Attitudes and Behavioral Intentions of Pet Amphibian Owners About Biosecurity Practices.

Global trade has been linked with the emergence of novel pathogens and declines in amphibian populations worldwide. The potential for pathogen transmission within and between collections of captive amphibians and spillover to wild populations makes it important to understand the motivations, knowledge, attitudes and behaviors of pet amphibian owners. We surveyed US pet amphibian owners to understand their characteristics and evaluated whether and how they were associated with behavioral intentions to adopt biosecurity practices. We found that the majority of pet amphibian owners are aware of the threat of emerging pathogens, concerned about potential spillover of pathogens from captive to wild populations and willing to adopt biosecurity practices to mitigate pathogen threats. Intentions to adopt such practices were driven more by psychosocial constructs such as attitudes, perceptions and beliefs than demographic characteristics. Pet amphibian owners also expressed a strong interest in acquiring, and willingness to pay a price premium for, certified disease-free animals. These findings advance our understanding of the characteristics, motivations and behaviors of pet owners, a key stakeholder in global amphibian trade, which could help to inform new policies and outreach strategies to engage them in mitigating pathogen threats. Moreover, our results imply the economic viability of a market-based program to promote pathogen-free, sustainable trade of amphibians.

Pooled samples and eDNA-based detection can facilitate the "clean trade" of aquatic animals.

The regional and international trade of live animals facilitates the movement, spillover, and emergence of zoonotic and epizootic pathogens around the world. Detecting pathogens in trade is critical for preventing their continued movement and introduction, but screening a sufficient fraction to ensure rare infections are detected is simply infeasible for many taxa and settings because of the vast numbers of animals involved-hundreds of millions of live animals are imported into the U.S.A. alone every year. Batch processing pools of individual samples or using environmental DNA (eDNA)-the genetic material shed into an organism's environment-collected from whole consignments of animals may substantially reduce the time and cost associated with pathogen surveillance. Both approaches, however, lack a framework with which to determine sampling requirements and interpret results. Here I present formulae for pooled individual samples (e.g,. swabs) and eDNA samples collected from finite populations and discuss key assumptions and considerations for their use with a focus on detecting Batrachochytrium salamandrivorans, an emerging pathogen that threatens global salamander diversity. While empirical validation is key, these formulae illustrate the potential for eDNA-based detection in particular to reduce sample sizes and help bring clean trade into reach for a greater number of taxa, places, and contexts.

Salinity stress increases the severity of ranavirus epidemics in amphibian populations.

The stress-induced susceptibility hypothesis, which predicts chronic stress weakens immune defences, was proposed to explain increasing infectious disease-related mass mortality and population declines. Previous work characterized wetland salinization as a chronic stressor to larval amphibian populations. Thus, we combined field observations with experimental exposures quantifying epidemiological parameters to test the role of salinity stress in the occurrence of ranavirus-associated mass mortality events. Despite ubiquitous pathogen presence (94%), populations exposed to salt runoff had slightly more frequent ranavirus related mass mortality events, more lethal infections, and 117-times greater pathogen environmental DNA. Experimental exposure to chronic elevated salinity (0.8-1.6 g l-1 Cl-) reduced tolerance to infection, causing greater mortality at lower doses. We found a strong negative relationship between splenocyte proliferation and corticosterone in ranavirus-infected larvae at a moderate elevation of salinity, supporting glucocorticoid-medicated immunosuppression, but not at high salinity. Salinity alone reduced proliferation further at similar corticosterone levels and infection intensities. Finally, larvae raised in elevated salinity had 10 times more intense infections and shed five times as much virus with similar viral decay rates, suggesting increased transmission. Our findings illustrate how a small change in habitat quality leads to more lethal infections and potentially greater transmission efficiency, increasing the severity of ranavirus epidemics.

Environmental Drivers of Questing Activity of Juvenile Black-Legged Ticks (Acari: Ixodidae): Temperature, Desiccation Risk, and Diel Cycles.

Vector feeding behavior can have a profound influence on the transmission of vector-borne diseases. In the case of black-legged ticks, Ixodes scapularis Say, which vectors the agents of Lyme disease, babesiosis, and other pathogens, the timing and propensity of questing can determine which hosts are fed upon as well as the risk of contact with humans. Yet we know little about the controls and constraints on tick host-finding behavior under natural conditions. Ticks must balance the need to quest for blood meal hosts with the risk of desiccation, all on a fixed energy budget. Prior research, primarily in the laboratory, has shown that questing activity varies with conditions (e.g., temperature, relative humidity), light-dark cycles, and energy reserves, but the findings have been idiosyncratic and the dominant factor(s) in nature remains unknown. We measured questing activity of nymphs and larvae throughout the day and night and over several weeks in enclosures across a range of suitable tick habitats within a site in the Northeast. Activity of nymphs increased slightly during dawn and dusk, opposite of larvae, and declined slightly with air temperature and rain, but these patterns were weak and inconsistent among replicate sites. Rather it appears a fraction of ticks were questing most of the time, regardless of conditions. Our study suggests neither climatic conditions or light-dark cycles have appreciable influence on tick questing behavior.

Ranavirus infection dynamics and shedding in American bullfrogs: consequences for spread and detection in trade.

American bullfrogs Lithobates catesbeianus are thought to be important in the global spread of ranaviruses-often lethal viruses of cold-blooded vertebrates-because they are commonly farmed, dominate international trade, and may be 'carriers' of ranavirus infections. However, whether American bullfrogs are easily infected and maintain long-lasting ranavirus infections, or are refractory to or rapidly clear infections, remains unknown. We tracked the dynamics of ranavirus in American bullfrogs through time and with temperature in multiple types of samples and also screened shipments from commercial suppliers to determine whether we could detect subclinical infections. Collectively, we found that tadpoles and juveniles were commonly infected at moderate doses, and while some died, others controlled and appeared to clear their infections. Some individuals, however, harbored subclinical infections for up to 49 d, suggesting that American bullfrogs may be important carriers. Indeed, tadpoles and metamorphosed frogs from 2 of 5 commercial suppliers harbored subclinicial infections. Juveniles at warmer temperatures had less intense but still persistent infections. Because diagnostic performance was strongly related to infection intensity, non-lethal samples (i.e. tail or toe clips, swabs, and environmental DNA) had only a moderate chance of detecting subclinical infections. Even internal tissues may fail to detect subclinical infections. However, viral shedding was correlated with the intensity of infection, so while subclinically infected tadpoles shed virus for 35-49 d, the low levels might lead to little transmission. We suggest that a quantitative focus on virus dynamics within hosts can provide a more nuanced view of ranavirus infections and the risk presented by American bullfrogs in trade.

Evaluating the Within-Host Dynamics of Ranavirus Infection with Mechanistic Disease Models and Experimental Data.

Mechanistic models are critical for our understanding of both within-host dynamics (i.e., pathogen replication and immune system processes) and among-host dynamics (i.e., transmission). Within-host models, however, are not often fit to experimental data, which can serve as a robust method of hypothesis testing and hypothesis generation. In this study, we use mechanistic models and empirical, time-series data of viral titer to better understand the replication of ranaviruses within their amphibian hosts and the immune dynamics that limit viral replication. Specifically, we fit a suite of potential models to our data, where each model represents a hypothesis about the interactions between viral replication and immune defense. Through formal model comparison, we find a parsimonious model that captures key features of our time-series data: The viral titer rises and falls through time, likely due to an immune system response, and that the initial viral dosage affects both the peak viral titer and the timing of the peak. Importantly, our model makes several predictions, including the existence of long-term viral infections, which can be validated in future studies.

Evaluating the Importance of Environmental Persistence for Ranavirus Transmission and Epidemiology.

Viruses persist outside their hosts in a variety of forms, from naked virions to virus protected in sloughed tissues or carcasses, and for a range of times, all of which affect the likelihood and importance of transmission from the environment. This review synthesizes the literature on environmental persistence of viruses in the genus Ranavirus (family Iridoviridae), which are large double-stranded DNA viruses of ectothermic, often aquatic or semiaquatic vertebrates. Ranaviruses have been associated with mass mortality events in natural and captive settings around the world, and with population and community-wide declines in Europe. Early work suggested ranaviruses are environmentally robust and transmission from the environment should be common. More recent work has shown a large effect of temperature and microbial action on persistence times, although other aspects of the environment (e.g., water chemistry) and aquatic communities (e.g., zooplankton) may also be important. Ranaviruses may persist in the carcasses of animals that have died of infection, and so decomposing organisms and invertebrate scavengers may reduce these persistence times. The question is, do persistence times vary enough to promote or preclude substantial transmission from the environment. We built an epidemiological model with transmission from contacts, free virus in water, and carcasses, to explore the conditions in which environmental persistence could be important for ranavirus epidemiology. Based on prior work, we expected a substantial amount of transmission from the water and that longer persistence times would make this route of transmission dominant. However, neither water-borne nor transmission from carcasses played an important role in the simulated epidemics except under fairly restrictive conditions, such as when there were high rates of virus shedding or high rates of scavenging on highly infectious carcasses. While many aspects of environmental persistence of ranaviruses are being resolved by experiments, key parameters such as viral shedding rates are virtually unknown and will need to be empirically constrained if we are to determine whether environmental persistence and transmission from the environment are essential or insignificant features of Ranavirus epidemiology. We conclude by emphasizing the need to place environmental persistence research in an epidemiological framework.

From fish to frogs and beyond: Impact and host range of emergent ranaviruses.

Ranaviruses are pathogens of ectothermic vertebrates, including amphibians. We reviewed patterns of host range and virulence of ranaviruses in the context of virus genotype and postulate that patterns reflect significant variation in the historical and current host range of three groups of Ranavirus: FV3-like, CMTV-like and ATV-like ranaviruses. Our synthesis supports previous hypotheses about host range and jumps: FV3s are amphibian specialists, while ATVs are predominantly fish specialists that switched once to caudate amphibians. The most recent common ancestor of CMTV-like ranaviruses and FV3-like forms appears to have infected amphibians but CMTV-like ranaviruses may circulate in both amphibian and fish communities independently. While these hypotheses are speculative, we hope that ongoing efforts to describe ranavirus genetics, increased surveillance of host species and targeted experimental assays of susceptibility to infection and/or disease will facilitate better tests of the importance of hypothetical evolutionary drivers of ranavirus virulence and host range.

Heterogeneities in the infection process drive ranavirus transmission.

Transmission is central to our understanding and efforts to control the spread of infectious diseases. Because transmission generally requires close contact, host movements and behaviors can shape transmission dynamics: random and complete mixing leads to the classic density-dependent model, but if hosts primarily interact locally (e.g., aggregate) or within groups, transmission may saturate. Manipulating host behavior may thus change both the rate and functional form of transmission. We used the ranavirus-wood frog (Lithobates sylvaticus) tadpole system to test whether transmission rates reflect contacts, and whether the functional form of transmission can be influenced by the distribution of food in mesocosms (widely dispersed, promoting random movement and mixing vs. a central pile, promoting aggregations). Contact rates increased with density, as expected, but transmission rapidly saturated. Observed rates of transmission were not explained by observed contact rates or the density-dependent model, but instead transmission in both treatments followed models allowing for heterogeneities in the transmission process. We argue that contacts were not generally limiting, but instead that our results are better explained by heterogeneities in host susceptibility. Moreover, manipulating host behavior to manage the spread of infectious disease may prove difficult to implement.

Evaluating environmental DNA-based quantification of ranavirus infection in wood frog populations.

A variety of challenges arise when monitoring wildlife populations for disease. Sampling tissues can be invasive to hosts, and obtaining sufficient sample sizes can be expensive and time-consuming, particularly for rare species and when pathogen prevalence is low. Environmental DNA (eDNA)-based detection of pathogens is an alternative approach to surveillance for aquatic communities that circumvents many of these issues. Ranaviruses are emerging pathogens of ectothermic vertebrates linked to die-offs of amphibian populations. Detecting ranavirus infections is critical, but nonlethal methods have the above issues and are prone to false negatives. We report on the feasibility and effectiveness of eDNA-based ranavirus detection in the field. We compared ranavirus titres in eDNA samples collected from pond water to titres in wood frog (Lithobates sylvaticus; n = 5) tadpoles in sites dominated by this one species (n = 20 pond visits). We examined whether ranavirus DNA can be detected in eDNA from pond water when infections are present in the pond and if viral titres detected in eDNA samples correlate with the prevalence or intensity of ranavirus infections in tadpoles. With three 250 mL water samples, we were able to detect the virus in all visits with infected larvae (0.92 diagnostic sensitivity). Also, we found a strong relationship between the viral eDNA titres and titres in larval tissues. eDNA titres increased prior to observed die-offs and declined afterwards, and were two orders of magnitude higher in ponds with a die-off. Our results suggest that eDNA is useful for detecting ranavirus infections in wildlife and aquaculture.

Geophysiology of Wood Frogs: Landscape Patterns of Prevalence of Disease and Circulating Hormone Concentrations across the Eastern Range.

One of the major challenges for conservation physiologists is to determine how current or future environmental conditions relate to the health of animals at the population level. In this study, we measured prevalence of disease, mean condition of the body, and mean resting levels of corticosterone and testosterone in a total of 28 populations across the years 2011 and 2012, and correlated these measures of health to climatic suitability of habitat, using estimates from a model of the ecological niche of the wood frog's geographic range. Using the core-periphery hypothesis as a theoretical framework, we predicted a higher prevalence and intensity of infection of Batrachochytrium dendrobatidis (Bd) and ranaviruses, two major amphibian pathogens causing disease, and higher resting levels of circulating corticosterone, an indicator of allostatic load incurred from living in marginal habitats. We found that Bd infections were rare (2% of individuals tested), while infections with ranavirus were much more common: ranavirus-infected individuals were found in 92% of ponds tested over the 2 years. Contrary to our predictions, rates of infection with ranaviruses were positively correlated with quality of the habitat with the highest prevalence at the core of the range, and plasma corticosterone concentrations measured when frogs were at rest were not correlated with quality of the habitat, the prevalence of ranavirus, or the intensity of infection. Prevalence and mean viral titers of ranavirus infection were higher in 2012 than in 2011, which coincided with lower levels of circulating corticosterone and testosterone and an extremely early time of breeding due to relatively higher temperatures during the winter. In addition, the odds of having a ranavirus infection increased with decreased body condition, and if animals had an infection, viral titers were positively correlated to levels of circulating testosterone concentration. By resolving these patterns, experiments can be designed to test hypotheses about the mechanisms that produce them, such as whether transmission of the ranavirus and tolerance of the host are greater or whether virulence is lower in populations within core habitats. While there is debate about which metrics serve as the best bioindicators of population health, the findings of this study demonstrate the importance of long-term monitoring of multiple physiological parameters to better understand the dynamic relationship between the environment and the health of wildlife populations over space and time.

Factors affecting larval tick feeding success: host, density and time.

BACKGROUND: Ectoparasites rely on blood-feeding to sustain activity, support development and produce offspring. Blood-feeding is also a route for transmission of diverse vector-borne pathogens. The likelihood of successfully feeding is thus an important aspect of ectoparasite population dynamics and pathogen transmission. Factors that affect blood-feeding include ectoparasite density, host defenses, and ages of the host and ectoparasite. How these factors interact to affect feeding success is not well understood. METHODS: We monitored blood-feeding success of larval Rocky Mountain wood ticks (RMWTs; Dermacentor andersoni) on deer mice (Peromyscus maniculatus) in several experiments to determine how tick density, host defense, and ages of mice and ticks interact to influence feeding success. In the first experiment, tick-naive deer mice were infested with one of several densities of RMWT larvae, while a second cohort of mice were infested with 50 larvae each. Two weeks after ticks dropped off, mice in the first cohort were re-exposed to 50 larvae each and mice in the second cohort were re-exposed to varying densities of larvae. In the second experiment mice of different ages (45-374 days old) were exposed to 50 larvae each. Two weeks later mice were re-exposed to 50 larvae each. We combined data from these and several similar experiments to test the generality of the patterns we observed. Lastly, we tested whether tick feeding success was consistent on individual mice that were challenged on four occasions. RESULTS: Mice acquired resistance such that feeding success declined dramatically from the first to the second infestation. Feeding success also declined with tick density and tick age. Mice, however, became more permissive with age. The sizes of these effects were similar and additive. Surprisingly, over successive infestations the relative resistance among mice changed among hosts within a cohort. CONCLUSIONS: We predict that larval blood-feeding success, and thus development to the nymph stage, will change due to variation in tick age and density, as well as the age and history of the host. Incorporating these biotic factors into modeling of tick population dynamics may improve predictions of tick-borne pathogen transmission.

Rapid Response to Evaluate the Presence of Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis) and Ranavirus in Wild Amphibian Populations in Madagascar.

We performed a rapid response investigation to evaluate the presence and distribution of amphibian pathogens in Madagascar following our identification of amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd) and ranavirus in commercially exported amphibians. This targeted risk-based field surveillance program was conducted from February to April 2014 encompassing 12 regions and 47 survey sites. We simultaneously collected amphibian and environmental samples to increase survey sensitivity and performed sampling both in wilderness areas and commercial amphibian trade facilities. Bd was not detected in any of 508 amphibian skin swabs or 68 water filter samples, suggesting pathogen prevalence was below 0.8%, with 95% confidence during our visit. Ranavirus was detected in 5 of 97 amphibians, including one adult Mantidactylus cowanii and three unidentified larvae from Ranomafana National Park, and one adult Mantidactylus mocquardi from Ankaratra. Ranavirus was also detected in water samples collected from two commercial amphibian export facilities. We also provide the first report of an amphibian mass-mortality event observed in wild amphibians in Madagascar. Although neither Bd nor ranavirus appeared widespread in Madagascar during this investigation, additional health surveys are required to disentangle potential seasonal variations in pathogen abundance and detectability from actual changes in pathogen distribution and rates of spread. Accordingly, our results should be conservatively interpreted until a comparable survey effort during winter months has been performed. It is imperative that biosecurity practices be immediately adopted to limit the unintentional increased spread of disease through the movement of contaminated equipment or direct disposal of contaminated material from wildlife trade facilities. The presence of potentially introduced strains of ranaviruses suggests that Madagascar's reptile species might also be threatened by disease. Standardized population monitoring of key amphibian and reptile species should be established with urgency to enable early detection of potential impacts of disease emergence in this global biodiversity hotspot.

Climate change and Ixodes tick-borne diseases of humans.

The evidence that climate warming is changing the distribution of Ixodes ticks and the pathogens they transmit is reviewed and evaluated. The primary approaches are either phenomenological, which typically assume that climate alone limits current and future distributions, or mechanistic, asking which tick-demographic parameters are affected by specific abiotic conditions. Both approaches have promise but are severely limited when applied separately. For instance, phenomenological approaches (e.g. climate envelope models) often select abiotic variables arbitrarily and produce results that can be hard to interpret biologically. On the other hand, although laboratory studies demonstrate strict temperature and humidity thresholds for tick survival, these limits rarely apply to field situations. Similarly, no studies address the influence of abiotic conditions on more than a few life stages, transitions or demographic processes, preventing comprehensive assessments. Nevertheless, despite their divergent approaches, both mechanistic and phenomenological models suggest dramatic range expansions of Ixodes ticks and tick-borne disease as the climate warms. The predicted distributions, however, vary strongly with the models' assumptions, which are rarely tested against reasonable alternatives. These inconsistencies, limited data about key tick-demographic and climatic processes and only limited incorporation of non-climatic processes have weakened the application of this rich area of research to public health policy or actions. We urge further investigation of the influence of climate on vertebrate hosts and tick-borne pathogen dynamics. In addition, testing model assumptions and mechanisms in a range of natural contexts and comparing their relative importance as competing models in a rigorous statistical framework will significantly advance our understanding of how climate change will alter the distribution, dynamics and risk of tick-borne disease.

Detection of the emerging amphibian pathogens Batrachochytrium dendrobatidis and ranavirus in Russia.

In a population of the European common toad Bufo bufo from a rural pond in the region of Lake Glubokoe Regional Reserve in Moscow province, Russia, unexplained mass mortality events involving larvae and metamorphs have been observed over a monitoring period of >20 yr. We tested toads from this and a nearby site for the emerging amphibian pathogens Batrachochytrium dendrobatidis (Bd) and ranavirus (Rv). Both pathogens were detected, and at the rural pond site, with the above-noted losses and decline in toad breeding success, 40% of B. bufo metamorphs were Bd positive, 46% were Rv positive and 20% were co-infected with both pathogens. Toad metamorphs from a neighbouring water body were also Bd and Rv positive (25 and 55%, respectively). This is the first confirmation of these pathogens in Russia. Questions remain as to the origins of these pathogens in Russia and their roles in documented mass mortality events.

Co-infection of blacklegged ticks with Babesia microti and Borrelia burgdorferi is higher than expected and acquired from small mammal hosts.

Humans in the northeastern and midwestern United States are at increasing risk of acquiring tickborne diseases--not only Lyme disease, but also two emerging diseases, human granulocytic anaplasmosis and human babesiosis. Co-infection with two or more of these pathogens can increase the severity of health impacts. The risk of co-infection is intensified by the ecology of these three diseases because all three pathogens (Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti) are transmitted by the same vector, blacklegged ticks (Ixodes scapularis), and are carried by many of the same reservoir hosts. The risk of exposure to multiple pathogens from a single tick bite and the sources of co-infected ticks are not well understood. In this study, we quantify the risk of co-infection by measuring infection prevalence in 4,368 questing nymphs throughout an endemic region for all three diseases (Dutchess County, NY) to determine if co-infections occur at frequencies other than predicted by independent assortment of pathogens. Further, we identify sources of co-infection by quantifying rates of co-infection on 3,275 larval ticks fed on known hosts. We find significant deviations of levels of co-infection in questing nymphs, most notably 83% more co-infection with Babesia microti and Borrelia burgdorferi than predicted by chance alone. Further, this pattern of increased co-infection was observed in larval ticks that fed on small mammal hosts, but not on meso-mammal, sciurid, or avian hosts. Co-infections involving A. phagocytophilum were less common, and fewer co-infections of A. phagocytophilum and B. microti than predicted by chance were observed in both questing nymphs and larvae fed on small mammals. Medical practitioners should be aware of the elevated risk of B. microti/B. burgdorferi co-infection.

Prevalence of human-active and variant 1 strains of the tick-borne pathogen Anaplasma phagocytophilum in hosts and forests of eastern North America.

Anaplasmosis is an emerging infectious disease caused by infection with the bacterium Anaplasma phagocytophilum. In the eastern United States, A. phagocytophilum is transmitted to hosts through the bite of the blacklegged tick, Ixodes scapularis. We determined the realized reservoir competence of 14 species of common vertebrate hosts for ticks by establishing the probability that each species transmits two important strains of A. phagocytophilum (A. phagocytophilum human-active, which causes human cases, and A. phagocytophilum variant 1, which does not) to feeding larval ticks. We also sampled questing nymphal ticks from ∼ 150 sites in a single county over 2 years and sampled over 6 years at one location. White-footed mice (Peromyscus leucopus) and Eastern chipmunks (Tamias striatus) were the most competent reservoirs for infection with the A. phagocytophilum human-active strain. Across the county, prevalence in ticks for both strains together was 8.3%; ticks were more than two times as likely to be infected with A. phagocytophilum human-active as A. phagocytophilum variant 1.

An Experimental Test of Competition among Mice, Chipmunks, and Squirrels in Deciduous Forest Fragments.

Mixed hardwood forests of the northeast United States support a guild of granivorous/omnivorous rodents including gray squirrels (Sciurus carolinensis), eastern chipmunks (Tamias striatus), and white-footed mice (Peromyscus leucopus). These species coincide geographically, co-occur locally, and consume similar food resources. Despite their idiosyncratic responses to landscape and patch variables, patch occupancy models suggest that competition may influence their respective distributions and abundances, and accordingly their influence on the rest of the forest community. Experimental studies, however, are wanting. We present the result of a large-scale experiment in which we removed white-footed mice or gray squirrels from small, isolated forest fragments in Dutchess County, New York, and added these mammals to other fragments in order to alter the abundance of these two species. We then used mark-recapture analyses to quantify the population-level and individual-level effects on resident mice, squirrels, and chipmunks. Overall, we found little evidence of competition. There were essentially no within-season numerical responses to changes in the abundance of putative competitors. Moreover, while individual-level responses (apparent survival and capture probability) did vary with competitor densities in some models, these effects were often better explained by site-specific parameters and were restricted to few of the 19 sites we studied. With only weak or nonexistent competition among these three common rodent species, we expect their patterns of habitat occupancy and population dynamics to be largely independent of one another.

Natural stressors and ranavirus susceptibility in larval wood frogs (Rana sylvatica).

Chronic exposure to stressors has been shown to suppress immune function in vertebrates, making them more susceptible to pathogens. It is less clear, however, whether many natural stressors are immunosuppressive. Moreover, whether stressors make disease more likely or more severe in populations is unclear because animals respond to stressors both behaviorally and physiologically. We tested whether chronic exposure to three natural stressors of wood frog tadpoles-high-densities, predator-cues, and low-food conditions-influence their susceptibility to a lethal ranavirus both individually in laboratory experiments, and collectively in outdoor mesocosms. Prior to virus exposure, we observed elevated corticosterone only in low-food treatments, although other treatments altered rates of growth and development as well as tadpole behavior. None of the treatments, however, increased susceptibility to ranavirus as measured by the proportion of tadpoles that became infected or died, or the time to death compared to controls. In fact, mortality in the mesocosms was actually lower in the high-density treatment even though most individuals became infected, largely because of increased rates of metamorphosis. Overall we find no support for the hypothesis that chronic exposure to common, ecologically relevant challenges necessarily elevates corticosterone levels in a population or leads to more severe ranaviral disease or epidemics. Conditions may, however, conspire to make ranavirus infection more common in metamorphosing amphibians.