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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., The pathogen Batrachochytrium dendrobatidis (BD) associated with widespread amphibian declines is present in Europe but has not consistently caused disease-induced declines in that region. Here, the authors suggest that an endemic strain of BD with low virulence may protect the hosts upon co-infection with more virulent strains.

The remoteness and isolation of South American tabletop mountain (tepui) summits may protect against infections that underpin global amphibian declines. Increases in recreational pressure in such unspoiled destinations, and in isolated ecosystems globally, pose a poorly understood risk of wildlife disease introduction, especially in supposedly immunologically naive communities. We here report the first observed chytrid infections in the Pantepui biogeographical region. Infections significantly correlate with proximity to the nearest basic tourist infrastructure in four endemic amphibians occurring on tepui summits and their slopes. Phylogenetic relationships and environmental context suggest a high risk of severe and irreversible population declines in unique, early branching amphibians. These findings advocate for an urgent control of recreational pressure in isolated, highly vulnerable ecosystems.

Significance Batrachochytrium dendrobatidis [Bd] is one of the most devastating wildlife pathogens ever documented. Most surveys for Bd report only the presence/absence of the pathogen. However, Bd has distinct genetic lineages that vary in geographic extent and virulence, thus reporting Bd presence alone is not particularly informative. Our study uses a custom method for genotyping degraded Bd DNA samples, such as those nondestructively collected from live animal or museum specimen skin swabs, and presents the discovery of a divergent lineage of Bd—BdASIA3. This study advances our understanding of the evolutionary origins of Bd, highlights areas of the world where Bd lineages are coming into contact, and opens the door to affordable, rapid genetic monitoring of this pathogen., Biodiversity loss is one major outcome of human-mediated ecosystem disturbance. One way that humans have triggered wildlife declines is by transporting disease-causing agents to remote areas of the world. Amphibians have been hit particularly hard by disease due in part to a globally distributed pathogenic chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Prior research has revealed important insights into the biology and distribution of Bd; however, there are still many outstanding questions in this system. Although we know that there are multiple divergent lineages of Bd that differ in pathogenicity, we know little about how these lineages are distributed around the world and where lineages may be coming into contact. Here, we implement a custom genotyping method for a global set of Bd samples. This method is optimized to amplify and sequence degraded DNA from noninvasive skin swab samples. We describe a divergent lineage of Bd, which we call BdASIA3, that appears to be widespread in Southeast Asia. This lineage co-occurs with the global panzootic lineage (BdGPL) in multiple localities. Additionally, we shed light on the global distribution of BdGPL and highlight the expanded range of another lineage, BdCAPE. Finally, we argue that more monitoring needs to take place where Bd lineages are coming into contact and where we know little about Bd lineage diversity. Monitoring need not use expensive or difficult field techniques but can use archived swab samples to further explore the history—and predict the future impacts—of this devastating pathogen.

The pandemic disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a major threat to amphibian biodiversity. For most species, the exact mechanisms of chytridiomycosis that lead to negative population dynamics remain uncertain, though mounting evidence suggests that sublethal effects could be an important driver. In this review, we propose that tree frog attachment is a promising case to study the sublethal effects of a Bd infection on amphibians. A synthesis of the current knowledge on the functional morphology of the adhesive toe pads of tree frogs, on the underlying mechanisms of tree frog attachment, and on the epidermal pathology of chytridiomycosis substantiates the hypothesis that Bd-induced epidermal alterations have the potential to disrupt tree frog attachment. We highlight a series of (biomechanical) experiments to test this hypothesis and to shed some light on the sublethal disease mechanisms of chytridiomycosis. The knowledge generated from such an approach could contribute to future research on Bd epidemiology and ultimately to the conservation of the biodiversity of arboreal anurans.

Emerging fungal pathogens pose a serious, global and growing threat to food supply systems, wild ecosystems, and human health. However, historic chronic underinvestment in their research has resulted in a limited understanding of their epidemiology relative to bacterial and viral pathogens. Therefore, the untargeted nature of genomics and, more widely, -omics approaches is particularly attractive in addressing the threats posed by and illuminating the biology of these pathogens. Typically, research into plant, human and wildlife mycoses have been largely separated, with limited dialogue between disciplines. However, many serious mycoses facing the world today have common traits irrespective of host species, such as plastic genomes; wide host ranges; large population sizes and an ability to persist outside the host. These commonalities mean that -omics approaches that have been productively applied in one sphere and may also provide important insights in others, where these approaches may have historically been underutilised. In this review, we consider the advances made with genomics approaches in the fields of plant pathology, human medicine and wildlife health and the progress made in linking genomes to other -omics datatypes and sets; we identify the current barriers to linking -omics approaches and how these are being underutilised in each field; and we consider how and which -omics methodologies it is most crucial to build capacity for in the near future.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide., S.J.O., T.W.J.G., L.Br., A.Lo., A.A.C., D.S.S., E.A.C., C.M., J.B., D.M.A., F.C., and M.C.F. were supported through NERC (standard grant NE/K014455/1). S.J.O. acknowledges a Microsoft Azure for Research Sponsorship (subscription ID: ab7cd695-49cf-4a83-910a-ef71603e708b). T.W.J.G., A.Lo., A.A.C., D.S.S., E.A.C., C.M., J.B., D.M.A., F.C., and M.C.F. were also supported by the EU BiodivERsA scheme (RACE, funded through NERC directed grant NE/G002193/1 and ANR08-Biodiversa-002-03) and NERC (standard grant NE/K012509/1). M.C.F., E.A.C., and C.M. acknowledge the Nouragues Travel Grant Program 2014. R.A.F. was supported by an MIT/Wellcome Trust Fellowship. T.W.J.G. was supported by the People’s Trust for Endangered Species and the Morris Animal Foundation (D12ZO002). J.M.G.S. and M.C.F. were supported by the Leverhulme Trust (RPG-2014-273) and the Morris Animal Foundation (D16ZO-022). F.B. was supported by the ERC (grant ERC 260801–Big_Idea). D.M.A. was funded by Wellcome Trust grant 099202. J.V. was supported by the Hungarian Scientific Research Fund (OTKA K77841) and Bolyai János Research Scholarship, Hungarian Academy of Sciences (BO/00579/14/8). D.J.G. was supported by the Conservation Leadership Programme (grant 0134010) with additional assistance from F. Gebresenbet, R. Kassahun, and S. P. Loader. C.S.-A. was supported by Fondecyt Nº11140902 and 1181758. T.M.D.-B. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland with assistance from M. Hirschfeld and the Budongo Conservation Field Station. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282). L.F.T. was supported by FAPESP (#2016/25358-3) and CNPq (#300896/2016-6). L.Be., L.F.S., and R.J.W. were supported by the Australian Research Council (FT100100375, DP120100811). A.A.C. was supported by a Royal Society Wolfson Research Merit award. J.H., A.La., and S.M. were funded by the Swedish Research Council Formas (grant no. 2013- 1389-26445-20). C.W. was funded by the National Research Foundation, South Africa. T.Y.J. and T.S.J. acknowledge NSF grant DEB-1601259. W.E.H. was funded by the NSERC Strategic and Discovery grant programs

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.

Amphibians face many threats leading to declines and extinctions, but the chytrid fungal skin pathogens Batrachochytriurn dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) have been identified as the causative factors leading to one of the greatest disease-driven losses of amphibian biodiversity worldwide. Infection may lead to different clinical outcomes, and lethal infections are commonly associated with unrestricted, exponential fungal growth in the amphibian epidermis. Mechanisms underpinning Bd and Bsal growth in the amphibian host are poorly understood. Here, we describe a quorum sensing mechanism that allows cell-to-cell communication by Bd and Bsal in order to regulate fungal densities and infection strategies. Addition of chytrid culture supernatant to chytrid cultures resulted in a concentration-dependent growth reduction and using dialysis, small metabolites were shown to be the causative factor. U-HPLC-MS/MS and in vitro growth tests identified the aromatic alcohol tryptophol as a key metabolite in regulating fungal growth. We determined tryptophol kinetics in both Bd and Bsal and confirmed the autostimulatory mode of action of this quorum sensing metabolite. Finally, we linked expression of genes that might be involved in tryptophol production, with in vitro and in vivo chytrid growth. Our results show that Bd and Bsal fungi use tryptophol to act as multicellular entities in order to regulate their growth.

Emerging fungal pathogens are a growing threat to global health, ecosystems, food security, and the world economy. Over the last century, environmental change and globalized transport, twinned with the increasing application of antifungal chemical drugs have led to increases in outbreaks of fungal diseases with sometimes catastrophic effects. In order to tackle contemporary epidemics and predemic threats, there is a pressing need for a unified approach in identification and monitoring of fungal pathogens. In this paper, we discuss current high throughput technologies, as well as new platforms capable of combining diverse data types to inform practical epidemiological strategies with a focus on emerging fungal pathogens of wildlife.

Symbiotic bacteria may dampen the impacts of infectious diseases on hosts by inhibiting pathogen growth. However, our understanding of the generality of pathogen inhibition by different bacterial taxa across pathogen genotypes and environmental conditions is limited. Bacterial inhibitory properties are of particular interest for the amphibian-killing fungal pathogens (Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans), for which probiotic applications as conservation strategies have been proposed. We quantified the inhibition strength of five putatively B. dendrobatidis-inhibitory bacteria isolated from woodland salamander skin against six Batrachochytrium genotypes at two temperatures (12 and 18°C). We selected six genotypes from across the Batrachochytrium phylogeny: B. salamandrivorans, B. dendrobatidis-Brazil and four genotypes of the B. dendrobatidis Global Panzootic Lineage (GPL1: JEL647, JEL404; GPL2: SRS810, JEL423). We performed 96-well plate challenge assays in a full factorial design. We detected a Batrachochytrium genotype by temperature interaction on bacterial inhibition score for all bacteria, indicating that bacteria vary in ability to inhibit Batrachochytrium depending on pathogen genotype and temperature. Acinetobacter rhizosphaerae moderately inhibited B. salamandrivorans at both temperatures (μ = 46–53%), but not any B. dendrobatidis genotypes. Chryseobacterium sp. inhibited three Batrachochytrium genotypes at both temperatures (μ = 5–71%). Pseudomonas sp. strain 1 inhibited all Batrachochytrium genotypes at 12°C and four Batrachochytrium genotypes at 18°C (μ = 5–100%). Pseudomonas sp. strain 2 and Stenotrophomonas sp. moderately to strongly inhibited all six Batrachochytrium genotypes at both temperatures (μ = 57–100%). All bacteria consistently inhibited B. salamandrivorans. Using cluster analysis of inhibition scores, we found that more closely related Batrachochytrium genotypes grouped together, suggesting that bacterial inhibition strength may be predictable based on Batrachochytrium relatedness. We conclude that bacterial inhibition capabilities change among bacterial strains, Batrachochytrium genotypes and temperatures. A comprehensive understanding of bacterial inhibitory function, across pathogen genotypes and temperatures, is needed to better predict the role of bacterial symbionts in amphibian disease ecology. For targeted conservation applications, we recommend using bacterial strains identified as strongly inhibitory as they are most likely to produce broad-spectrum antimicrobial agents at a range of temperatures.

Emerging diseases have been increasingly associated with population declines, with co-infections exhibiting many types of interactions. The chytrid fungus (Batrachochytrium dendrobatidis) and ranaviruses have extraordinarily broad host ranges, however co-infection dynamics have been largely overlooked. We investigated the pattern of co-occurrence of these two pathogens in an amphibian assemblage in Serra da Estrela (Portugal). The detection of chytridiomycosis in Portugal was linked to population declines of midwife-toads (Alytes obstetricans). The asynchronous and subsequent emergence of a second pathogen - ranavirus - caused episodes of lethal ranavirosis. Chytrid effects were limited to high altitudes and a single host, while ranavirus was highly pathogenic across multiple hosts, life-stages and altitudinal range. This new strain (Portuguese newt and toad ranavirus - member of the CMTV clade) caused annual mass die-offs, similar in host range and rapidity of declines to other locations in Iberia affected by CMTV-like ranaviruses. However, ranavirus was not always associated with disease, mortality and declines, contrasting with previous reports on Iberian CMTV-like ranavirosis. We found little evidence that pre-existing chytrid emergence was associated with ranavirus and the emergence of ranavirosis. Despite the lack of cumulative or amplified effects, ranavirus drove declines of host assemblages and changed host community composition and structure, posing a grave threat to all amphibian populations., JB was supported by Spanish Ministry of Economy and Competitiveness grant CGL2015-70070-R.

Aquatic chytrid fungi threaten amphibian biodiversity worldwide owing to their ability to rapidly expand their geographical distributions and to infect a wide range of hosts. Combating this risk requires an understanding of chytrid host range to identify potential reservoirs of infection and to safeguard uninfected regions through enhanced biosecurity. Here we extend our knowledge on the host range of the chytrid Batrachochytrium dendrobatidis by demonstrating infection of a non-amphibian vertebrate host, the zebrafish. We observe dose-dependent mortality and show that chytrid can infect and proliferate on zebrafish tissue. We also show that infection phenotypes (fin erosion, cell apoptosis and muscle degeneration) are direct symptoms of infection. Successful infection is dependent on disrupting the zebrafish microbiome, highlighting that, as is widely found in amphibians, commensal bacteria confer protection against this pathogen. Collectively, our findings greatly expand the limited tool kit available to study pathogenesis and host response to chytrid infection., New infections of the amphibian chytrid fungus could arise from other animal reservoirs in the environment. Here, Liew et al. demonstrate that zebrafish can be infected by chytrid similarly to amphibians, expanding our understanding of how this pathogen can parasitize its hosts.

All multicellular organisms are host to microbial symbionts that constitute the microbiome and can have significant impacts on the host, including altering development, behavior, and health [1]. In turn, aspects of the host and their environment can influence the microbiome [2]. Here, we briefly summarize current knowledge of the amphibian skin microbiome and its role in heath and disease. Given the increase in fungal diseases that now threaten amphibians and other wildlife—including bees, bats, snakes, and corals, as well as a variety of economically important crops [3]—we hope that lessons learned from amphibian host–microbe interactions can also ultimately be applied in other systems (Fig 1). Funding was provided by the National Science Foundation (DEB-1136640). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

International audience; Health monitoring is a crucial aspect of the management of any research animal house. RESAMA is a network strong of 60 academic and private partners acting in France since the end of 2012. The network aims to increase awareness of animal caretakers and researchers on health management issues in facilities holding aquatic model species (zebrafish, Xenopus, medaka, Mexican tetra). To do so, each partner research facility will be visited at least once. The visiting team is composed at least of one veterinarian and one zootechnician specialized in aquatic species. The visit results in a health-monitoring assessment of the facility, which includes a sampling for histo-pathological, bacteriological, and molecular pathogen detection. During the visit, rearing practices are also reviewed through an interview of animal caretakers. However, the present report essentially focuses on the health-monitoring aspect. The ultimate goal of the project is to provide a network-wide picture of health issues in aquatic facilities. Performed in parallel, the rearing practice assessment will ultimately help to establish rational relationship between handling practices and animal health in aquatic facilities. The study is still in progress. Here, we describe the results to be drawn from an analysis of the 23 facilities that had been visited so far. We sampled 720 fish and 127 amphibians and performed a little less than 1400 individual tests.

Epidemiological studies have suggested that increasing connectivity in metapopulations usually facilitates pathogen transmission. However, these studies focusing on single-host systems usually neglect that increasing connectivity can increase species diversity which might reduce pathogen transmission via the ‘dilution effect’, a hypothesis whose generality is still disputed. On the other hand, studies investigating the generality of the dilution effect were usually conducted without considering habitat structure, which is surprising as species loss is often driven by habitat fragmentation. Using a simple general model to link fragmentation to the dilution effect, we determined the effect of connectivity on disease risk and explored when the dilution effect can be detected. We showed that landscape structure can largely modify the diversity–disease relationship. The net impact of connectivity on disease risk can be either positive or negative, depending on the relative importance of the facilitation effect (through increasing contact rates among patches) versus the dilution effect (via increasing species richness). We also demonstrated that different risk indices (i.e. infection prevalence and abundance of infected hosts) react differently to increasing connectivity and species richness. Our study may contribute to the current debate on the dilution effect, and a better understanding of the impacts of fragmentation on disease risks.

The amphibian chytrid fungus, Batrachochytrium dendrobatidis, is one of the main factors in global amphibian decline. Accurate knowledge of its presence and prevalence in an area is needed to trigger conservation actions. However, imperfect capture rates determine the number of individuals caught and tested during field surveys, and contribute to the uncertainty surrounding estimates of prevalence. Screening programs should be planned with the objective of minimizing such uncertainty. We show how this can be achieved by using predictive models that incorporate information about population size and capture rates. Using as a case study an existing screening program for three populations of the yellow-bellied toad (Bombina variegata pachypus) in northern Italy, we sought to quantify the effect of seasonal variation in individual capture rates on the uncertainty surrounding estimates of chytrid prevalence. We obtained estimates of population size and capture rates from mark-recapture data, and found wide seasonal variation in the individual recapture rates. We then incorporated this information in a binomial model to predict the estimates of prevalence that would be obtained by sampling at different times in the season, assuming no infected individuals were found. Sampling during the period of maximum capture probability was predicted to decrease upper 95% credible intervals by a maximum of 36%, compared with least suitable periods, with greater gains when using uninformative priors. We evaluated model predictions by comparing them with the results of screening surveys in 2012. The observed results closely matched the predicted figures for all populations, suggesting that this method can be reliably used to maximize the sampling size of surveillance programs, thus improving their efficiency.

The Apennine Yellow-bellied toad Bombina variegata pachypus, a small anuran endemic to peninsular Italy, has been declining throughout its range over the last 30 years. Although mortality by chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis, was first reported for the species in 2004, its role in the decline has not yet been assessed. Between 2011 and 2012 we sampled eight populations of B. v. pachypus in Liguria, northern Italy, swabbing 86 and 143 individuals respectively, corresponding to between 24 and 80% of the estimated individuals within each population. We did not detect chytrid in any the samples collected. For the three largest populations in the region, we can rule out infections of prevalence greater than 10% with at least 98% confidence. Research at a larger scale is urgently needed to clarify the role of B. dendrobatidis in the decline of this and other amphibians in Italy., Acta Herpetologica, Vol 8 No 1 (2013)

The skin secretion of many amphibians contains an arsenal of bioactive molecules, including hormone-like peptides (HLPs) acting as defense toxins against predators, and antimicrobial peptides (AMPs) providing protection against infectious microorganisms. Several amphibian taxa seem to have independently acquired the genes to produce skin-secreted peptide arsenals, but it remains unknown how these originated from a non-defensive ancestral gene and evolved diverse defense functions against predators and pathogens. We conducted transcriptome, genome, peptidome and phylogenetic analyses to chart the full gene repertoire underlying the defense peptide arsenal of the frog Silurana tropicalis and reconstruct its evolutionary history. Our study uncovers a cluster of 13 transcriptionally active genes, together encoding up to 19 peptides, including diverse HLP homologues and AMPs. This gene cluster arose from a duplicated gastrointestinal hormone gene that attained a HLP-like defense function after major remodeling of its promoter region. Instead, new defense functions, including antimicrobial activity, arose by mutation of the precursor proteins, resulting in the proteolytic processing of secondary peptides alongside the original ones. Although gene duplication did not trigger functional innovation, it may have subsequently facilitated the convergent loss of the original function in multiple gene lineages (subfunctionalization), completing their transformation from HLP gene to AMP gene. The processing of multiple peptides from a single precursor entails a mechanism through which peptide-encoding genes may establish new functions without the need for gene duplication to avoid adaptive conflicts with older ones., Author Summary Many amphibians defend themselves against predation and infections by secreting a mixture of gene-encoded toxins and antimicrobials. How does such an integrated defense weapon arise and how does it diversify to gain distinct antipredatory and antimicrobial functions? We took advantage of the availability of a sequenced genome for the African clawed frog Silurana tropicalis to provide the first comprehensive overview of an amphibian peptide defense arsenal, from its underlying genes to its bioactive components. A reconstruction of the evolutionary history of this gene repertoire allows us to elucidate the timing and mode of evolution of distinct defense functions. Our study shows that the basal transition from a gastrointestinal hormone function to a skin-secretory defense function was accompanied by major restructuring of regulatory sequences in the ancestral gene. Instead, subsequently diversifying defense genes underwent functional shifts by entering a bifunctional stage (by cleavage of two distinct defense peptides from a single precursor protein) and occasionally losing the original defense function (by loss of the original defense peptide). This pattern provides an evolutionary explanation for the processing of structurally or functionally unrelated toxins from the same or closely related precursor proteins in other poisonous and venomous animals.

The Andes have experienced an unprecedented wave of amphibian declines and extinctions that are linked to a combination of habitat reduction and the spread of the fungal pathogen, Batrachochytrium dendrobatidis (Bd). In the present study, a range of high-altitude habitats in Southern Ecuador were surveyed for the presence of Bd. With a particular focus on Yacuri National Park, infection data are presented from across the resident amphibian community. This community contains a once putatively extinct species which was rediscovered in 2016, the Podocarpus Stubfoot Toad (Atelopus podocarpus). Across species, local Bd prevalence was 73% in tadpoles (n = 41 individuals from three species) and 14% in adults (n = 43 individuals from 14 species). Strikingly, 93% (14/15) of tested tadpoles of the recently described local endemic, Gastrotheca yacuri, were infected with a high pathogen load, suggesting that this species likely acts as a reservoir of infection in Yacuri. These fndings show that the threat of disease for A. podocarpus still exists, and that this species requires urgent action to ensure its survival.

BackgroundThe establishment of safe and effective protocols to treat chytridiomycosis in amphibians is urgently required. In this study, the usefulness of antibacterial agents to clear chytridiomycosis from infected amphibians was evaluated.ResultsFlorfenicol, sulfamethoxazole, sulfadiazine and the combination of trimethoprim and sulfonamides were activein vitroagainst cultures of fiveBatrachochytrium dendrobatidisstrains containing sporangia and zoospores, with minimum inhibitory concentrations (MIC) of 0.5-1.0 μg/ml for florfenicol and 8.0 μg/ml for the sulfonamides. Trimethoprim was not capable of inhibiting growth but, combined with sulfonamides, reduced the time to visible growth inhibition by the sulfonamides. Growth inhibition ofB. dendrobatidiswas not observed after exposure to clindamycin, doxycycline, enrofloxacin, paromomycin, polymyxin E and tylosin. Cultures of sporangia and zoospores ofB. dendrobatidisstrains JEL423 and IA042 were killed completely after 14 days of exposure to 100 μg/ml florfenicol or 16 μg/ml trimethoprim combined with 80 μg/ml sulfadiazine. These concentrations were, however, not capable of efficiently killing zoospores within 4 days after exposure as assessed using flow cytometry. Florfenicol concentrations remained stable in a bathing solution during a ten day period. Exposure ofDiscoglossus scovazzitadpoles for ten days to 100 μg/ml but not to 10 μg florfenicol /ml water resulted in toxicity. In anin vivotrial, post metamorphicAlytes muletensis, experimentally inoculated withB. dendrobatidis,were treated topically with a solution containing 10 μg/ml of florfenicol during 14 days. Although a significant reduction of theB. dendrobatidisload was obtained, none of the treated animals cleared the infection.ConclusionsWe thus conclude that, despite marked antiB. dendrobatidisactivityin vitro, the florfenicol treatment used is not capable of eliminatingB. dendrobatidisinfections from amphibians.