Are novel or locally adapted pathogens more devastating and why? Resolving opposing hypotheses.

There is a rich literature highlighting that pathogens are generally better adapted to infect local than novel hosts, and a separate seemingly contradictory literature indicating that novel pathogens pose the greatest threat to biodiversity and public health. Here, using Batrachochytrium dendrobatidis, the fungus associated with worldwide amphibian declines, we test the hypothesis that there is enough variance in "novel" (quantified by geographic and phylogenetic distance) host-pathogen outcomes to pose substantial risk of pathogen introductions despite local adaptation being common. Our continental-scale common garden experiment and global-scale meta-analysis demonstrate that local amphibian-fungal interactions result in higher pathogen prevalence, pathogen growth, and host mortality, but novel interactions led to variable consequences with especially virulent host-pathogen combinations still occurring. Thus, while most pathogen introductions are benign, enough variance exists in novel host-pathogen outcomes that moving organisms around the planet greatly increases the chance of pathogen introductions causing profound harm.

Voriconazole successfully treats chytridiomycosis in frogs.

Chytridiomycosis is a devastating disease and is a key cause of amphibian population declines around the world. Despite active research on this amphibian disease system for over 2 decades, we still do not have treatment methods that are safe and that can be broadly used across species. Here, we show evidence that voriconazole is a successful method of treatment for 1 species of amphibian in captivity and that this treatment could offer benefits over other treatment options like heat or itraconazole, which are not able to be used for all species and life stages. We conducted 2 treatments of chytridiomycosis using voriconazole. The treatment was effective and resulted in 100% pathogen clearance, and mortality ceased. Additionally, treating frogs with voriconazole requires less handling than treatment methods like itraconazole and requires no specialized equipment, like heat treatment. We highlight that clinical treatment trials should be conducted to identify an optimum dosage and treatment time and that trials should test whether this treatment is safe and effective for tadpoles and other species.

Limited impact of chytridiomycosis on juvenile frogs in a recovered species.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused catastrophic frog declines on several continents, but disease outcome is mediated by a number of factors. Host life stage is an important consideration and many studies have highlighted the vulnerability of recently metamorphosed or juvenile frogs compared to adults. The majority of these studies have taken place in a laboratory setting, and there is a general paucity of longitudinal field studies investigating the influence of life stage on disease outcome. In this study, we assessed the effect of endemic Bd on juvenile Mixophyes fleayi (Fleay's barred frog) in subtropical eastern Australian rainforest. Using photographic mark-recapture, we made 386 captures of 116 individuals and investigated the effect of Bd infection intensity on the apparent mortality rates of frogs using a multievent model correcting for infection state misclassification. We found that neither Bd infection status nor infection intensity predicted mortality in juvenile frogs, counter to the expectation that early life stages are more vulnerable to disease, despite average high infection prevalence (0.35, 95% HDPI [0.14, 0.52]). Additionally, we found that observed infection prevalence and intensity were somewhat lower for juveniles than adults. Our results indicate that in this Bd-recovered species, the realized impacts of chytridiomycosis on juveniles were apparently low, likely resulting in high recruitment contributing to population stability. We highlight the importance of investigating factors relating to disease outcome in a field setting and make recommendations for future studies.

Comparison of methods for determining Batrachochytrium dendrobatidis zoospore viability.

The emerging fungal pathogen Batrachochytrium dendrobatidis (Bd) threatens hundreds of amphibian species globally. During laboratory-based experiments it is often essential to quantify live Bd cells, but a comparison of the effectiveness of methods for counting and assessing the viability of the infectious zoospore life stage has not been done. A direct comparison of staining methods that assess viability will ensure that the most accurate and efficient method is used. Here, we compared the use of 2 relatively cheap common stains, trypan blue and methylene blue, and assessed their accuracy and precision for estimating the viability of Bd zoospores during both manual counting and colorimetric assays. We stained known proportions of killed Bd zoospores (0, 0.25, 0.50, 0.75, and 1.00) with each stain and estimated the proportion of stained (dead) and unstained (viable) cells in each sample using both manual counting and colorimetric assays. Trypan blue was found to be a much more effective stain than methylene blue for both microscopy and colorimetric assays. Additionally, counting zoospores via microscopy was both a more accurate and precise technique. We recommend using manual counts via microscopy using the trypan blue stain for assessing Bd zoospore viability.

Sperm parameters following hormonal induction of spermiation in an endangered frog [the alpine tree frog] (Litoria verreauxii alpina).

CONTEXT: With global amphibian biodiversity rapidly declining, improving reproductive technology outcomes has become essential. Captive breeding programs have struggled because amphibian breeding physiology often requires specific environmental cues that reproductive technologies can circumvent. AIMS: This study tests the efficiency of hormonal induction by evaluating sperm quality in the endangered Litoria verreauxii alpina . METHODS: We assessed the effects of exogenous hormones - gonadotrophin-releasing hormone agonist (GnRH-a, Lucrin), and human chorionic gonadotrophin (hCG, Chorulon) - on sperm quality. KEY RESULTS: Hormone induction with hCG showed high efficacy while GnRH-a yielded a low response in producing sperm. Sperm quantity was affected by time post injection, with the greatest quantity at 1h post injection. Sperm quality was also affected by time, where the sperm head size decreased by 11% at 7h post injection. CONCLUSIONS: Based on the results from this study, we recommend that that sperm be collected soon after induction, and not more than 4h post induction in L. v. alpina . More work needs to be completed before recommending an optimal hormone induction method and dose, but 120IU of hCG per male was successful for inducing spermiation. IMPLICATIONS: This study represents a useful starting point for developing assisted reproductive techniques for non-model organisms.

Mechanisms underlying host persistence following amphibian disease emergence determine appropriate management strategies.

Emerging infectious diseases have caused many species declines, changes in communities and even extinctions. There are also many species that persist following devastating declines due to disease. The broad mechanisms that enable host persistence following declines include evolution of resistance or tolerance, changes in immunity and behaviour, compensatory recruitment, pathogen attenuation, environmental refugia, density-dependent transmission and changes in community composition. Here we examine the case of chytridiomycosis, the most important wildlife disease of the past century. We review the full breadth of mechanisms allowing host persistence, and synthesise research on host, pathogen, environmental and community factors driving persistence following chytridiomycosis-related declines and overview the current evidence and the information required to support each mechanism. We found that for most species the mechanisms facilitating persistence have not been identified. We illustrate how the mechanisms that drive long-term host population dynamics determine the most effective conservation management strategies. Therefore, understanding mechanisms of host persistence is important because many species continue to be threatened by disease, some of which will require intervention. The conceptual framework we describe is broadly applicable to other novel disease systems.

Susceptibility of frogs to chytridiomycosis correlates with increased levels of immunomodulatory serotonin in the skin.

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a skin disease responsible for the global decline of amphibians. Frog species and populations can vary in susceptibility, but this phenomenon remains poorly understood. Here, we investigated serotonin in the skin of infected and uninfected frogs. In more susceptible frog populations, skin serotonin rose with increasing infection intensity, but decreased in later stages of the disease. The more resistant population maintained a basal level of skin serotonin. Serotonin inhibited both Bd sporangial growth and Jurkat lymphocyte proliferation in vitro. However, serotonin accumulates in skin granular glands, and this compartmentalisation may prevent inhibition of Bd growth in vivo. We suggest that skin serotonin increases in susceptible frogs due to pathogen excretion of precursor tryptophan, but that resistant frogs are able to control the levels of serotonin. Overall, the immunosuppressive effects of serotonin may contribute to the susceptibility of frogs to chytridiomycosis.

Evaluating environmental DNA as a tool for detecting an amphibian pathogen using an optimized extraction method.

Environmental DNA (eDNA) detection is a valuable conservation tool that can be used to identify and monitor imperiled or invasive species and wildlife pathogens. Batrachochytrium pathogens are of global conservation concern because they are a leading cause of amphibian decline. While eDNA techniques have been used to detect Batrachochytrium DNA in the environment, a systematic comparison of extraction methods across environmental samples is lacking. In this study, we first compared eDNA extraction methods and found that a soil extraction kit (Qiagen PowerSoil) was the most effective for detecting Batrachochytrium dendrobatidis in water samples. The PowerSoil extraction had a minimum detection level of 100 zoospores and had a two- to four-fold higher detection probability than other commonly used extraction methods (e.g., QIAamp extraction, DNeasy+Qiashredder extraction method, respectively). Next, we used this extraction method on field-collected water and sediment samples and were able to detect pathogen DNA in both. While field-collected water filters were equivalent to amphibian skin swab samples in detecting the presence of pathogen DNA, the seasonal patterns in pathogen quantity were different between skin swabs and water samples. Detection rate was lowest in sediment samples. We also found that detection probability increases with the volume of water filtered. Our results indicate that water filter eDNA samples can be accurate in detecting pathogen presence at the habitat scale but their utility for quantifying pathogen loads in the environment appears limited. We suggest that eDNA techniques be used for early warning detection to guide animal sampling efforts.

Optimized Batrachochytrium dendrobatidis DNA extraction of swab samples results in imperfect detection particularly when infection intensities are low.

Accurate detection of the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) is critical for wildlife disease research; however, false negatives in detection do occur. Here we compared different DNA extraction methods to determine the threshold for Bd detection and identify an optimal extraction method to improve detection and quantification of the pathogen. We extracted both lab-created cell suspension standards using PrepMan Ultra, Chelex resin, and 3 spin column DNA extraction kits (Qiagen DNeasy Blood and Tissue, Zymo Quick DNA miniprep, and IBI gMAX mini kit), and further compared extraction methods using field-collected samples. We found that when extracting Bd DNA from cells in lab-created culture, the spin column extraction methods and PrepMan Ultra were equivalent, while the resin method detected higher Bd DNA quantities, especially at higher loads. However, when swabs from live animals were analyzed, low Bd quantities were more than twice as likely to be detected using a spin column extraction than with the PrepMan Ultra extraction method. All tested spin column extraction methods performed similarly across both field and lab samples. Samples containing low Bd quantities yielded inconsistent detection and quantification of Bd DNA copies regardless of extraction method. To manage imperfect detection of Bd, we suggest that presence/absence analyses are more informative than attempting to quantify Bd DNA when quantities are low. Overall, we recommend that a cost-benefit analysis of target species susceptibility and epidemiology be taken into consideration when designing an experiment to determine the most appropriate DNA extraction method to be used, because sometimes detecting low Bd quantities is imperative to the study, whereas in other situations, detecting low DNA quantities is less important.

The efficacy and pharmacokinetics of terbinafine against the frog-killing fungus (Batrachochytrium dendrobatidis).

Captive and wild amphibians are under threat of extinction from the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd). The antifungal drug terbinafine (TBF) is used by pet owners to treat Bd-infected frogs; however, it is not widely used in academic or zoological institutions due to limited veterinary clinical trials. To assess TBF's efficacy, we undertook treatment trials and pharmacokinetic studies to investigate drug absorption and persistence in frog skin; and then we correlated these data to the minimal lethal concentrations (MLC) against Bd. Despite an initial reduction in zoospore load, the recommended treatment (five daily 5 min 0.01% TBF baths) was unable to cure experimentally infected alpine tree frogs and naturally infected common eastern froglets. In vitro and in vivo pharmacokinetics showed that absorbed TBF accumulates in frog skin with increased exposure, indicating its suitability for treating cutaneous pathogens via direct application. The MLC of TBF for zoosporangia was 100 µg/ml for 2 h, while the minimal inhibitory concentration was 2 µg/ml, suggesting that the drug concentration absorbed during 5 min treatments is not sufficient to cure high Bd burdens. With longer treatments of five daily 30 min baths, Bd clearance improved from 12.5% to 50%. A higher dose of 0.02% TBF resulted in 78% of animals cured; however, clearance was not achieved in all individuals due to low TBF skin persistence, as the half-life was less than 2 h. Therefore, the current TBF regime is not recommended as a universal treatment against Bd until protocols are optimized, such as with increased exposure frequency.

Fungal infection has sublethal effects in a lowland subtropical amphibian population.

BACKGROUND: The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), has been implicated as a primary cause of decline in many species around the globe. However, there are some species and populations that are known to become infected in the wild, yet declines have not been observed. Here we conducted a yearlong capture-mark-recapture study and a 2-year long disease monitoring study of northern cricket frogs, Acris crepitans, in the lowland subtropical forests of Louisiana. RESULTS: We found little evidence for an impact of Bd infection on survival; however, Bd infection did appear to cause sublethal effects, including increased capture probability in the field. CONCLUSIONS: Our study suggests that even in apparently stable populations, where Bd does not appear to cause mortality, there may be sublethal effects of infection that can impact a host population's dynamics and structure. Understanding and documenting such sublethal effects of infection on wild, seemingly stable populations is important, particularly for predicting future population declines.

Age- and size-dependent resistance to chytridiomycosis in the invasive cane toad Rhinella marina.

In Australia, the cane toad Rhinella marina and chytrid fungus Batrachochytrium dendrobatidis (Bd) are examples of invasive species that have had dramatic impacts on native fauna. However, little is known about the interaction between Bd and cane toads. We aimed to explore the interaction of these 2 species in 3 parts. First, we collated data from the literature on Bd infection in wild cane toads. Second, we tested the susceptibility of recently metamorphosed cane toads to Bd infection. Finally, we modelled the distribution of the 2 species in Australia to identify where they overlap and, therefore, might interact. Through our data collation, we found that adult cane toads are infrequently infected and do not carry high infection burdens; however, our infection experiment showed that metamorphs are highly susceptible to infection and disease, but resistance appears to increase with increasing toad size. Niche modelling revealed overlapping distributions and the potential for cane toads to be affected by chytridiomycosis in the wild. While Bd can cause mortality in small juveniles in the laboratory, warm microhabitats used by wild toads likely prevent infection, and furthermore, high mortality of juveniles is unlikely to affect the adult populations because they are highly fecund. However, to demonstrate the impact of Bd on wild cane toad populations, targeted field studies are required to assess (1) the overall impact of chytridiomycosis on recruitment especially in cooler areas more favourable for Bd and (2) whether cane toad juveniles can amplify Bd exposure of native amphibian species in these areas.

Characterization of MHC class IA in the endangered southern corroboree frog.

Southern corroboree frogs (Pseudophryne corroboree) have declined to near extinction in the wild after the emergence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in southeastern Australia in the 1980s. A major captive breeding and reintroduction program is underway to preserve this iconic species, but improving resistance to B. dendrobatidis would help the wild population to be self-sustaining. Using 3' and 5' rapid amplification of complementary DNA ends (RACE), we characterized the major histocompatibility complex (MHC) class IA locus in this species. We then used sequences generated from RACE to design primers to amplify the peptide-binding region (PBR) of this functional genetic marker. Finally, we analysed the diversity, phylogeny, and selection patterns of PBR sequences from four P. corroboree populations and compared this with other amphibian species. We found moderately high MHC class IA genetic diversity in this species and evidence of strong positive and purifying selection at sites that are associated with putative PBR pockets in other species, indicating that this gene region may be under selection for resistance to Bd. Future studies should focus on identifying alleles associated with Bd resistance in P. corroboree by performing a Bd laboratory challenge study to confirm the functional importance of our genetic findings and explore their use in artificial selection or genetic engineering to increase resistance to chytridiomycosis.

Reservoir-host amplification of disease impact in an endangered amphibian.

Emerging wildlife pathogens are an increasing threat to biodiversity. One of the most serious wildlife diseases is chytridiomycosis, caused by the fungal pathogen, Batrachochytrium dendrobatidis (Bd), which has been documented in over 500 amphibian species. Amphibians vary greatly in their susceptibility to Bd; some species tolerate infection, whereas others experience rapid mortality. Reservoir hosts-species that carry infection while maintaining high abundance but are rarely killed by disease-can increase extinction risk in highly susceptible, sympatric species. However, whether reservoir hosts amplify Bd in declining amphibian species has not been examined. We investigated the role of reservoir hosts in the decline of the threatened northern corroboree frog (Pseudophryne pengilleyi) in an amphibian community in southeastern Australia. In the laboratory, we characterized the response of a potential reservoir host, the (nondeclining) common eastern froglet (Crinia signifera), to Bd infection. In the field, we conducted frog abundance surveys and Bd sampling for both P. pengilleyi and C. signifera. We built multinomial logistic regression models to test whether Crinia signifera and environmental factors were associated with P. pengilleyi decline. C. signifera was a reservoir host for Bd. In the laboratory, many individuals maintained intense infections (>1000 zoospore equivalents) over 12 weeks without mortality, and 79% of individuals sampled in the wild also carried infections. The presence of C. signifera at a site was strongly associated with increased Bd prevalence in sympatric P. pengilleyi. Consistent with disease amplification by a reservoir host, P. pengilleyi declined at sites with high C. signifera abundance. Our results suggest that when reservoir hosts are present, population declines of susceptible species may continue long after the initial emergence of Bd, highlighting an urgent need to assess extinction risk in remnant populations of other declined amphibian species.

Chytrid fungus Batrachochytrium dendrobatidis has nonamphibian hosts and releases chemicals that cause pathology in the absence of infection.

Batrachochytrium dendrobatidis, a pathogenic chytrid fungus implicated in worldwide amphibian declines, is considered an amphibian specialist. Identification of nonamphibian hosts could help explain the virulence, heterogeneous distribution, variable rates of spread, and persistence of B. dendrobatidis in freshwater ecosystems even after amphibian extirpations. Here, we test whether mosquitofish (Gambusia holbrooki) and crayfish (Procambarus spp. and Orconectes virilis), which are syntopic with many amphibian species, are possible hosts for B. dendrobatidis. Field surveys in Louisiana and Colorado revealed that zoosporangia occur within crayfish gastrointestinal tracts, that B. dendrobatidis prevalence in crayfish was up to 29%, and that crayfish presence in Colorado wetlands was a positive predictor of B. dendrobatidis infections in cooccurring amphibians. In experiments, crayfish, but not mosquitofish, became infected with B. dendrobatidis, maintained the infection for at least 12 wk, and transmitted B. dendrobatidis to amphibians. Exposure to water that previously held B. dendrobatidis also caused significant crayfish mortality and gill recession. These results indicate that there are nonamphibian hosts for B. dendrobatidis and suggest that B. dendrobatidis releases a chemical that can cause host pathology, even in the absence of infection. Managing these biological reservoirs for B. dendrobatidis and identifying this chemical might provide new hope for imperiled amphibians.

Susceptibility of amphibians to chytridiomycosis is associated with MHC class II conformation.

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal.

Batrachochytrium dendrobatidis in natural and farmed Louisiana crayfish populations: prevalence and implications.

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) has been linked to global declines and extinctions of amphibians, making it one of the most devastating wildlife pathogens known. Understanding the factors that affect disease dynamics in this system is critical for mitigating infection and protecting threatened species. Crayfish are hosts of this pathogen and can transmit Bd to amphibians. Because they co-occur with susceptible amphibian communities, crayfish may be important alternative hosts for Bd. Understanding the prevalence and seasonal dynamics of crayfish infections is of agricultural and ecological interest in areas where crayfish are farmed and traded for human consumption. We conducted a survey of Bd in farmed and natural crayfish (Procambarus spp.) populations in Louisiana, USA. We found that Bd prevalence and infection intensity was low in both farmed and native populations and that prevalence varied seasonally in wild Louisiana crayfish. This seasonal pattern mirrors that seen in local amphibians. As crayfish are an important globally traded freshwater taxon, even with low prevalence, they could be an important vector in the spread of Bd.

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.

Baseline haematological parameters in three common Australian frog species.

Amphibians are experiencing declines globally, with emerging infectious diseases as one of the main causes. Haematological parameters present a useful method for determining the health status of animals and the effects of particular diseases, but the interpretation of differential cell counts relies on knowing the normal ranges for the species and factors that can affect these counts. However, there is very little data on either normal haematological parameters or guides for blood cell types for free-ranging frog species across the world. This study aims to 1) create a visual guide for three different Australian frog species: Litoria paraewingi, Limnodynastes dumerilii, and Crinia signifera, 2) determine the proportions of erythrocytes to leukocytes and 3) differential leukocytes within blood smears from these three species and 4) assess the association between parasites and differential counts. We collected blood samples from free-ranging frogs and analysed blood smears. We also looked for ectoparasites and tested for the fungal disease chytridiomycosis. Overall, we found that the differentials of erythrocytes to leukocytes were not affected by species, but the proportions of different leukocytes did vary across species. For example, while lymphocytes were the most common type of leukocyte across the three species, eosinophils were relatively common in Limnodynastes dumerilii but rarely present in the other two species. We noted chytridiomycosis infection as well as ectoparasites present in some individuals but found no effect of parasites on blood parameters. Our results add baseline haematological parameters for three Australian frog species and provide an example of how different frog species can vary in their differential blood cell counts. More information is needed on frog haematological data before these parameters can be used to determine the health status of wild or captive frogs.

Advances in Managing Chytridiomycosis for Australian Frogs: Gradarius Firmus Victoria.

Extensive knowledge gains from research worldwide over the 25 years since the discovery of chytridiomycosis can be used for improved management. Strategies that have saved populations in the short term and/or enabled recovery include captive breeding, translocation into disease refugia, translocation from resistant populations, disease-free exclosures, and preservation of disease refuges with connectivity to previous habitat, while antifungal treatments have reduced mortality rates in the wild. Increasing host resistance is the goal of many strategies under development, including vaccination and targeted genetic interventions. Pathogen-directed strategies may be more challenging but would have broad applicability. While the search for the silver bullet solution continues, we should value targeted local interventions that stop extinction and buy time for evolution of resistance or development of novel solutions. As for most invasive species and infectious diseases, we need to accept that ongoing management is necessary. For species continuing to decline, proactive deployment and assessment of promising interventions are more valid than a hands-off, do-no-harm approach that will likely allow further extinctions.