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Transmission is an essential process that contributes to the survival of pathogens. Ranaviruses are known to infect different classes of lower vertebrates including amphibians, fishes and reptiles. Differences in the likelihood of infection among ectothermic vertebrate hosts could explain the successful yearlong persistence of ranaviruses in aquatic environments. The goal of this study was to determine if transmission of a Frog Virus 3 (FV3)-like ranavirus was possible among three species from different ectothermic vertebrate classes: Cope's gray treefrog (Hyla chrysoscelis) larvae, mosquito fish (Gambusia affinis), and red-eared slider (Trachemys scripta elegans). We housed individuals previously exposed to the FV3-like ranavirus with naïve (unexposed) individuals in containers divided by plastic mesh screen to permit water flow between subjects. Our results showed that infected gray treefrog larvae were capable of transmitting ranavirus to naïve larval conspecifics and turtles (60% and 30% infection, respectively), but not to fish. Also, infected turtles and fish transmitted ranavirus to 50% and 10% of the naïve gray treefrog larvae, respectively. Nearly all infected amphibians experienced mortality, whereas infected turtles and fish did not die. Our results demonstrate that ranavirus can be transmitted through water among ectothermic vertebrate classes, which has not been reported previously. Moreover, fish and reptiles might serve as reservoirs for ranavirus given their ability to live with subclinical infections. Subclinical infections of ranavirus in fish and aquatic turtles could contribute to the pathogen's persistence, especially when highly susceptible hosts like amphibians are absent as a result of seasonal fluctuations in relative abundance.

Environmental temperature is a key factor driving various biological processes, including immune defenses and host-pathogen interactions. Here, we evaluated the effects of environmental temperature on the pathogenicity of the emerging fungal pathogen, Batrachochytrium salamandrivorans ( Bsal ), using controlled laboratory experiments, and measured components of host immune defense to identify regulating mechanisms. We found that adult and juvenile Notophthalmus viridescens died faster due to Bsal chytridiomycosis at 14°C than at 6 and 22°C. Pathogen replication rates, total available proteins on the skin, and microbiome composition likely drove these relationships. Temperature-dependent skin microbiome composition in our laboratory experiments matched seasonal trends in wild N . viridescens , adding validity to these results. We also found that hydrophobic peptide production after two months post-exposure to Bsal was reduced in infected animals compared to controls, perhaps due to peptide release earlier in infection or impaired granular gland function in diseased animals. Using our temperature-dependent susceptibility results, we performed a geographic analysis that revealed N . viridescens populations in the northeastern United States and southeastern Canada are at greatest risk for Bsal invasion, which shifted risk north compared to previous assessments. Our results indicate that environmental temperature will play a key role in the epidemiology of Bsal and provide evidence that temperature manipulations may be a viable disease management strategy.

Batrachochytrium salamandrivorans ( Bsal ) is a fungal pathogen of amphibians that is emerging in Europe and could be introduced to North America through international trade or other pathways. To evaluate the risk of Bsal invasion to amphibian biodiversity, we performed dose-response experiments on 35 North American species from 10 families, including larvae from five species. We discovered that Bsal caused infection in 74% and mortality in 35% of species tested. Both salamanders and frogs became infected and developed Bsal chytridiomycosis. Based on our host susceptibility results, environmental suitability conditions for Bsal , and geographic ranges of salamanders in the United States, predicted biodiversity loss is expected to be greatest in the Appalachian Region and along the West Coast. Indices of infection and disease susceptibility suggest that North American amphibian species span a spectrum of vulnerability to Bsal chytridiomycosis and most amphibian communities will include an assemblage of resistant, carrier, and amplification species. Predicted salamander losses could exceed 80 species in the United States and 140 species in North America. The emerging fungal pathogen Bsal is a potential threat for amphibians. Here, the authors use data from dose-response experiments on 35 species to assess vulnerability of North American amphibians to Bsal and identify species and areas potentially at risk.

Ranaviruses have caused die-offs of amphibians across the globe. In North America, these pathogens cause more amphibian mortality events than any other pathogen. Field observations suggest that ranavirus epizootics in amphibian communities are common during metamorphosis, presumably due to changes in immune function. However, few controlled studies have compared the relative susceptibility of amphibians to ranaviruses across life stages. Our objectives were to measure differences in mortality and infection prevalence following exposure to ranavirus at four developmental stages and determine whether the differences were consistent among seven anuran species. Based on previous studies, we hypothesized that susceptibility to ranavirus would be greatest at metamorphosis. Our results did not support this hypothesis, as four of the species were most susceptible to ranavirus during the larval or hatchling stages. The embryo stage had the lowest susceptibility among species probably due to the protective membranous layers of the egg. Our results indicate that generalizations should be made cautiously about patterns of susceptibility to ranaviruses among amphibian developmental stages and species. Further, if early developmental stages of amphibians are susceptible to ranaviruses, the impact of ranavirus epizootic events may be greater than realized due to the greater difficulty of detecting morbid hatchlings and larvae compared to metamorphs.

Hellbenders ( Cryptobranchus alleganiensis ) are large, aquatic salamanders from the eastern United States. Both subspecies, eastern and Ozark hellbenders, have experienced declines resulting in federal listing of Ozark hellbenders. The globally distributed chytrid fungus, Batrachochytrium dendrobatidis (Bd) has been detected in both subspecies, and Batrachochytrium salamandrivorans ( Bsal ) poses a new threat if introduced into North America. Ozark hellbenders also suffer a high prevalence of toe lesions of unknown etiology, with changes in host immunocompetence hypothesized to contribute. Antimicrobial peptides (AMPs) secreted from dermal granular glands may play a role in hellbender health. We collected skin secretions from free-ranging hellbenders and enriched them for small cationic peptides used for growth inhibition assays against Bd and Bsal . Generalized linear mixed models revealed the presence of active toe lesions as the strongest and only significant predictor of decreased Bd inhibition by skin peptides. We also found skin secretions were more inhibitory of Bsal than Bd . MALDI-TOF mass spectrometry revealed candidate peptides responsible for anti-chytrid activity. Results support the hypothesis that hellbender skin secretions are important for innate immunity against chytrid pathogens, and decreased production or release of skin peptides may be linked to other sub-lethal effects of disease associated with toe lesions.

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.

The world’s reptiles and amphibians are experiencing dramatic and ongoing losses in biodiversity, changes that can have substantial effects on ecosystems and human health. In 2022, the first Global Amphibian and Reptile Disease Conference was held, using One Health as a guiding principle. The conference showcased knowledge on numerous reptile and amphibian pathogens from several standpoints, including epidemiology, host immune defenses, wild population effects, and mitigation. The conference also provided field experts the opportunity to discuss and identify the most urgent herpetofaunal disease research directions necessary to address current and future threats to reptile and amphibian biodiversity.

Ranaviruses are an important wildlife pathogen of fish, amphibians, and reptiles. Previous studies have shown that susceptibility and severity of infection can vary with age, host species, virus strain, temperature, population density, and presence of environmental stressors. Experiments are limited with respect to interactions between this pathogen and environmental stressors in reptiles. In this study, we exposed hatchling red-eared slider turtles (Trachemys scripta elegans) to herbicide and ranavirus treatments to examine direct effects and interactions on growth, morbidity, and mortality. Turtles were assigned to one of three herbicide treatments or a control group. Turtles were exposed to atrazine, Roundup ProMax®, or Rodeo® via water bath during the first 3 weeks of the experiment. After 1 week, turtles were exposed to either a control (cell culture medium) or ranavirus-infected cell lysate via injection into the pectoral muscles. Necropsies were performed upon death or upon euthanasia after 5 weeks. Tissues were collected for histopathology and detection of ranavirus DNA via quantitative PCR. Only 57.5% of turtles exposed to ranavirus tested positive for ranaviral DNA at the time of death. Turtles exposed to ranavirus died sooner and lost more mass and carapace length, but not plastron length, than did controls. Exposure to environmentally relevant concentrations of herbicides did not impact infection rate, morbidity, or mortality of hatchling turtles due to ranavirus exposure. We also found no direct effects of herbicide or interactions with ranavirus exposure on growth or survival time. Results of this study should be interpreted in the context of the modest ranavirus infection rate achieved, the general lack of growth, and the unplanned presence of an additional pathogen in our study.

The consumption of cocoa and dark chocolate is associated with a lower risk of CVD, and improvements in endothelial function may mediate this relationship. Less is known about the effects of cocoa/chocolate on the augmentation index (AI), a measure of vascular stiffness and vascular tone in the peripheral arterioles. We enrolled thirty middle-aged, overweight adults in a randomised, placebo-controlled, 4-week, cross-over study. During the active treatment (cocoa) period, the participants consumed 37 g/d of dark chocolate and a sugar-free cocoa beverage (total cocoa = 22 g/d, total flavanols (TF) = 814 mg/d). Colour-matched controls included a low-flavanol chocolate bar and a cocoa-free beverage with no added sugar (TF = 3 mg/d). Treatments were matched for total fat, saturated fat, carbohydrates and protein. The cocoa treatment significantly increased the basal diameter and peak diameter of the brachial artery by 6 % (+2 mm) and basal blood flow volume by 22 %. Substantial decreases in the AI, a measure of arterial stiffness, were observed in only women. Flow-mediated dilation and the reactive hyperaemia index remained unchanged. The consumption of cocoa had no effect on fasting blood measures, while the control treatment increased fasting insulin concentration and insulin resistance (P= 0·01). Fasting blood pressure (BP) remained unchanged, although the acute consumption of cocoa increased resting BP by 4 mmHg. In summary, the high-flavanol cocoa and dark chocolate treatment was associated with enhanced vasodilation in both conduit and resistance arteries and was accompanied by significant reductions in arterial stiffness in women.

The emerging fungal pathogen, Batrachochytrium salamandrivorans (Bsal), is a significant conservation threat to salamander biodiversity in Europe, although its potential to affect North American species is poorly understood. We tested the susceptibility of two genera (Eurycea and Pseudotriton) and three populations of lungless salamanders (Plethodontidae) to Bsal. All species became infected with Bsal and two (Pseudotriton ruber and Eurycea wilderae) developed chytridiomycosis. We also documented that susceptibility of E. wilderae differed among populations. Regardless of susceptibility, all species reduced feeding when exposed to Bsal at the highest zoospore dose, and P. ruber and one population of E. wilderae used cover objects less. Our results indicate that Bsal invasion in eastern North America could have significant negative impacts on endemic lungless salamander populations. Future conservation efforts should include surveillance for Bsal in the wild and in captivity, and championing legislation that requires and subsidizes pathogen‐free trade of amphibians.