd isolates and underscore the importance of understanding the evolution of virulence in amphibian chytridiomycosis.
doi:10.1002/ece3.1199
PMCID: PMC4224537  PMID: 25478154
Amphibian chytridiomycosis; amphibian declines; Batrachochytrium dendrobatidis; evolution of virulence; experimental evolution; host–pathogen interactions; serial passage experiments
8.  Inhibition of Local Immune Responses by the Frog-Killing Fungus Batrachochytrium dendrobatidis 
Infection and Immunity  2014;82(11):4698-4706.
Amphibians are suffering unprecedented global declines. A leading cause is the infectious disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis. Chytridiomycosis is a skin disease which disrupts transport of essential ions leading to death. Soluble factors produced by B. dendrobatidis impair amphibian and mammalian lymphocytes in vitro, but previous studies have not shown the effects of these inhibitory factors in vivo. To demonstrate in vivo inhibition of immunity by B. dendrobatidis, a modified delayed-type-hypersensitivity (DTH) protocol was developed to induce innate and adaptive inflammatory swelling in the feet of Xenopus laevis by injection of killed bacteria or phytohemagglutinin (PHA). Compared to previous protocols for PHA injection in amphibians, this method induced up to 20-fold greater inflammatory swelling. Using this new protocol, we measured DTH responses induced by killed bacteria or PHA in the presence of B. dendrobatidis supernatants. Swelling induced by single injection of PHA or killed bacteria was not significantly affected by B. dendrobatidis supernatants. However, swelling caused by a secondary injection of PHA, was significantly reduced by B. dendrobatidis supernatants. As previously described in vitro, factors from B. dendrobatidis appear to inhibit lymphocyte-mediated inflammatory swelling but not swelling caused by an inducer of innate leukocytes. This suggests that B. dendrobatidis is capable of inhibiting lymphocytes in a localized response to prevent adaptive immune responses in the skin. The modified protocol used to induce inflammatory swelling in the present study may be more effective than previous methods to investigate amphibian immune competence, particularly in nonmodel species.
doi:10.1128/IAI.02231-14
PMCID: PMC4249309  PMID: 25156734
9.  Evaluation of Amphotericin B and Chloramphenicol as Alternative Drugs for Treatment of Chytridiomycosis and Their Impacts on Innate Skin Defenses 
Applied and Environmental Microbiology  2014;80(13):4034-4041.
Chytridiomycosis, an amphibian skin disease caused by the emerging fungal pathogen Batrachochytrium dendrobatidis, has been implicated in catastrophic global amphibian declines. The result is an alarming decrease in amphibian diversity that is a great concern for the scientific community. Clinical trials testing potential antifungal drugs are needed to identify alternative treatments for amphibians infected with this pathogen. In this study, we quantified the MICs of chloramphenicol (800 μg/ml), amphotericin B (0.8 to 1.6 μg/ml), and itraconazole (Sporanox) (20 ng/ml) against B. dendrobatidis. Both chloramphenicol and amphotericin B significantly reduced B. dendrobatidis infection in naturally infected southern leopard frogs (Rana [Lithobates] sphenocephala), although neither drug was capable of complete fungal clearance. Long-term exposure of R. sphenocephala to these drugs did not inhibit antimicrobial peptide (AMP) synthesis, indicating that neither drug is detrimental to this important innate skin defense. However, we observed that chloramphenicol, but not amphotericin B or itraconazole, inhibited the growth of multiple R. sphenocephala skin bacterial isolates in vitro at concentrations below the MIC against B. dendrobatidis. These results indicate that treatment with chloramphenicol might dramatically alter the protective natural skin microbiome when used as an antifungal agent. This study represents the first examination of the effects of alternative antifungal drug treatments on amphibian innate skin defenses, a crucial step to validating these treatments for practical applications.
doi:10.1128/AEM.04171-13
PMCID: PMC4054225  PMID: 24771024
10.  Resistance to Chytridiomycosis in European Plethodontid Salamanders of the Genus Speleomantes 
PLoS ONE  2013;8(5):e63639.
North America and the neotropics harbor nearly all species of plethodontid salamanders. In contrast, this family of caudate amphibians is represented in Europe and Asia by two genera, Speleomantes and Karsenia, which are confined to small geographic ranges. Compared to neotropical and North American plethodontids, mortality attributed to chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) has not been reported for European plethodontids, despite the established presence of Bd in their geographic distribution. We determined the extent to which Bd is present in populations of all eight species of European Speleomantes and show that Bd was undetectable in 921 skin swabs. We then compared the susceptibility of one of these species, Speleomantes strinatii, to experimental infection with a highly virulent isolate of Bd (BdGPL), and compared this to the susceptible species Alytes muletensis. Whereas the inoculated A. muletensis developed increasing Bd-loads over a 4-week period, none of five exposed S. strinatii were colonized by Bd beyond 2 weeks post inoculation. Finally, we determined the extent to which skin secretions of Speleomantes species are capable of killing Bd. Skin secretions of seven Speleomantes species showed pronounced killing activity against Bd over 24 hours. In conclusion, the absence of Bd in Speleomantes combined with resistance to experimental chytridiomycosis and highly efficient skin defenses indicate that the genus Speleomantes is a taxon unlikely to decline due to Bd.
doi:10.1371/journal.pone.0063639
PMCID: PMC3659026  PMID: 23703511
11.  Experimental evidence for a cost of resistance to the fungal pathogen, Batrachochytrium dendrobatidis, for the palmate newt, Lissotriton helveticus 
BMC Ecology  2013;13:27.
Background
Batrachochytrium dendrobatidis (Bd), the causative agent of chytridiomycosis, is decimating amphibians worldwide. Unsurprisingly, the majority of studies have therefore concentrated on documenting morbidity and mortality of susceptible species and projecting population consequences as a consequence of this emerging infectious disease. Currently, there is a paucity of studies investigating the sub-lethal costs of Bd in apparently asymptomatic species, particularly in controlled experimental conditions. Here we report the consequences of a single dose of B. dendrobatidis zoospores on captive adult palmate newts (Lissotriton helveticus) for morphological and behavioural traits that associate with reproductive success.
Results
A single exposure to ~2000 zoospores induced a subclinical Bd infection. One week after inoculation 84% of newts tested positive for Bd, and of those, 98% had apparently lost the infection by the day 30. However, exposed newts suffered significant mass loss compared with control newts, and those experimental newts removing higher levels of Bd lost most mass. We found no evidence to suggest that three secondary sexual characteristics (areas of dorsal crest and rear foot webbing, and length of tail filament) were reduced between experimental versus control newts; in fact, rear foot webbing was 26% more expansive at the end of the experiment in exposed newts. Finally, compared with unexposed controls, exposure to Bd was associated with a 50% earlier initiation of the non-reproductive terrestrial phase.
Conclusions
Our results suggest that Bd has measureable, but sub-lethal effects, on adult palmate newts, at least under the laboratory conditions presented. We conclude that the effects reported are most likely to be mediated through the initiation of costly immune responses and/or tissue repair mechanisms. Although we found no evidence of hastened secondary sexual trait regression, through reducing individual body condition and potentially, breeding season duration, we predict that Bd exposure might have negative impacts on populations of palmate newts through reducing individual reproductive success and adult recruitment.
doi:10.1186/1472-6785-13-27
PMCID: PMC3722082  PMID: 23866033
Body condition; Cost of immunity; Chytridiomycosis; Emerging infectious disease; Resistance; Secondary sexual traits
12.  Swabbing Often Fails to Detect Amphibian Chytridiomycosis under Conditions of Low Infection Load 
PLoS ONE  2014;9(10):e111091.
The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (denoted Bd), causes large-scale epizootics in naïve amphibian populations. Intervention strategies to rapidly respond to Bd incursions require sensitive and accurate diagnostic methods. Chytridiomycosis usually is assessed by quantitative polymerase chain reaction (qPCR) amplification of amphibian skin swabs. Results based on this method, however, sometimes yield inconsistent results on infection status and inaccurate scores of infection intensity. In Asia and other regions where amphibians typically bear low Bd loads, swab results are least reliable. We developed a Bd-sampling method that collects zoospores released by infected subjects into an aquatic medium. Bd DNA is extracted by filters and amplified by nested PCR. Using laboratory colonies and field populations of Bombina orientalis, we compare results with those obtained on the same subjects by qPCR of DNA extracted from swabs. Many subjects, despite being diagnosed as Bd-negative by conventional methods, released Bd zoospores into collection containers and thus must be considered infected. Infection loads determined from filtered water were at least 1000 times higher than those estimated from swabs. Subjects significantly varied in infection load, as they intermittently released zoospores, over a 5-day period. Thus, the method might be used to compare the infectivity of individuals and study the periodicity of zoospore release. Sampling methods based on water filtration can dramatically increase the capacity to accurately diagnose chytridiomycosis and contribute to a better understanding of the interactions between Bd and its hosts.
doi:10.1371/journal.pone.0111091
PMCID: PMC4205094  PMID: 25333363
13.  Sodium Chloride Inhibits the Growth and Infective Capacity of the Amphibian Chytrid Fungus and Increases Host Survival Rates 
PLoS ONE  2012;7(5):e36942.
The amphibian chytrid fungus Batrachochytrium dendrobatidis is a recently emerged pathogen that causes the infectious disease chytridiomycosis and has been implicated as a contributing factor in the global amphibian decline. Since its discovery, research has been focused on developing various methods of mitigating the impact of chytridiomycosis on amphibian hosts but little attention has been given to the role of antifungal agents that could be added to the host's environment. Sodium chloride is a known antifungal agent used routinely in the aquaculture industry and this study investigates its potential for use as a disease management tool in amphibian conservation. The effect of 0–5 ppt NaCl on the growth, motility and survival of the chytrid fungus when grown in culture media and its effect on the growth, infection load and survivorship of infected Peron's tree frogs (Litoria peronii) in captivity, was investigated. The results reveal that these concentrations do not negatively affect the survival of the host or the pathogen. However, concentrations greater than 3 ppt significantly reduced the growth and motility of the chytrid fungus compared to 0 ppt. Concentrations of 1–4 ppt NaCl were also associated with significantly lower host infection loads while infected hosts exposed to 3 and 4 ppt NaCl were found to have significantly higher survival rates. These results support the potential for NaCl to be used as an environmentally distributed antifungal agent for the prevention of chytridiomycosis in susceptible amphibian hosts. However, further research is required to identify any negative effects of salt exposure on both target and non-target organisms prior to implementation.
doi:10.1371/journal.pone.0036942
PMCID: PMC3349647  PMID: 22590639
14.  A Fungal Pathogen of Amphibians, Batrachochytrium dendrobatidis, Attenuates in Pathogenicity with In Vitro Passages 
PLoS ONE  2013;8(10):e77630.
Laboratory investigations into the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), have accelerated recently, given the pathogen’s role in causing the global decline and extinction of amphibians. Studies in which host animals were exposed to Bd have largely assumed that lab-maintained pathogen cultures retained the infective and pathogenic properties of wild isolates. Attenuated pathogenicity is common in artificially maintained cultures of other pathogenic fungi, but to date, it is unknown whether, and to what degree, Bd might change in culture. We compared zoospore production over time in two samples of a single Bd isolate having different passage histories: one maintained in artificial media for more than six years (JEL427-P39), and one recently thawed from cryopreserved stock (JEL427-P9). In a common garden experiment, we then exposed two different amphibian species, Eleutherodactylus coqui and Atelopus zeteki, to both cultures to test whether Bd attenuates in pathogenicity with in vitro passages. The culture with the shorter passage history, JEL427-P9, had significantly greater zoospore densities over time compared to JEL427-P39. This difference in zoospore production was associated with a difference in pathogenicity for a susceptible amphibian species, indicating that fecundity may be an important virulence factor for Bd. In the 130-day experiment, Atelopus zeteki frogs exposed to the JEL427-P9 culture experienced higher average infection intensity and 100% mortality, compared with 60% mortality for frogs exposed to JEL427-P39. This effect was not observed with Eleutherodactylus coqui, which was able to clear infection. We hypothesize that the differences in phenotypic performance observed with Atelopus zeteki are rooted in changes of the Bd genome. Future investigations enabled by this study will focus on the underlying mechanisms of Bd pathogenicity.
doi:10.1371/journal.pone.0077630
PMCID: PMC3795048  PMID: 24130895
15.  Persistence of the emerging pathogen Batrachochytrium dendrobatidis outside the amphibian host greatly increases the probability of host extinction 
Pathogens do not normally drive their hosts to extinction; however, Batrachochytrium dendrobatidis, which causes amphibian chytridiomycosis, has been able to do so. Theory predicts that extinction can be caused by long-lived or saprobic free-living stages. The hypothesis that such a stage occurs in B. dendrobatidis is supported by the recent discovery of an apparently encysted form of the pathogen. To investigate the effect of a free-living stage of B. dendrobatidis on host population dynamics, a mathematical model was developed to describe the introduction of chytridiomycosis into a breeding population of Bufo bufo, parametrized from laboratory infection and transmission experiments. The model predicted that the longer that B. dendrobatidis was able to persist in water, either due to an increased zoospore lifespan or saprobic reproduction, the more likely it was that it could cause local B. bufo extinction (defined as decrease below a threshold level). Establishment of endemic B. dendrobatidis infection in B. bufo, with severe host population depression, was also possible, in agreement with field observations. Although this model is able to predict clear trends, more precise predictions will only be possible when the life history of B. dendrobatidis, including free-living stages of the life cycle, is better understood.
doi:10.1098/rspb.2007.1356
PMCID: PMC2593721  PMID: 18048287
mathematical modelling; epidemiology; Batrachochytrium dendrobatidis; chytridiomycosis; Bufo bufo; amphibian declines
16.  Whether the Weather Drives Patterns of Endemic Amphibian Chytridiomycosis: A Pathogen Proliferation Approach 
PLoS ONE  2013;8(4):e61061.
The pandemic amphibian disease chytridiomycosis often exhibits strong seasonality in both prevalence and disease-associated mortality once it becomes endemic. One hypothesis that could explain this temporal pattern is that simple weather-driven pathogen proliferation (population growth) is a major driver of chytridiomycosis disease dynamics. Despite various elaborations of this hypothesis in the literature for explaining amphibian declines (e.g., the chytrid thermal-optimum hypothesis) it has not been formally tested on infection patterns in the wild. In this study we developed a simple process-based model to simulate the growth of the pathogen Batrachochytrium dendrobatidis (Bd) under varying weather conditions to provide an a priori test of a weather-linked pathogen proliferation hypothesis for endemic chytridiomycosis. We found strong support for several predictions of the proliferation hypothesis when applied to our model species, Litoria pearsoniana, sampled across multiple sites and years: the weather-driven simulations of pathogen growth potential (represented as a growth index in the 30 days prior to sampling; GI30) were positively related to both the prevalence and intensity of Bd infections, which were themselves strongly and positively correlated. In addition, a machine-learning classifier achieved ∼72% success in classifying positive qPCR results when utilising just three informative predictors 1) GI30, 2) frog body size and 3) rain on the day of sampling. Hence, while intrinsic traits of the individuals sampled (species, size, sex) and nuisance sampling variables (rainfall when sampling) influenced infection patterns obtained when sampling via qPCR, our results also strongly suggest that weather-linked pathogen proliferation plays a key role in the infection dynamics of endemic chytridiomycosis in our study system. Predictive applications of the model include surveillance design, outbreak preparedness and response, climate change scenario modelling and the interpretation of historical patterns of amphibian decline.
doi:10.1371/journal.pone.0061061
PMCID: PMC3629077  PMID: 23613783
17.  Temperature alters reproductive life history patterns in Batrachochytrium dendrobatidis, a lethal pathogen associated with the global loss of amphibians 
Ecology and Evolution  2012;2(9):2241-2249.
Understanding how pathogens respond to changing environmental conditions is a central challenge in disease ecology. The environmentally sensitive fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the amphibian disease chytridiomycosis, has spread globally causing amphibian extirpations in a wide variety of climatic regions. To gain an in-depth understanding of Bd's responses to temperature, we used an integrative approach, combining empirical laboratory experiments with mathematical modeling. First, we selected a single Bd isolate and serially propagated two lineages of the isolate for multiple generations in two stable thermal conditions: 4°C (cold-adapted lineage) and 23°C (warm-adapted lineage). We quantified the production of infectious zoospores (fecundity), the timing of zoospore release, and zoospore activity in reciprocal temperature transplant experiments in which both Bd lineages were grown in either high or low temperature conditions. We then developed population growth models for the Bd lineages under each set of temperature conditions. We found that Bd had lower population growth rates, but longer periods of zoospore activity in the low temperature treatment (4°C) compared to the high temperature treatment (23°C). This effect was more pronounced in Bd lineages that were propagated in the low temperature treatment (4°C), suggesting a shift in Bd's response to low temperature conditions. Our results provide novel insights into the mechanisms by which Bd can thrive in a wide variety of temperature conditions, potentially altering the dynamics of chytridiomycosis and thus, the propensity for Bd to cause amphibian population collapse. We also suggest that the adaptive responses of Bd to thermal conditions warrant further investigation, especially in the face of global climate change.
doi:10.1002/ece3.334
PMCID: PMC3488674  PMID: 23139882
Amphibian declines; Batrachochytrium dendrobatidis; chytridiomycosis; climate change; emerging infectious disease; temperature
18.  Induction of the Carrier State in Pigeons Infected with Salmonella enterica Subspecies enterica Serovar Typhimurium PT99 by Treatment with Florfenicol: a Matter of Pharmacokinetics▿  
Paratyphoid caused by Salmonella enterica subsp. enterica serovar Typhimurium is the main bacterial disease in pigeons. The ability of Salmonella serovar Typhimurium to persist intracellularly inside pigeon macrophages results in the development of chronic carriers, which maintain the infection in the flock. In this study, the effect of drinking-water medication with florfenicol on Salmonella infection in pigeons was examined. The pharmacokinetics of florfenicol in pigeons revealed a relatively high volume of distribution of 2.02 liters/kg of body weight and maximum concentrations in plasma higher than the MICs for the Salmonella strain used (4 μg/ml) but quick clearance of florfenicol due to a short half-life of 1.73 h. Together with highly variable bioavailability and erratic drinking-water uptake, these parameters resulted in the inability to reach a steady-state concentration through the continuous administration of florfenicol in the drinking water. Florfenicol was capable of reducing only moderately the number of intracellular salmonellae in infected pigeon macrophages in vitro. Only at high extracellular concentrations (>16 μg/ml) was a more-than-10-fold reduction of the number of intracellular bacteria noticed. Florfenicol treatment of pigeons via the drinking water from 2 days after experimental inoculation with Salmonella serovar Typhimurium until euthanasia at 16 days postinoculation resulted in a reduction of Salmonella shedding and an improvement in the fecal consistency. However, internal organs in florfenicol-treated pigeons were significantly more heavily colonized than those in untreated pigeons. In conclusion, the oral application of florfenicol for the treatment of pigeon paratyphoid contributes to the development of carrier animals through sub-MIC concentrations in plasma that do not inhibit intracellular persistency.
doi:10.1128/AAC.00575-07
PMCID: PMC2258533  PMID: 18180355
19.  Genome-Wide Transcriptional Response of Silurana (Xenopus) tropicalis to Infection with the Deadly Chytrid Fungus 
PLoS ONE  2009;4(8):e6494.
Emerging infectious diseases are of great concern for both wildlife and humans. Several highly virulent fungal pathogens have recently been discovered in natural populations, highlighting the need for a better understanding of fungal-vertebrate host-pathogen interactions. Because most fungal pathogens are not fatal in the absence of other predisposing conditions, host-pathogen dynamics for deadly fungal pathogens are of particular interest. The chytrid fungus Batrachochytrium dendrobatidis (hereafter Bd) infects hundreds of species of frogs in the wild. It is found worldwide and is a significant contributor to the current global amphibian decline. However, the mechanism by which Bd causes death in amphibians, and the response of the host to Bd infection, remain largely unknown. Here we use whole-genome microarrays to monitor the transcriptional responses to Bd infection in the model frog species, Silurana (Xenopus) tropicalis, which is susceptible to chytridiomycosis. To elucidate the immune response to Bd and evaluate the physiological effects of chytridiomycosis, we measured gene expression changes in several tissues (liver, skin, spleen) following exposure to Bd. We detected a strong transcriptional response for genes involved in physiological processes that can help explain some clinical symptoms of chytridiomycosis at the organismal level. However, we detected surprisingly little evidence of an immune response to Bd exposure, suggesting that this susceptible species may not be mounting efficient innate and adaptive immune responses against Bd. The weak immune response may be partially explained by the thermal conditions of the experiment, which were optimal for Bd growth. However, many immune genes exhibited decreased expression in Bd-exposed frogs compared to control frogs, suggesting a more complex effect of Bd on the immune system than simple temperature-mediated immune suppression. This study generates important baseline data for ongoing efforts to understand differences in response to Bd between susceptible and resistant frog species and the effects of chytridiomycosis in natural populations.
doi:10.1371/journal.pone.0006494
PMCID: PMC2727658  PMID: 19701481
20.  Heterogeneous Occupancy and Density Estimates of the Pathogenic Fungus Batrachochytrium dendrobatidis in Waters of North America 
PLoS ONE  2014;9(9):e106790.
Biodiversity losses are occurring worldwide due to a combination of stressors. For example, by one estimate, 40% of amphibian species are vulnerable to extinction, and disease is one threat to amphibian populations. The emerging infectious disease chytridiomycosis, caused by the aquatic fungus Batrachochytrium dendrobatidis (Bd), is a contributor to amphibian declines worldwide. Bd research has focused on the dynamics of the pathogen in its amphibian hosts, with little emphasis on investigating the dynamics of free-living Bd. Therefore, we investigated patterns of Bd occupancy and density in amphibian habitats using occupancy models, powerful tools for estimating site occupancy and detection probability. Occupancy models have been used to investigate diseases where the focus was on pathogen occurrence in the host. We applied occupancy models to investigate free-living Bd in North American surface waters to determine Bd seasonality, relationships between Bd site occupancy and habitat attributes, and probability of detection from water samples as a function of the number of samples, sample volume, and water quality. We also report on the temporal patterns of Bd density from a 4-year case study of a Bd-positive wetland. We provide evidence that Bd occurs in the environment year-round. Bd exhibited temporal and spatial heterogeneity in density, but did not exhibit seasonality in occupancy. Bd was detected in all months, typically at less than 100 zoospores L−1. The highest density observed was ∼3 million zoospores L−1. We detected Bd in 47% of sites sampled, but estimated that Bd occupied 61% of sites, highlighting the importance of accounting for imperfect detection. When Bd was present, there was a 95% chance of detecting it with four samples of 600 ml of water or five samples of 60 mL. Our findings provide important baseline information to advance the study of Bd disease ecology, and advance our understanding of amphibian exposure to free-living Bd in aquatic habitats over time.
doi:10.1371/journal.pone.0106790
PMCID: PMC4164359  PMID: 25222122
21.  Seasonal Pattern of Batrachochytrium dendrobatidis Infection and Mortality in Lithobates areolatus: Affirmation of Vredenburg's “10,000 Zoospore Rule” 
PLoS ONE  2011;6(3):e16708.
To fully comprehend chytridiomycosis, the amphibian disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), it is essential to understand how Bd affects amphibians throughout their remarkable range of life histories. Crawfish Frogs (Lithobates areolatus) are a typical North American pond-breeding species that forms explosive spring breeding aggregations in seasonal and semipermanent wetlands. But unlike most species, when not breeding Crawfish Frogs usually live singly—in nearly total isolation from conspecifics—and obligately in burrows dug by crayfish. Crayfish burrows penetrate the water table, and therefore offer Crawfish Frogs a second, permanent aquatic habitat when not breeding. Over the course of two years we sampled for the presence of Bd in Crawfish Frog adults. Sampling was conducted seasonally, as animals moved from post-winter emergence through breeding migrations, then back into upland burrow habitats. During our study, 53% of Crawfish Frog breeding adults tested positive for Bd in at least one sample; 27% entered breeding wetlands Bd positive; 46% exited wetlands Bd positive. Five emigrating Crawfish Frogs (12%) developed chytridiomycosis and died. In contrast, all 25 adult frogs sampled while occupying upland crayfish burrows during the summer tested Bd negative. One percent of postmetamorphic juveniles sampled were Bd positive. Zoospore equivalents/swab ranged from 0.8 to 24,436; five out of eight frogs with zoospore equivalents near or >10,000 are known to have died. In summary, Bd infection rates in Crawfish Frog populations ratchet up from near zero during the summer to over 25% following overwintering; rates then nearly double again during and just after breeding—when mortality occurs—before the infection wanes during the summer. Bd-negative postmetamorphic juveniles may not be exposed again to this pathogen until they take up residence in crayfish burrows, or until their first breeding, some years later.
doi:10.1371/journal.pone.0016708
PMCID: PMC3053364  PMID: 21423745
22.  The invasive chytrid fungus of amphibians paralyzes lymphocyte responses 
Science (New York, N.Y.)  2013;342(6156):366-369.
The chytrid fungus, Batrachochytrium dendrobatidis, causes chytridiomycosis and is a major contributor to global amphibian declines. Although amphibians have robust immune defenses, clearance of this pathogen is impaired. Because inhibition of host immunity is a common survival strategy of pathogenic fungi, we hypothesized that B. dendrobatidis evades clearance by inhibiting immune functions. We found that B. dendrobatidis cells and supernantants impaired lymphocyte proliferation and induced apoptosis; however, fungal recognition and phagocytosis by macrophages and neutrophils was not impaired. Fungal inhibitory factors were resistant to heat, acid, and protease. Their production was absent in zoospores and reduced by nikkomycin Z, suggesting that they may be components of the cell wall. Evasion of host immunity may explain why this pathogen has devastated amphibian populations worldwide.
doi:10.1126/science.1243316
PMCID: PMC3956111  PMID: 24136969
23.  Amphibians acquire resistance to live and dead fungus overcoming fungal immunosuppression 
Nature  2014;511(7508):224-227.
Emerging fungal pathogens pose a greater threat to biodiversity than any other parasitic group1, causing declines of many taxa, including bats, corals, bees, snakes and amphibians1–4. Currently, there is little evidence that wild animals can acquire resistance to these pathogens5. Batrachochytrium dendrobatidis is a pathogenic fungus implicated in the recent global decline of amphibians6. Here we demonstrate that three species of amphibians can acquire behavioural or immunological resistance to B. dendrobatidis. Frogs learned to avoid the fungus after just one B. dendrobatidis exposure and temperature-induced clearance. In subsequent experiments in which B. dendrobatidis avoidance was prevented, the number of previous exposures was a negative predictor of B. dendrobatidis burden on frogs and B. dendrobatidis-induced mortality, and was a positive predictor of lymphocyte abundance and proliferation. These results suggest that amphibians can acquire immunity to B. dendrobatidis that overcomes pathogen-induced immunosuppression7–9 and increases their survival. Importantly, exposure to dead fungus induced a similar magnitude of acquired resistance as exposure to live fungus. Exposure of frogs to B. dendrobatidis antigens might offer a practical way to protect pathogen-naive amphibians and facilitate the reintroduction of amphibians to locations in the wild where B. dendrobatidis persists. Moreover, given the conserved nature of vertebrate immune responses to fungi5 and the fact that many animals are capable of learning to avoid natural enemies10, these results offer hope that other wild animal taxa threatened by invasive fungi might be rescued by management approaches based on herd immunity.
doi:10.1038/nature13491
PMCID: PMC4464781  PMID: 25008531
24.  Survival of Batrachochytrium dendrobatidis in Water: Quarantine and Disease Control Implications 
Emerging Infectious Diseases  2003;9(8):922-925.
Amphibian chytridiomycosis is an emerging infectious disease of amphibians thought to be moved between countries by trade in infected amphibians. The causative fungus, Batrachochytrium dendrobatidis, produces aquatic, motile zoospores; infections have been achieved in experiments by exposing amphibians to water containing zoospores. However, the ability of this fungus to survive in the environment in the absence of an amphibian host is unknown. We show that B. dendrobatidis will survive in tap water and in deionized water for 3 and 4 weeks, respectively. In lake water, infectivity was observed for 7 weeks after introduction. The knowledge that water can remain infective for up to 7 weeks is important for the formulation of disease control and quarantine strategies for the management of water that has been in contact with amphibians.
doi:10.3201/eid0908.030145
PMCID: PMC3020615  PMID: 12967488
Batrachochytrium dendrobatidis; chytridiomycosis; amphibian disease; research
25.  More than Skin Deep: Functional Genomic Basis for Resistance to Amphibian Chytridiomycosis 
Genome Biology and Evolution  2014;7(1):286-298.
The amphibian-killing chytrid fungus Batrachochytrium dendrobatidis (Bd) is one of the most generalist pathogens known, capable of infecting hundreds of species globally and causing widespread population declines and extinctions. However, some host species are seemingly unaffected by Bd, tolerating or clearing infections without clinical signs of disease. Variation in host immune responses is commonly evoked for these resistant or tolerant species, yet to date, we have no direct comparison of amphibian species responses to infection at the level of gene expression. In this study, we challenged four Central American frog species that vary in Bd susceptibility, with a sympatric virulent strain of the pathogen. We compared skin and spleen orthologous gene expression using differential expression tests and coexpression gene network analyses. We found that resistant species have reduced skin inflammatory responses and increased expression of genes involved in skin integrity. In contrast, only highly susceptible species exhibited suppression of splenic T-cell genes. We conclude that resistance to chytridiomycosis may be related to a species’ ability to escape the immunosuppressive activity of the fungus. Moreover, our results indicate that within-species differences in splenic proteolytic enzyme gene expression may contribute to intraspecific variation in survival. This first comparison of amphibian functional immunogenomic architecture in response to Bd provides insights into key genetic mechanisms underlying variation in disease outcomes among amphibian species.
doi:10.1093/gbe/evu285
PMCID: PMC4316636  PMID: 25539724
Batrachochytrium dendrobatidis; immunogenomics; comparative transcriptomics; immunosuppression; amphibian immunity

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