Matthew J. Gray

Ecology and pathology of amphibian ranaviruses

Mass mortality of amphibians has occurred globally since at least the early 1990s from viral pathogens that are members of the genus Ranavirus, family Iridoviridae. The pathogen infects multiple amphibian hosts, larval and adult cohorts, and may persist in herpetofaunal and osteichthyan reservoirs. Environmental persistence of ranavirus virions outside a host may be several weeks or longer in aquatic systems. Transmission occurs by indirect and direct routes, and includes exposure to contaminated water or soil, casual or direct contact with infected individuals, and ingestion of infected tissue during predation, cannibalism, or necrophagy. Some gross lesions include swelling of the limbs or body, erythema, swollen friable livers, and hemorrhage. Susceptible amphibians usually die from chronic cell death in multiple organs, which can occur within a few days following infection or may take several weeks. Amphibian species differ in their susceptibility to ranaviruses, which may be related to their co-evolutionary history with the pathogen. The occurrence of recent widespread amphibian population die-offs from ranaviruses may be an interaction of suppressed and naïve host immunity, anthropogenic stressors, and novel strain introduction. This review summarizes the ecological research on amphibian ranaviruses, discusses possible drivers of emergence and conservation strategies, and presents ideas for future research directions. We also discuss common pathological signs of ranaviral disease, methods for diagnostic evaluation, and ranavirus surveillance methods. In as much as ranaviral disease is listed as a notifiable disease by the World Organization for Animal Health and is a threat to amphibian survival, we recommend that biosecurity precautions are implemented by nations to reduce the likelihood of transporting ranavirus virions among populations. Biosecurity precautions include disinfecting footwear and equipment that comes in contact with surface water inhabited by amphibians and testing commercially shipped amphibians for the pathogen. We also encourage natural resource organizations to establish routine surveillance programs for ranaviruses in wild amphibian populations.

Ecopathology of Ranaviruses Infecting Amphibians

Ranaviruses are capable of infecting amphibians from at least 14 families and over 70 individual species. Ranaviruses infect multiple cell types, often culminating in organ necrosis and massive hemorrhaging. Subclinical infections have been documented, although their role in ranavirus persistence and emergence remains unclear. Water is an effective transmission medium for ranaviruses, and survival outside the host may be for significant duration. In aquatic communities, amphibians, reptiles and fish may serve as reservoirs. Controlled studies have shown that susceptibility to ranavirus infection and disease varies among amphibian species and developmental stages, and likely is impacted by host-pathogen coevolution, as well as, exogenous environmental factors. Field studies have demonstrated that the likelihood of epizootics is increased in areas of cattle grazing, where aquatic vegetation is sparse and water quality is poor. Translocation of infected amphibians through commercial trade (e.g., food, fish bait, pet industry) contributes to the spread of ranaviruses. Such introductions may be of particular concern, as several studies report that ranaviruses isolated from ranaculture, aquaculture, and bait facilities have greater virulence (i.e., ability to cause disease) than wild-type isolates. Future investigations should focus on the genetic basis for pathogen virulence and host susceptibility, ecological and anthropogenic mechanisms contributing to emergence, and vaccine development for use in captive populations and species reintroduction programs.

Phylogeny, Life History, and Ecology Contribute to Differences in Amphibian Susceptibility to Ranaviruses

Research that identifies the potential host range of generalist pathogens as well as variation in host susceptibility is critical for understanding and predicting the dynamics of infectious diseases within ecological communities. Ranaviruses have been linked to amphibian die-off events worldwide with the greatest number of reported mortality events occurring in the United States. While reports of ranavirus-associated mortality events continue to accumulate, few data exist comparing the relative susceptibility of different species. Using a series of laboratory exposure experiments and comparative phylogenetics, we compared the susceptibilities of 19 amphibian species from two salamander families and five anurans families for two ranavirus isolates: frog virus 3 (FV3) and an FV3-like isolate from an American bullfrog culture facility. We discovered that ranaviruses were capable of infecting 17 of the 19 larval amphibian species tested with mortality ranging from 0 to 100%. Phylogenetic comparative methods demonstrated that species within the anuran family Ranidae were generally more susceptible to ranavirus infection compared to species from the other five families. We also found that susceptibility to infection was associated with species that breed in semi-permanent ponds, develop rapidly as larvae, and have limited range sizes. Collectively, these results suggest that phylogeny, life history characteristics, and habitat associations of amphibians have the potential to impact susceptibility to ranaviruses.

Anuran susceptibilities to ranaviruses: role of species identity, exposure route, and a novel virus isolate

Ranaviruses are responsible for widespread amphibian die-offs, particularly with larval anurans. To understand the factors that may be contributing to the emergence of ranaviruses, we conducted 3 experiments that exposed 3 species of larval anurans to either endemic frog virus 3 (FV3) or an FV3-like isolate from a ranaculture facility. Our goals were to (1) determine the susceptibility of each species to each virus, (2) determine whether direct ingestion of virions or exposure to virions in a water bath were similarly lethal routes of transmission, and (3) quantify the effects of exposure duration on disease outcomes. We conducted our research in a controlled aquatic laboratory using a factorial combination of virus isolates, transmission routes, and exposure durations. While ranaviruses can affect many species, we found that larval anurans differ greatly in susceptibility to ranaviruses. Average mortality rates of Copes gray tree frogs (66%) and pickerel frogs (68%) were similar but 3-fold higher than for eastern narrow-mouthed toads. Direct ingestion of the viruses increased mean infection and mortality rates by 30% and caused death about 2 times faster compared to water bath exposure. However, exposure duration did not impact mean infection or mortality rates. We also found that the ranaculture isolate increased mortality by > 34% compared to FV3. Our results suggest that ranaviruses can rapidly infect and cause disease in multiple amphibian species. Given the risk associated with introducing novel ranaviruses from ranaculture facilities, we recommend that all nations adopt the protocol set forth by the World Organization for Animal Health for testing and certifying that amphibians that are commercially shipped are negative for ranavirus infection.

Influence of agricultural landscape structure on a Southern High Plains, USA, amphibian assemblage

Landscape structure can influence demographics of spatially structured populations, particularly less vagile organisms such as amphibians. We examined the influence of agricultural landscape structure on community composition and relative abundance of the 4 most common amphibians in the Southern High Plains of central USA. Amphibian populations were monitored using pitfall traps and drift fence at 16 playa wetlands (8 playas/year) in 1999 and 2000. We quantified landscape structure surrounding each playa via estimating 13 spatial metrics that indexed playa isolation and inter-playa landscape complexity. Multivariate ordination and univariate correlations and regressions indicated that landscape structure was associated with community composition and relative abundance for 2 of the 4 amphibians. Spadefoots (Spea multiplicata, S. bombifrons) generally were positively associated with decreasing inter-playa distance and increasing inter-playa landscape complexity. Great Plains toads (Bufo cognatus) and barred tiger salamanders (Ambystoma tigrinum mavortium) usually were negatively associated with spadefoots but not influenced by landscape structure. Composition and relative abundance patterns were related to amphibian body size, which can influence species vagility and perception to landscape permeability. Spatial separation of these species in the multivariate ordination also may have been a consequence of differential competitive ability among species. These results suggest agricultural landscape structure may influence abundance and composition of spatially structured amphibian populations. This also is the first applied documentation that inter-patch landscape complexity can affect intra-patch community composition of amphibians as predicted by metapopulation theory. In the Southern High Plains, landscape complexity is positively associated with agricultural cultivation. Agricultural cultivation increases sedimentation, decreases hydroperiod, alters amphibian community dynamics, and negatively impacts postmetamorphic body size of amphibians in playa wetlands. Thus, conservation efforts should focus on preserving or restoring native landscape structure, hydroperiod, and connectivity among playas to maintain native amphibian populations and historic inter-playa movement.

Development and disease: how susceptibility to an emerging pathogen changes through anuran development.

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.

INFLUENCE OF LAND USE ON POSTMETAMORPHIC BODY SIZE OF PLAYA LAKE AMPHIBIANS

Abstract Agricultural land use may indirectly affect the body size of amphibians by altering the hydroperiods of nearby wetlands and influencing amphibian densities—both factors which can limit the larval and postmetamorphic growth rates of amphibians. We measured postmetamorphic body size for 4 species (Spea multiplicata, S. bombifrons, Bufo cognatus, Ambystoma tigrinum mavortium) and 3 age classes (metamorph, subadult, adult) of amphibians captured at playa wetlands surrounded by one of 2 general land-use types (cultivation, grassland) in the Southern High Plains. Sixteen playas (4 per land-use type in 1999 and 2000) were partially enclosed with drift fence and pitfall traps, and mass and snout-vent length (SVL) were measured from a subsample of captured individuals. Mass and SVL were 10–148% greater for amphibians captured at grassland wetlands than at cropland wetlands for most species and age classes. Mass and SVL also were 3–124% greater in 1999 than in 2000 for most species and age classes. We attribute differences in body size between land-use types to differences in the hydroperiods of the associated wetlands, and potentially to variation in the density of terrestrial conspecifics and aquatic predators. We attribute differences in body size between years to differences in rainfall. Body size is positively related to the probability of survival, reproduction, and evolutionary fitness in amphibians. Thus, if cultivation of landscapes surrounding wetlands negatively influences postmetamorphic body size of amphibians, restoration of native grasslands surrounding playa wetlands may help prevent local amphibian declines.

Ranavirus: past, present and future

Emerging infectious diseases are a significant threat to global biodiversity. While historically overlooked, a group of iridoviruses in the genus Ranavirus has been responsible for die-offs in captive and wild amphibian, reptile and fish populations around the globe over the past two decades. In order to share contemporary information on ranaviruses and identify critical research directions, the First International Symposium on Ranaviruses was held in July 2011 in Minneapolis, MN, USA. Twenty-three scientists and veterinarians from nine countries examined the ecology and evolution of ranavirus–host interactions, potential reservoirs, transmission dynamics, as well as immunological and histopathological responses to infection. In addition, speakers discussed possible mechanisms for die-offs, and conservation strategies to control outbreaks.

Agricultural Seed Biomass for Migrating and Wintering Waterfowl in the Southeastern United States

Abstract Waterfowl frequently acquire high-energy agricultural seeds in harvested and unharvested croplands during migration and winter. Estimates of agricultural seed biomass in harvested and unharvested corn, soybean, and grain sorghum fields do not exist or are outdated for the southeastern United States. Therefore, we estimated seed biomass in 105 harvested and 59 unharvested corn, soybean, and grain sorghum fields across 4 climate regions in Tennessee, USA, from September through January 2006 and 2007. We also used estimates of seed biomass to calculate duck-energy days (DEDs) in December and January when migratory waterfowl abundance peaks in the southeastern United States. Mean biomass of corn, soybean, and grain sorghum seed in harvested fields declined 239 kg/ha to 39 kg/ha, 118 kg/ha to 26 kg/ha, and 392 kg/ha to 19 kg/ha, respectively, from postharvest to January. Continuous monthly rates of decline were 64% for corn, 84% for soybean, and 74% for grain sorghum. Agricultural seed biomass in harvested corn and grain sorghum fields dropped below the waterfowl giving-up density (i.e., 50 kg/ha) in 3 months; soybean dropped below this threshold 1 month postharvest. Mean DEDs/ha in harvested corn, soybean, and grain sorghum fields were low (274, 90, and 27, respectively) in January, and DEDs were zero in >85% of fields. In unharvested corn, soybean, and grain sorghum fields, mean DEDs/ha in January were high (69,000, 18,000, and 26,000, respectively), and continuous rates of decline (3%, 7%, and 18%, respectively) were much lower than for harvested crops. Waterfowl biologists in the Southeast should use our estimates of agricultural seed biomass in DED calculations. We also recommend that biologists provide unharvested grain fields and natural wetlands for migrating and wintering waterfowl because seed resources are low in harvested agricultural fields.

Differential Responses of Postmetamorphic Amphibians to Cattle Grazing in Wetlands

Abstract Global amphibian declines have been linked to various anthropogenic land uses. Recent studies have documented negative impacts of cropland agriculture and deforestation on amphibians; however, few have examined potential impacts of cattle grazing in wetlands on resident amphibians. Therefore, we measured differences in number of captures and body size of postmetamorphic amphibians, egg mass abundance, and shoreline vegetation structure and composition between 4 wetlands with direct cattle access and 4 wetlands from which cattle were excluded on the Cumberland Plateau in Tennessee, USA. We captured amphibians at wetlands from March to August 2005 and 2006 using pitfall traps. Number of green frog (Rana clamitans) metamorphs captured at nonaccess wetlands was 2.5 times and 9.8 times greater than at wetlands with cattle access in 2005 and 2006, respectively. However, number of American toads (Bufo americanus) captured was 68 times and 76 times greater at cattle-access wetlands in 2005 and 2006, respectively. In general, metamorph body size was negatively correlated with species-specific capture rate. We detected no differences in egg mass abundance between cattle land-use types. Height, percent horizontal cover, and percent vertical cover of shoreline vegetation were 74%, 25%, and 84% greater, respectively, in nonaccess wetlands in 2005; vegetation trends were similar in 2006. Our results suggest that cattle impact amphibian populations but effects vary by species. Differences in postmetamorphic capture rate may be related to less emergent vegetation at cattle-access wetlands. Although body size differed between land uses for metamorphs, these trends probably were short-lived, because we did not detect differences in juvenile and adult body size between land uses for most species. Based on our findings, we suggest that fencing cattle from wetlands may be a prudent conservation strategy for some amphibian species (e.g., ranids), whereas other species (e.g., bufonids) may benefit from controlled grazing.