The impacts of water quality on the amphibian chytrid fungal pathogen: A systematic review.

The pathogenic fungus Batrachochytrium dendrobatidis has caused declines of amphibians worldwide. Yet our understanding of how water quality influences fungal pathogenicity is limited. Here, we reviewed experimental studies on the effect of water quality on this pathogen to determine which parameters impacted disease dynamics consistently. The strongest evidence for protective effects is salinity which shows strong antifungal properties in hosts at natural levels. Although many fungicides had detrimental effects on the fungal pathogen in vitro, their impact on the host is variable and they can worsen infection outcomes. However, one fungicide, epoxiconazole, reduced disease effects experimentally and likely in the field. While heavy metals are frequently studied, there is weak evidence that they influence infection outcomes. Nitrogen and phosphorous do not appear to impact pathogen growth or infection in the amphibian host. The effects of other chemicals, like pesticides and disinfectants on infection were mostly unclear with mixed results or lacking an in vivo component. Our study shows that water chemistry does impact disease dynamics, but the effects of specific parameters require more investigation. Improving our understanding of how water chemistry influences disease dynamics will help predict the impact of chytridiomycosis, especially in amphibian populations affected by land use changes.

Fate of hepatotoxin microcystin during infection of cyanobacteria by fungal chytrid parasites.

Chytrid parasites are increasingly recognized as ubiquitous and potent control agents of phytoplankton, including bloom-forming toxigenic cyanobacteria. In order to explore the fate of the cyanobacterial toxin microcystins (MCs) and assess potential upregulation of their production under parasite attack, a laboratory experiment was conducted to evaluate short- and long-term variation in extracellular and intracellular MC in the cyanobacteria Planktothrix agardhii and P. rubescens, both under chytrid infection and in the presence of lysates of previously infected cyanobacteria. MCs release under parasite infection was limited and not different to uninfected cyanobacteria, with extracellular toxin shares never exceeding 10%, substantially below those caused by mechanical lysis induced by a cold-shock. Intracellular MC contents in P. rubescens under infection were not significantly different from uninfected controls, whereas infected P. agardhii showed a 1.5-fold increase in intracellular MC concentrations, but this was detected within the first 48 hours after parasite inoculation and not later, indicating no substantial MC upregulation in cells being infected. The presence of lysates of previously infected cyanobacteria did not elicit higher intracellular MC contents in exposed cyanobacteria, speaking against a putative upregulation of toxin production induced via quorum sensing in response to parasite attack. These results indicate that chytrid epidemics can constitute a bloom decay mechanism that is not accompanied by massive release of toxins into the medium.

Is overwintering mortality driving enigmatic declines? Evaluating the impacts of trematodes and the amphibian chytrid fungus on an anuran from hatching through overwintering.

Emerging infectious diseases are increasing globally and are an additional challenge to species dealing with native parasites and pathogens. Therefore, understanding the combined effects of infectious agents on hosts is important for species' conservation and population management. Amphibians are hosts to many parasites and pathogens, including endemic trematode flatworms (e.g., Echinostoma spp.) and the novel pathogenic amphibian chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Our study examined how exposure to trematodes during larval development influenced the consequences of Bd pathogen exposure through critical life events. We found that prior exposure to trematode parasites negatively impacted metamorphosis but did not influence the effect of Bd infection on terrestrial growth and survival. Bd infection alone, however, resulted in significant mortality during overwintering-an annual occurrence for most temperate amphibians. The results of our study indicated overwintering mortality from Bd could provide an explanation for enigmatic declines and highlights the importance of examining the long-term consequences of novel parasite exposure.

Tracking Batrachochytrium dendrobatidis Infection Across the Globe.

Infection records of Batrachochytrium dendrobatidis (Bd), a pathogen that has devastated amphibian populations worldwide, have rapidly increased since the pathogen's discovery. Dealing with so many records makes it difficult to (a) know where, when and in which species infections have been detected, (b) understand how widespread and pervasive Bd is and (c) prioritize study and management areas. We conducted a systematic review of papers and compiled a database with Bd infection records. Our dataset covers 71 amphibian families and 119 countries. The data revealed how widespread and adaptable Bd is, being able to infect over 50% of all tested amphibian species, with over 1000 confirmed host species and being present in 86 countries. The distribution of infected species is uneven among and within countries. Areas where the distributions of many infected species overlap are readily visible; these are regions where Bd likely develops well. Conversely, areas where the distributions of species that tested negative overlap, such as the Atlantic Coast in the USA, suggest the presence of Bd refuges. Finally, we report how the number of tested and infected species has changed through time, and provide a list of oldest detection records per country.

Instant killing of pathogenic chytrid fungi by disposable nitrile gloves prevents disease transmission between amphibians.

To prevent transmission of the pathogenic chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), hygiene protocols prescribe the single use of disposable gloves for handling amphibians. We discovered that rinse water from nitrile gloves instantly kills 99% of Bd and Bsal zoospores. Transmission experiments using midwife toads (Alytes obstetricans) and Bd, and Alpine newts (Ichthyosaura alpestris) and Bsal, show that the use of the same pair of gloves for 2 subsequent individuals does not result in significant transmission of any chytrid fungus. In contrast, handling infected amphibians bare-handed caused transmission of Bsal in 4 out of 10 replicates, but did not result in transmission of Bd. Based on the manufacturer's information, high resolution mass spectrometry (HRMS) and colorimetric tests, calcium lactate and calcium nitrate were identified as compounds with antifungal activity against both Bd and Bsal. These findings corroborate the importance of wearing gloves as an important sanitary measure in amphibian disease prevention. If the highly recommended single use of gloves is not possible, handling multiple post-metamorphic amphibians with the same pair of nitrile gloves should still be preferred above bare-handed manipulation.

Presence of low virulence chytrid fungi could protect European amphibians from more deadly strains.

Wildlife diseases are contributing to the current Earth's sixth mass extinction; one disease, chytridiomycosis, has caused mass amphibian die-offs. While global spread of a hypervirulent lineage of the fungus Batrachochytrium dendrobatidis (BdGPL) causes unprecedented loss of vertebrate diversity by decimating amphibian populations, its impact on amphibian communities is highly variable across regions. Here, we combine field data with in vitro and in vivo trials that demonstrate the presence of a markedly diverse variety of low virulence isolates of BdGPL in northern European amphibian communities. Pre-exposure to some of these low virulence isolates protects against disease following subsequent exposure to highly virulent BdGPL in midwife toads (Alytes obstetricans) and alters infection dynamics of its sister species B. salamandrivorans in newts (Triturus marmoratus), but not in salamanders (Salamandra salamandra). The key role of pathogen virulence in the complex host-pathogen-environment interaction supports efforts to limit pathogen pollution in a globalized world.

Experimental methodologies can affect pathogenicity of Batrachochytrium salamandrivorans infections.

Controlled experiments are one approach to understanding the pathogenicity of etiologic agents to susceptible hosts. The recently discovered fungal pathogen, Batrachochytrium salamandrivorans (Bsal), has resulted in a surge of experimental investigations because of its potential to impact global salamander biodiversity. However, variation in experimental methodologies could thwart knowledge advancement by introducing confounding factors that make comparisons difficult among studies. Thus, our objective was to evaluate if variation in experimental methods changed inferences made on the pathogenicity of Bsal. We tested whether passage duration of Bsal culture, exposure method of the host to Bsal (water bath vs. skin inoculation), Bsal culturing method (liquid vs. plated), host husbandry conditions (aquatic vs. terrestrial), and skin swabbing frequency influenced diseased-induced mortality in a susceptible host species, the eastern newt (Notophthalmus viridescens). We found that disease-induced mortality was faster for eastern newts when exposed to a low passage isolate, when newts were housed in terrestrial environments, and if exposure to zoospores occurred via water bath. We did not detect differences in disease-induced mortality between culturing methods or swabbing frequencies. Our results illustrate the need to standardize methods among Bsal experiments. We provide suggestions for future Bsal experiments in the context of hypothesis testing and discuss the ecological implications of our results.

High-efficiency electroporation of chytrid fungi.

Two species of parasitic fungi from the phylum Chytridiomycota (chytrids) are annihilating global amphibian populations. These chytrid species-Batrachochytrium dendrobatidis and B. salamandrivorans-have high rates of mortality and transmission. Upon establishing infection in amphibians, chytrids rapidly multiply within the skin and disrupt their hosts' vital homeostasis mechanisms. Current disease models suggest that chytrid fungi locate and infect their hosts during a motile, unicellular 'zoospore' life stage. Moreover, other chytrid species parasitize organisms from across the tree of life, making future epidemics in new hosts a likely possibility. Efforts to mitigate the damage and spread of chytrid disease have been stymied by the lack of knowledge about basic chytrid biology and tools with which to test molecular hypotheses about disease mechanisms. To overcome this bottleneck, we have developed high-efficiency delivery of molecular payloads into chytrid zoospores using electroporation. Our electroporation protocols result in payload delivery to between 75 and 97% of living cells of three species: B. dendrobatidis, B. salamandrivorans, and a non-pathogenic relative, Spizellomyces punctatus. This method lays the foundation for molecular genetic tools needed to establish ecological mitigation strategies and answer broader questions in evolutionary and cell biology.

Infection dynamics, dispersal, and adaptation: understanding the lack of recovery in a remnant frog population following a disease outbreak.

Emerging infectious diseases can cause dramatic declines in wildlife populations. Sometimes, these declines are followed by recovery, but many populations do not recover. Studying differential recovery patterns may yield important information for managing disease-afflicted populations and facilitating population recoveries. In the late 1980s, a chytridiomycosis outbreak caused multiple frog species in Australia's Wet Tropics to decline. Populations of some species (e.g., Litoria nannotis) subsequently recovered, while others (e.g., Litoria dayi) did not. We examined the population genetics and current infection status of L. dayi, to test several hypotheses regarding the failure of its populations to recover: (1) a lack of individual dispersal abilities has prevented recolonization of previously occupied locations, (2) a loss of genetic variation has resulted in limited adaptive potential, and (3) L. dayi is currently adapting to chytridiomycosis. We found moderate-to-high levels of gene flow and diversity (Fst range: <0.01-0.15; minor allele frequency (MAF): 0.192-0.245), which were similar to previously published levels for recovered L. nannotis populations. This suggests that dispersal ability and genetic diversity do not limit the ability of L. dayi to recolonize upland sites. Further, infection intensity and prevalence increased with elevation, suggesting that chytridiomycosis is still limiting the elevational range of L. dayi. Outlier tests comparing infected and uninfected individuals consistently identified 18 markers as putatively under selection, and several of those markers matched genes that were previously implicated in infection. This suggests that L. dayi has genetic variation for genes that affect infection dynamics and may be undergoing adaptation.

Tropical snake diversity collapses after widespread amphibian loss.

Biodiversity is declining at unprecedented rates worldwide. Yet cascading effects of biodiversity loss on other taxa are largely unknown because baseline data are often unavailable. We document the collapse of a Neotropical snake community after the invasive fungal pathogen Batrachochytrium dendrobatidis caused a chytridiomycosis epizootic leading to the catastrophic loss of amphibians, a food source for snakes. After mass mortality of amphibians, the snake community contained fewer species and was more homogeneous across the study site, with several species in poorer body condition, despite no other systematic changes in the environment. The demise of the snake community after amphibian loss demonstrates the repercussive and often unnoticed consequences of the biodiversity crisis and calls attention to the invisible declines of rare and data-deficient species.

Chytrid fungi and global amphibian declines.

Discovering that chytrid fungi cause chytridiomycosis in amphibians represented a paradigm shift in our understanding of how emerging infectious diseases contribute to global patterns of biodiversity loss. In this Review we describe how the use of multidisciplinary biological approaches has been essential to pinpointing the origins of amphibian-parasitizing chytrid fungi, including Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans, as well as to timing their emergence, tracking their cycles of expansion and identifying the core mechanisms that underpin their pathogenicity. We discuss the development of the experimental methods and bioinformatics toolkits that have provided a fuller understanding of batrachochytrid biology and informed policy and control measures.

Distribution of P1(D1) wart disease resistance in potato germplasm and GWAS identification of haplotype-specific SNP markers.

KEY MESSAGE: A Genome-Wide Association Study using 330 commercial potato varieties identified haplotype specific SNP markers associated with pathotype 1(D1) wart disease resistance. Synchytrium endobioticum is a soilborne obligate biotrophic fungus responsible for wart disease. Growing resistant varieties is the most effective way to manage the disease. This paper addresses the challenge to apply molecular markers in potato breeding. Although markers linked to Sen1 were published before, the identification of haplotype-specific single-nucleotide polymorphisms may result in marker assays with high diagnostic value. To identify hs-SNP markers, we performed a genome-wide association study (GWAS) in a panel of 330 potato varieties representative of the commercial potato gene pool. SNP markers significantly associated with pathotype 1 resistance were identified on chromosome 11, at the position of the previously identified Sen1 locus. Haplotype specificity of the SNP markers was examined through the analysis of false positives and false negatives and validated in two independent full-sib populations. This paper illustrates why it is not always feasible to design markers without false positives and false negatives for marker-assisted selection. In the case of Sen1, founders could not be traced because of a lack of identity by descent and because of the decay of linkage disequilibrium between Sen1 and flanking SNP markers. Sen1 appeared to be the main source of pathotype 1 resistance in potato varieties, but it does not explain all the resistance observed. Recombination and introgression breeding may have introduced new, albeit rare haplotypes involved in pathotype 1 resistance. The GWAS approach, in such case, is instrumental to identify SNPs with the best possible diagnostic value for marker-assisted breeding.

Hybridization Facilitates Adaptive Evolution in Two Major Fungal Pathogens.

Hybridization is increasingly recognized as an important force impacting adaptation and evolution in many lineages of fungi. During hybridization, divergent genomes and alleles are brought together into the same cell, potentiating adaptation by increasing genomic plasticity. Here, we review hybridization in fungi by focusing on two fungal pathogens of animals. Hybridization is common between the basidiomycete yeast species Cryptococcusneoformans × Cryptococcusdeneoformans, and hybrid genotypes are frequently found in both environmental and clinical settings. The two species show 10-15% nucleotide divergence at the genome level, and their hybrids are highly heterozygous. Though largely sterile and unable to mate, these hybrids can propagate asexually and generate diverse genotypes by nondisjunction, aberrant meiosis, mitotic recombination, and gene conversion. Under stress conditions, the rate of such genetic changes can increase, leading to rapid adaptation. Conversely, in hybrids formed between lineages of the chytridiomycete frog pathogen Batrachochytriumdendrobatidis (Bd), the parental genotypes are considerably less diverged (0.2% divergent). Bd hybrids are formed from crosses between lineages that rarely undergo sex. A common theme in both species is that hybrids show genome plasticity via aneuploidy or loss of heterozygosity and leverage these mechanisms as a rapid way to generate genotypic/phenotypic diversity. Some hybrids show greater fitness and survival in both virulence and virulence-associated phenotypes than parental lineages under certain conditions. These studies showcase how experimentation in model species such as Cryptococcus can be a powerful tool in elucidating the genotypic and phenotypic consequences of hybridization.

Host thermoregulatory constraints predict growth of an amphibian chytrid pathogen (Batrachochytrium dendrobatidis).

1. The course and outcome of many wildlife diseases are context-dependent, and therefore change depending on the behaviour of hosts and environmental response of the pathogen. 2. Contemporary declines in amphibian populations are widely attributed to chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis. Despite the thermal sensitivity of the pathogen and its amphibian hosts, we do not understand how host thermal regimes experienced by frogs in the wild directly influence pathogen growth. 3. We tested how thermal regimes experienced by the rainforest frog Litoria rheocola in the wild influence pathogen growth in the laboratory, and whether these responses differ from pathogen growth under available environmental thermal regimes. 4. Frog thermal regimes mimicked in the laboratory accelerated pathogen growth during conditions representative of winter at high elevations more so than if temperatures matched air or stream water temperatures. By contrast, winter frog thermal regimes at low elevations slowed pathogen growth relative to air temperatures, but not water temperatures. 5. The growth pattern of the fungus under frog thermal regimes matches field prevalence and intensity of infections for this species (high elevation winter > high elevation summer > low elevation winter > low elevation summer), whereas pathogen growth trajectories under environmental temperatures did not match these patterns. 6. If these laboratory results translate into field responses, tropical frogs may be able to reduce disease impacts by regulating their body temperatures to limit pathogen growth (e.g., by using microhabitats that facilitate basking to reach high temperatures); in other cases, the environment may limit the ability of frogs to thermoregulate such that individuals are more vulnerable to this pathogen (e.g., in dense forests at high elevations). 7. Species-specific thermoregulatory behaviour, and interactions with and constraints imposed by the environment, are therefore essential to understanding and predicting the spatial and temporal impacts of this global disease.

Batrachochytrium dendrobatidis infection in amphibians predates first known epizootic in Costa Rica.

Emerging infectious diseases are a growing threat to biodiversity worldwide. Outbreaks of the infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), are implicated in the decline and extinction of numerous amphibian species. In Costa Rica, a major decline event occurred in 1987, more than two decades before this pathogen was discovered. The loss of many species in Costa Rica is assumed to be due to Bd-epizootics, but there are few studies that provide data from amphibians in the time leading up to the proposed epizootics. In this study, we provide new data on Bd infection rates of amphibians collected throughout Costa Rica, in the decades prior to the epizootics. We used a quantitative PCR assay to test for Bd presence in 1016 anuran museum specimens collected throughout Costa Rica. The earliest specimen that tested positive for Bd was collected in 1964. Across all time periods, we found an overall infection rate (defined as the proportion of Bd-positive individuals) of 4%. The number of infected individuals remained relatively low across all species tested and the range of Bd-positive specimens was shown to be geographically constrained up until the 1980s; when epizootics are hypothesized to have occurred. After that time, infection rate increased three-fold, and the range of specimens tested positive for Bd increased, with Bd-positive specimens collected across the entire country. Our results suggest that Bd dynamics in Costa Rica are more complicated than previously thought. The discovery of Bd's presence in the country preceding massive declines leads to a number of different hypotheses: 1) Bd invaded Costa Rica earlier than previously known, and spread more slowly than previously reported; 2) Bd invaded multiple times and faded out; 3) an endemic Bd lineage existed; 4) an earlier Bd lineage evolved into the current Bd lineage or hybridized with an invasive lineage; or 5) an earlier Bd lineage went extinct and a new invasion event occurred causing epizootics. To help visualize areas where future studies should take place, we provide a Bd habitat suitability model trained with local data. Studies that provide information on genetic lineages of Bd are needed to determine the most plausible spatial-temporal, host-pathogen dynamics that could best explain the epizootics resulting in amphibian declines in Costa Rica and throughout Central America.

Widespread chytrid infection across frogs in the Peruvian Amazon suggests critical role for low elevation in pathogen spread and persistence.

Outbreaks of emerging infectious diseases are becoming more frequent as climate changes wildlife communities at unprecedented rates, driving population declines and raising concerns for species conservation. One critical disease is the global pandemic of chytridiomycosis in frogs, which can be caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Although there is clear evidence for Bd-induced mortality across high-elevation frog communities, little attention is given to the role of lowlands in Bd's persistence and spread because low elevations are assumed to be too warm to harbor significant levels of Bd. Here, we report widespread Bd infection across 80 frog species from three sites in the lowland Peruvian Amazon, an area with no documented Bd-related amphibian declines. Despite observing no clinical signs of infection in the field, we found that 24-46% of individuals were infected per site (up to ≈105,000 zoospore equivalents per frog) by three Bd strains from the global pandemic lineage (Bd-GPL). We also found collection site and seasonal effects to be only weak predictors of Bd prevalence and load, with lower elevation and drier habitats marginally decreasing both prevalence and load. We found no further effect of host phylogeny, ecotype, or body size. Our results showing high and widespread prevalence across a lowland tropical ecosystem contradict the expectations based on the global pattern of pathogenicity of Bd that is largely restricted to higher elevations and colder temperatures. These findings imply that the lowlands may play a critical role in the spread and persistence of Bd over time and space.

Skin-associated lactic acid bacteria from North American bullfrogs as potential control agents of Batrachochytrium dendrobatidis.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis and has been a key driver in the catastrophic decline of amphibians globally. While many strategies have been proposed to mitigate Bd outbreaks, few have been successful. In recent years, the use of probiotic formulations that protect an amphibian host by killing or inhibiting Bd have shown promise as an effective chytridiomycosis control strategy. The North American bullfrog (Lithobates catesbeianus) is a common carrier of Bd and harbours a diverse skin microbiota that includes lactic acid bacteria (LAB), a microbial group containing species classified as safe and conferring host benefits. We investigated beneficial/probiotic properties: anti-Bd activity, and adhesion and colonisation characteristics (hydrophobicity, biofilm formation and exopolysaccharide-EPS production) in two confirmed LAB (cLAB-Enterococcus gallinarum CRL 1826, Lactococcus garvieae CRL 1828) and 60 presumptive LAB (pLAB) [together named as LABs] isolated from bullfrog skin.We challenged LABs against eight genetically diverse Bd isolates and found that 32% of the LABs inhibited at least one Bd isolate with varying rates of inhibition. Thus, we established a score of sensitivity from highest (BdGPL AVS7) to lowest (BdGPL C2A) for the studied Bd isolates. We further reveal key factors underlying host adhesion and colonisation of LABs. Specifically, 90.3% of LABs exhibited hydrophilic properties that may promote adhesion to the cutaneous mucus, with the remaining isolates (9.7%) being hydrophobic in nature with a surface polarity compatible with colonisation of acidic, basic or both substrate types. We also found that 59.7% of LABs showed EPS synthesis and 66.1% produced biofilm at different levels: 21% weak, 29% moderate, and 16.1% strong. Together all these properties enhance colonisation of the host surface (mucus or epithelial cells) and may confer protective benefits against Bd through competitive exclusion. Correspondence analysis indicated that biofilm synthesis was LABs specific with high aggregating bacteria correlating with strong biofilm producers, and EPS producers being correlated to negative biofilm producing LABs. We performed Random Amplified Polymorphic DNA (RAPD)-PCR analysis and demonstrated a higher degree of genetic diversity among rod-shaped pLAB than cocci. Based on the LAB genetic analysis and specific probiotic selection criteria that involve beneficial properties, we sequenced 16 pLAB which were identified as Pediococcus pentosaceus, Enterococcus thailandicus, Lactobacillus pentosus/L. plantarum, L. brevis, and L. curvatus. Compatibility assays performed with cLAB and the 16 species described above indicate that all tested LAB can be included in a mixed probiotic formula. Based on our analyses, we suggest that E. gallinarum CRL 1826, L. garvieae CRL 1828, and P. pentosaceus 15 and 18B represent optimal probiotic candidates for Bd control and mitigation.

Shifts in temperature influence how Batrachochytrium dendrobatidis infects amphibian larvae.

Many climate change models predict increases in frequency and magnitude of temperature fluctuations that might impact how ectotherms are affected by disease. Shifts in temperature might especially affect amphibians, a group with populations that have been challenged by several pathogens. Because amphibian hosts invest more in immunity at warmer than cooler temperatures and parasites might acclimate to temperature shifts faster than hosts (creating lags in optimal host immunity), researchers have hypothesized that a temperature shift from cold-to-warm might result in increased amphibian sensitivity to pathogens, whereas a shift from warm-to-cold might result in decreased sensitivity. Support for components of this climate-variability based hypothesis have been provided by prior studies of the fungus Batrachochytrium dendrobatidis (Bd) that causes the disease chytridiomycosis in amphibians. We experimentally tested whether temperature shifts before exposure to Batrachochytrium dendrobatidis (Bd) alters susceptibility to the disease chytridiomycosis in the larval stage of two amphibian species-western toads (Anaxyrus boreas) and northern red legged frogs (Rana aurora). Both host species harbored elevated Bd infection intensities under constant cold (15° C) temperature in comparison to constant warm (20° C) temperature. Additionally, both species experienced an increase in Bd infection abundance after shifted from 15° C to 20° C, compared to a constant 20° C but they experienced a decrease in Bd after shifted from 20° C to 15° C, compared to a constant 15° C. These results are in contrast to prior studies of adult amphibians highlighting the potential for species and stage differences in the temperature-dependence of chytridiomycosis.

Ancestral chytrid pathogen remains hypervirulent following its long coevolution with amphibian hosts.

Many amphibian species around the world, except in Asia, suffer morbidity and mortality when infected by the emerging infectious pathogen Batrachochytrium dendrobatidis (Bd). A lineage of the amphibian chytrid fungus isolated from South Korean amphibians (BdAsia-1) is evolutionarily basal to recombinant global pandemic lineages (BdGPL) associated with worldwide amphibian population declines. In Asia, the Bd pathogen and its amphibian hosts have coevolved over 100 years or more. Thus, resilience of Asian amphibian populations to infection might result from attenuated virulence of endemic Bd lineages, evolved immunity to the pathogen or both. We compared susceptibilities of an Australasian amphibian, Litoria caerulea, known to lack resistance to BdGPL, with those of three Korean species, Bufo gargarizans, Bombina orientalis and Hyla japonica, after inoculation with BdAsia-1, BdGPL or a blank solution. Subjects became infected in all experimental treatments but Korean species rapidly cleared themselves of infection, regardless of Bd lineage. They survived with no apparent secondary effects. By contrast, L. caerulea, after infection by either BdAsia-1 or BdGPL, suffered deteriorating body condition and carried progressively higher Bd loads over time. Subsequently, most subjects died. Comparing their effects on L. caerulea, BdAsia-1 induced more rapid disease progression than BdGPL. The results suggest that genomic recombination with other lineages was not necessary for the ancestral Bd lineage to evolve hypervirulence over its long period of coevolution with amphibian hosts. The pathogen's virulence may have driven strong selection for immune responses in endemic Asian amphibian host species.

Treatment of Chytridiomycosis in Laboratory Axolotls (Ambystoma mexicanum) and Rough-skinned Newts (Taricha granulosa).

Chytridiomycosis is an infectious disease of amphibians caused by the fungal species Batrachochytrium dendrobatidis and B. salamandrivorans and has been implicated in the population decline of amphibian species worldwide. This case report describes a successful treatment protocol for chytridiomycosis in laboratory-maintained colonies of axolotls (Ambystoma mexicanum) and rough-skinned newts (Taricha granulosa). Over 12 mo, axolotls (n = 12) in a laboratory-reared colony developed multifocal erythematous dermatitis, mainly on the distal limbs and tails. Wild-caught newts handled by the same lab personnel were housed in an adjacent room and occasionally presented with abdominal distension and lethargy. Differentials included poor water quality, pathogen infection, parasitic infestation, and trauma. Antibiotic treatment of animals according to results of bacterial culture and sensitivity, combined with bleach disinfection of aquaria, did not resolve clinical signs. Skin swabs from clinically affected axolotls submitted for a newly available commercial screen were positive for B. dendrobatidis. Additional PCR and sequencing analysis revealed chytrid-positive animals among group-housed newts in 2 clinically unaffected aquaria and suspected PCR-positives for 2 affected newt aquaria and an additional axolotl. Axolotls with skin lesions (n = 2) and newts with abdominal distension and lethargy (n = 2) underwent experimental treatment with itraconazole submersion (0.002% to 0.0025%; 5 min daily for 10 d). This pilot treatment was well tolerated and led to clinical resolution. Subsequent itraconazole treatment of the entire colony led to regrowth of extremities and restoration of normal coloration among axolotls. During treatment, the facility was decontaminated, and additional biosecurity measures were developed. PCR results after the pilot treatment and subsequent full-colony treatments (at 1 wk, 1 mo, and 6 mo after treatment) were negative for the presence of B. dendrobatidis. Because chytridiomycosis is a reportable animal disease in our state, colonies officially remained quarantined until negative PCR results were obtained at least 6 mo after treatment.