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Agricultural practices have a profound impact on watershed dynamics, water quality, and the well-being of aquatic life. One major concern is agricultural pollution, particularly the excess of nutrients, which can elevate disease risks in various host-pathogen relationships. However, the exact mechanisms driving this effect remain uncertain. Elevated nutrient levels are believed to significantly influence populations of aquatic environmental bacteria, potentially reshaping the microbiomes of aquatic organisms and affecting their vulnerability to disease. Despite this, the impact of nutrient enrichment on host microbiomes as a link to diseases in aquatic organisms has been largely overlooked. In this study, we investigated the impact of nutrient enrichment on the skin-associated microbial communities of the American bullfrogLithobates catesbeianus. We observed a significant shift in bacterial richness and community composition in nutrient-enriched ponds compared with reference ponds. Although the proportion of the community inhibitory towardsBatrachochytrium dendrobatidis(Bd) did not change significantly,Bdloads were markedly higher in nutrient-enriched ponds. Nutrient enrichment significantly altered carbon utilization patterns as measured by Biolog EcoPlates, and antibiotic resistance was prevalent across all ponds and samples, with resistance to trimethoprim, sulfamethazine, and chloramphenicol significantly higher in nutrient-enriched ponds. Our findings indicate that nutrient enrichment affects the structure and function of skin-associated microbial communities in American bullfrogs, influencing bothBdload and antibiotic resistance. 

Detecting and quantifying pathogens with quick, cost-efficient and sensitive methods is needed across disease systems for addressing pertinent epidemiological questions. Typical methods rely on extracting DNA from collected samples. Here we develop and test an extraction-free method from water bath samples that is both sensitive and efficient for 2 major amphibian pathogens— Batrachochytrium dendrobatidis and B . salamandrivorans . We tested mock samples with known pathogen quantities as well as comparatively assessed detection from skin swabs and water baths from field sampled amphibians. Quantitative PCR (qPCR) directly on lyophilized water baths was able to reliably detect low loads of 10 and 1 zoospores for both pathogens, and detection rates were greater than those of swabs from field samples. Further concentration of samples did not improve detection, and collection container type did not influence pathogen load estimates. This method of lyophilization (i.e. freeze-drying) followed by direct qPCR offers an effective and efficient tool from detecting amphibian pathogens, which is crucial for surveillance efforts and estimating shedding rates for robust epidemiological understanding of transmission dynamics. Furthermore, water bath samples have multiple functions and can be used to evaluate mucosal function against pathogens and characterize mucosal components. The multifunctionality of water bath samples and reduced monetary costs and time expenditures make this method an optimal tool for amphibian disease research and may also prove to be useful in other wildlife disease systems. 

The increasing study of emerging wildlife pathogens and a lack of policy or legislation regulating their translocation and use has heightened concerns about laboratory escape, species spillover, and subsequent epizootics among animal populations. Responsible self-regulation by research laboratories, in conjunction with institutional-level safeguards, has an important role in mitigating pathogen transmission and spillover, as well as potential interspecies pathogenesis. A model system in disease ecology that highlights these concerns and related amelioration efforts is research focused on amphibian emerging infectious diseases. Whereas laboratory escape of amphibian pathogens has not been reported and may be rare compared with introduction events from trade or human globalization, the threat that novel disease outbreaks with mass mortality effects pose to wild populations warrants thorough biosecurity measures to ensure containment and prevent spillover. Here, we present a case study of the laboratory biosecurity concerns for the emerging amphibian fungal pathogen Batrachochytrium salamandrivorans . We conclude that proactive biosecurity strategies are needed to integrate researcher and institutional oversight of aquatic wildlife pathogens generally, and we call for increased national and international policy and legislative enforcement. Furthermore, taking professional responsibility beyond current regulations is needed as development of legal guidance can be slow at national and international levels. We outline the need for annual laboratory risk assessments, comprehensive training for all laboratory personnel, and appropriate safeguards specific to pathogens under study. These strategies are critical for upholding the integrity and credibility of the scientific community and maintaining public support for research on wildlife diseases. 

Discovered in 2013, the chytrid fungus Batrachochytrium salamandrivorans ( Bsal ) is an emerging amphibian pathogen that causes ulcerative skin lesions and multifocal erosion. A closely related pathogen, B. dendrobatidis ( Bd ), has devastated amphibian populations worldwide, suggesting that Bsal poses a significant threat to global salamander biodiversity. To expedite research into this emerging threat, we seek to standardize protocols across the field so that results of laboratory studies are reproducible and comparable. We have collated data and experience from multiple labs to standardize culturing practices of Bsal . Here we outline common culture practices including a medium for standardized Bsal growth, standard culturing protocols, and a method for isolating Bsal from infected tissue.