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1.  Interacting Symbionts and Immunity in the Amphibian Skin Mucosome Predict Disease Risk and Probiotic Effectiveness 
PLoS ONE  2014;9(4):e96375.
Pathogenesis is strongly dependent on microbial context, but development of probiotic therapies has neglected the impact of ecological interactions. Dynamics among microbial communities, host immune responses, and environmental conditions may alter the effect of probiotics in human and veterinary medicine, agriculture and aquaculture, and the proposed treatment of emerging wildlife and zoonotic diseases such as those occurring on amphibians or vectored by mosquitoes. Here we use a holistic measure of amphibian mucosal defenses to test the effects of probiotic treatments and to assess disease risk under different ecological contexts. We developed a non-invasive assay for antifungal function of the skin mucosal ecosystem (mucosome function) integrating host immune factors and the microbial community as an alternative to pathogen exposure experiments. From approximately 8500 amphibians sampled across Europe, we compared field infection prevalence with mucosome function against the emerging fungal pathogen Batrachochytrium dendrobatidis. Four species were tested with laboratory exposure experiments, and a highly susceptible species, Alytes obstetricans, was treated with a variety of temperature and microbial conditions to test the effects of probiotic therapies and environmental conditions on mucosome function. We found that antifungal function of the amphibian skin mucosome predicts the prevalence of infection with the fungal pathogen in natural populations, and is linked to survival in laboratory exposure experiments. When altered by probiotic therapy, the mucosome increased antifungal capacity, while previous exposure to the pathogen was suppressive. In culture, antifungal properties of probiotics depended strongly on immunological and environmental context including temperature, competition, and pathogen presence. Functional changes in microbiota with shifts in temperature provide an alternative mechanistic explanation for patterns of disease susceptibility related to climate beyond direct impact on host or pathogen. This nonlethal management tool can be used to optimize and quickly assess the relative benefits of probiotic therapies under different climatic, microbial, or host conditions.
doi:10.1371/journal.pone.0096375
PMCID: PMC4005770  PMID: 24789229
2.  Microbial community dynamics and effect of environmental microbial reservoirs on red-backed salamanders (Plethodon cinereus) 
The ISME Journal  2013;8(4):830-840.
Beneficial cutaneous bacteria on amphibians can protect against the lethal disease chytridiomycosis, which has devastated many amphibian species and is caused by the fungus Batrachochytrium dendrobatidis. We describe the diversity of bacteria on red-backed salamanders (Plethodon cinereus) in the wild and the stability of these communities through time in captivity using culture-independent Illumina 16S rRNA gene sequencing. After field sampling, salamanders were housed with soil from the field or sterile media. The captive conditions led to different trajectories of bacterial communities. Eight OTUs present on >90% of salamanders in the field, through time, and in both treatments were defined as the core community, suggesting that some bacteria are closely associated with the host and are independent of an environmental reservoir. One of these taxa, a Pseudomonas sp., was previously cultured from amphibians and found to be antifungal. As all host-associated bacteria were found in the soil reservoir, environmental microbes strongly influence host–microbial diversity and likely regulate the core community. Using PICRUSt, an exploratory bioinformatics tool to predict gene functions, we found that core skin bacteria provided similar gene functions to the entire community. We suggest that future experiments focus on testing whether core bacteria on salamander skin contribute to the observed resistance to chytridiomycosis in this species even under hygenic captive conditions. For disease-susceptible hosts, providing an environmental reservoir with defensive bacteria in captive-rearing programs may improve outcomes by increasing bacterial diversity on threatened amphibians or increasing the likelihood that defensive bacteria are available for colonization.
doi:10.1038/ismej.2013.200
PMCID: PMC3960541  PMID: 24335825
amphibians; bacterial reservoirs; Batrachochytrium dendrobatidis; community dynamics; host–bacteria interactions; symbiosis
3.  Stability of Microbiota Facilitated by Host Immune Regulation: Informing Probiotic Strategies to Manage Amphibian Disease 
PLoS ONE  2014;9(1):e87101.
Microbial communities can augment host immune responses and probiotic therapies are under development to prevent or treat diseases of humans, crops, livestock, and wildlife including an emerging fungal disease of amphibians, chytridiomycosis. However, little is known about the stability of host-associated microbiota, or how the microbiota is structured by innate immune factors including antimicrobial peptides (AMPs) abundant in the skin secretions of many amphibians. Thus, conservation medicine including therapies targeting the skin will benefit from investigations of amphibian microbial ecology that provide a model for vertebrate host-symbiont interactions on mucosal surfaces. Here, we tested whether the cutaneous microbiota of Panamanian rocket frogs, Colostethus panamansis, was resistant to colonization or altered by treatment. Under semi-natural outdoor mesocosm conditions in Panama, we exposed frogs to one of three treatments including: (1) probiotic - the potentially beneficial bacterium Lysinibacillus fusiformis, (2) transplant – skin washes from the chytridiomycosis-resistant glass frog Espadarana prosoblepon, and (3) control – sterile water. Microbial assemblages were analyzed by a culture-independent T-RFLP analysis. We found that skin microbiota of C. panamansis was resistant to colonization and did not differ among treatments, but shifted through time in the mesocosms. We describe regulation of host AMPs that may function to maintain microbial community stability. Colonization resistance was metabolically costly and microbe-treated frogs lost 7–12% of body mass. The discovery of strong colonization resistance of skin microbiota suggests a well-regulated, rather than dynamic, host-symbiont relationship, and suggests that probiotic therapies aiming to enhance host immunity may require an approach that circumvents host mechanisms maintaining equilibrium in microbial communities.
doi:10.1371/journal.pone.0087101
PMCID: PMC3906108  PMID: 24489847
4.  Chromosomal Copy Number Variation, Selection and Uneven Rates of Recombination Reveal Cryptic Genome Diversity Linked to Pathogenicity 
PLoS Genetics  2013;9(8):e1003703.
Pathogenic fungi constitute a growing threat to both plant and animal species on a global scale. Despite a clonal mode of reproduction dominating the population genetic structure of many fungi, putatively asexual species are known to adapt rapidly when confronted by efforts to control their growth and transmission. However, the mechanisms by which adaptive diversity is generated across a clonal background are often poorly understood. We sequenced a global panel of the emergent amphibian pathogen, Batrachochytrium dendrobatidis (Bd), to high depth and characterized rapidly changing features of its genome that we believe hold the key to the worldwide success of this organism. Our analyses show three processes that contribute to the generation of de novo diversity. Firstly, we show that the majority of wild isolates manifest chromosomal copy number variation that changes over short timescales. Secondly, we show that cryptic recombination occurs within all lineages of Bd, leading to large regions of the genome being in linkage equilibrium, and is preferentially associated with classes of genes of known importance for virulence in other pathosystems. Finally, we show that these classes of genes are under directional selection, and that this has predominantly targeted the Global Panzootic Lineage (BdGPL). Our analyses show that Bd manifests an unusually dynamic genome that may have been shaped by its association with the amphibian host. The rates of variation that we document likely explain the high levels of phenotypic variability that have been reported for Bd, and suggests that the dynamic genome of this pathogen has contributed to its success across multiple biomes and host-species.
Author Summary
Pathogenic fungi constitute a growing threat to both plant and animal species on a global scale. However, many features of the fungal genome that enable them to successfully adapt to infect diverse hosts and ecological niches remain cryptic, especially for newly evolved emerging lineages. In this paper, we report three novel features of genome diversity linked to pathogenicity in the emerging amphibian pathogen, Batrachochytrium dendrobatidis (Bd). Firstly, we identified widespread chromosome copy number variation (CCNV) across our lineages, with individual isolates harboring between 2 to 5 copies of each chromosome and rapid rates of CCNV occurring in culture. In addition, by using in vitro divergence of replicate lines of Bd, we showed that changes in ploidy can occur within as few as 40 generations. Secondly, we identified uneven rates of recombination across the genomes and lineages, revealing hot spots in known classes of virulence factors. Finally we identified significant evidence of diversifying selection across the secretome of Bd, and showed that selection also targets putative virulence factors. These findings add to our knowledge of genome-dynamicity and modes of evolution manifested by eukaryote microbial pathogens, and may explain the varied phenotypic responses observed in Bd.
doi:10.1371/journal.pgen.1003703
PMCID: PMC3744429  PMID: 23966879
5.  Tolerance of fungal infection in European water frogs exposed to Batrachochytrium dendrobatidis after experimental reduction of innate immune defenses 
Background
While emerging diseases are affecting many populations of amphibians, some populations are resistant. Determining the relative contributions of factors influencing disease resistance is critical for effective conservation management. Innate immune defenses in amphibian skin are vital host factors against a number of emerging pathogens such as ranaviruses and the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). Adult water frogs from Switzerland (Pelophylax esculentus and P. lessonae) collected in the field with their natural microbiota intact were exposed to Bd after experimental reduction of microbiota, skin peptides, both, or neither to determine the relative contributions of these defenses.
Results
Naturally-acquired Bd infections were detected in 10/51 P. lessonae and 4/19 P. esculentus, but no disease outbreaks or population declines have been detected at this site. Thus, this population was immunologically primed, and disease resistant. No mortality occurred during the 64 day experiment. Forty percent of initially uninfected frogs became sub-clinically infected upon experimental exposure to Bd. Reduction of both skin peptide and microbiota immune defenses caused frogs to gain less mass when exposed to Bd than frogs in other treatments. Microbiota-reduced frogs increased peptide production upon Bd infection. Ranavirus was undetectable in all but two frogs that appeared healthy in the field, but died within a week under laboratory conditions. Virus was detectable in both toe-clips and internal organs.
Conclusion
Intact skin microbiota reduced immune activation and can minimize subclinical costs of infection. Tolerance of Bd or ranavirus infection may differ with ecological conditions.
doi:10.1186/1746-6148-8-197
PMCID: PMC3485127  PMID: 23088169
Amphibian; Antimicrobial peptide; Chytridiomycosis; MALDI-MS; Microbiota; Pelophylax; Ranavirus
6.  Mitigating amphibian disease: strategies to maintain wild populations and control chytridiomycosis 
Background
Rescuing amphibian diversity is an achievable conservation challenge. Disease mitigation is one essential component of population management. Here we assess existing disease mitigation strategies, some in early experimental stages, which focus on the globally emerging chytrid fungus Batrachochytrium dendrobatidis. We discuss the precedent for each strategy in systems ranging from agriculture to human medicine, and the outlook for each strategy in terms of research needs and long-term potential.
Results
We find that the effects of exposure to Batrachochytrium dendrobatidis occur on a spectrum from transient commensal to lethal pathogen. Management priorities are divided between (1) halting pathogen spread and developing survival assurance colonies, and (2) prophylactic or remedial disease treatment. Epidemiological models of chytridiomycosis suggest that mitigation strategies can control disease without eliminating the pathogen. Ecological ethics guide wildlife disease research, but several ethical questions remain for managing disease in the field.
Conclusions
Because sustainable conservation of amphibians in nature is dependent on long-term population persistence and co-evolution with potentially lethal pathogens, we suggest that disease mitigation not focus exclusively on the elimination or containment of the pathogen, or on the captive breeding of amphibian hosts. Rather, successful disease mitigation must be context specific with epidemiologically informed strategies to manage already infected populations by decreasing pathogenicity and host susceptibility. We propose population level treatments based on three steps: first, identify mechanisms of disease suppression; second, parameterize epizootiological models of disease and population dynamics for testing under semi-natural conditions; and third, begin a process of adaptive management in field trials with natural populations.
doi:10.1186/1742-9994-8-8
PMCID: PMC3098159  PMID: 21496358
7.  Immune Defenses against Batrachochytrium dendrobatidis, a Fungus Linked to Global Amphibian Declines, in the South African Clawed Frog, Xenopus laevis▿  
Infection and Immunity  2010;78(9):3981-3992.
Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections.
doi:10.1128/IAI.00402-10
PMCID: PMC2937463  PMID: 20584973
8.  Variations in the expressed antimicrobial peptide repertoire of northern leopard frog (Rana pipiens) populations suggest intraspecies differences in resistance to pathogens 
The northern leopard frog (Rana pipiens or Lithobates pipiens) is historically found in most of the provinces of Canada and the northern and southwest states of the United States. In the last 50 years, populations have suffered significant losses, especially in the western regions of the species range. Using a peptidomics approach, we show that the pattern of expressed antimicrobial skin peptides of frogs from three geographically separated populations are distinct, and we report the presence of four peptides (brevinin-1Pg, brevinin-1Pl, ranatuerin-2Pb, and ranatuerin-2Pc) that have not previously been found in skin secretions. The differences in expressed peptides reflect differences in the distribution of alleles for the newly described Brevinin1.1 locus in the three populations. When enriched peptide mixtures were tested for their ability to inhibit growth of the pathogenic amphibian chytrid (Batrachochytrium dendrobatidis), peptides from Minnesota or Vermont frogs were more effective that peptides from Michigan frogs. Four of the purified peptides were tested for their ability to inhibit growth of two bacterial pathogens (Aeromonas hydrophila and Staphylococcus epidermidis) and B. dendrobatidis. Three of the four were effective inhibitors of B. dendrobatidis and S. epidermidis, but none inhibited A. hydrophila. We interpret these differences in expression and activity of antimicrobial peptides as evidence to suggest that each population may have been selected to express a suite of peptides that reflects current and past encounters with skin microbes.
doi:10.1016/j.dci.2009.07.004
PMCID: PMC2927990  PMID: 19622371
Antimicrobial peptides; Aeromonas hydrophila; Batrachochytrium dendrobatidis; Brevinin-1; Lithobates; Mass spectrometry; Peptidomics; Rana palustris; Rana pipiens; Ranatuerin-2; Staphylococcus epidermidis; Temporin

Results 1-8 (8)