Evidence for emergence of an amphibian iridoviral disease because of human-enhanced spread

Our understanding of origins and spread of emerging infectious diseases has increased dramatically because of recent applications of phylogenetic theory. Iridoviruses are emerging pathogens that cause global amphibian epizootics, including tiger salamander (Ambystoma tigrinum) die-offs throughout western North America. To explain phylogeographical relationships and potential causes for emergence of western North American salamander iridovirus strains, we sequenced major capsid protein and DNA methyltransferase genes, as well as two noncoding regions from 18 geographically widespread isolates. Phylogenetic analyses of sequence data from the capsid protein gene showed shallow genetic divergence (< 1%) among salamander iridovirus strains and monophyly relative to available fish, reptile, and other amphibian iridovirus strains from the genus Ranavirus, suggesting a single introduction and radiation. Analysis of capsid protein sequences also provided support for a closer relationship of tiger salamander virus strains to those isolated from sport fish (e.g. rainbow trout) than other amphibian isolates. Despite monophyly based on capsid protein sequences, there was low genetic divergence among all strains (< 1.1%) based on a supergene analysis of the capsid protein and the two noncoding regions. These analyses also showed polyphyly of strains from Arizona and Colorado, suggesting recent spread. Nested clade analyses indicated both range expansion and long-distance colonization in clades containing virus strains isolated from bait salamanders and the Indiana University axolotl (Ambystoma mexicanum) colony. Human enhancement of viral movement is a mechanism consistent with these results. These findings suggest North American salamander ranaviruses cause emerging disease, as evidenced by apparent recent spread over a broad geographical area.     

Influence of temperature on Ranavirus infection in larval salamanders Ambystoma tigrinum

Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV). Most salamanders survived when exposed at 26 degrees C, whereas all died at 18 degrees C and nearly all died at 10 degrees C. Some asymptomatic salamanders that survived 60 d at 10 or 26 degrees C were found to be carrying virus. Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamanders but was found to be less sensitive than cell culture in detecting ATV at low concentrations. PCR products were 100% identical to ATV in the major capsid protein sequence. Virus titer was higher in salamanders held at 10 degrees C than at 18 degrees C but little virus, if any, was present in the small number of salamanders that died at 26 degrees C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely.     

Mortality Rates Differ Among Amphibian Populations Exposed to Three Strains of a Lethal Ranavirus

Infectious diseases are a growing threat to biodiversity, in many cases because of synergistic effects with habitat loss, environmental contamination, and climate change. Emergence of pathogens as new threats to host populations can also arise when novel combinations of hosts and pathogens are unintentionally brought together, for example, via commercial trade or wildlife relocations and reintroductions. Chytrid fungus (Batrachochytrium dendrobatidis) and amphibian ranaviruses (family Iridoviridae) are pathogens implicated in global amphibian declines. The emergence of disease associated with these pathogens appears to be at least partly related to recent translocations over large geographic distances. We experimentally examined the outcomes of novel combinations of host populations and pathogen strains using the amphibian ranavirus Ambystoma tigrinum virus (ATV) and barred tiger salamanders (Ambystoma mavortium, formerly considered part of the Ambystoma tigrinum complex). One salamander population was highly resistant to lethal infections by all ATV strains, including its own strain, and mortality rates differed among ATV strains according to salamander population. Mortality rates in novel pairings of salamander population and ATV strain were not predictable based on knowledge of mortality rates when salamander populations were exposed to their own ATV strain. The underlying cause(s) for the differences in mortality rates are unknown, but local selection pressures on salamanders, viruses, or both, across the range of this widespread host–pathogen system are a plausible hypothesis. Our study highlights the need to minimize translocations of amphibian ranaviruses, even among conspecifc host populations, and the importance of considering intraspecific variation in endeavors to manage wildlife diseases.     

Isolation and characterisation of a novel Bohle-like virus from two frog species in the Darwin rural area, Australia

Twelve captive magnificent tree frogs Litoria splendida and 2 green tree frogs L. caerulea on a property in the Darwin rural area (Northern Territory, Australia) either died or were euthanased after becoming lethargic or developing skin lesions. Samples from both species of frog were submitted for histopathology and virus isolation. An irido-like virus was cultured from tissue samples taken from both species and was characterised using electron microscopy, restriction enzyme digests and nucleic acid amplification and sequencing. The isolates were determined to belong to the genus Ranavirus, were indistinguishable from each other and shared a 98.62% nucleotide similarity and a 97.32% deduced amino acid homology with the Bohle iridovirus over a 1161 bp region of the major capsid gene. This is the first isolation of a ranavirus from amphibians in the Northern Territory and the first report of natural infection in these 2 species of native frog. The virus is tentatively named Mahaffey Road virus (MHRV).     

First case of ranavirus-associated morbidity and mortality in natural populations of the South American frog Atelognathus patagonicus

Atelognathus patagonicus is an endangered leptodactylid frog endemic to a small region in and around Laguna Blanca National Park in northern Patagonia, Argentina. All of the lakes and small ponds of the region (except Laguna Blanca itself) contain A. patagonicus and in all but one of these lakes the species shows clinical signs of a previously undiagnosed disease, the characteristics of which suggested a ranavirus. We collected symptomatic and asymptomatic A. patagonicus frogs and tadpoles from 4 small lakes and analyzed tissues for ranavirus and the chytrid fungus Batrachochytrium dendrobatidis using PCR amplification of pathogen DNA. Of the 32 specimens tested, 25 were positive for ranavirus major capsid protein (MCP). Sequence alignments of the ranavirus MCP from these specimens showed 100% similarity with published FV3 and FV3-like viruses from anurans, 98 to 99 % similarity with Bohle iridovirus, and 95 % similarity with Ambystoma tigrinum virus (ATV) and Regina ranavirus (RRV). A search of the NCBI Blast nucleotide database using the 500 base pair MCP sequence obtained from these samples did not suggest any homology to any other pathogen. In addition, 1 sample (3 pooled individuals) from 1 lake tested positive for B. dendrobatidis. The clinical signs observed primarily in late-stage tadpoles and recent metamorphs, which have reoccurred each year since at least 2001, are consistent with ranaviral disease, but until histopathology of diseased individuals is carried out, chytridiomycosis or other diseases cannot be ruled out.     

Diagnostic and molecular evaluation of three iridovirus-associated salamander mortality events

In 1998 viruses were isolated from tiger salamander larvae (Ambystoma tigrinum diaboli and A. tigrinum melanostictum) involved in North Dakota and Utah (USA) mortality events and spotted salamander (A. maculatum) larvae in a third event in Maine (USA). Although sympatric caudates and anurans were present at all three sites only ambystomid larvae appeared to be affected. Mortality at the North Dakota site was in the thousands while at the Utah and Maine sites mortality was in the hundreds. Sick larvae were lethargic and slow moving. They swam in circles with obvious buoyancy problems and were unable to remain upright. On the ventral surface, near the gills and hind limbs, red spots or swollen areas were noted. Necropsy findings included: hemorrhages and ulceration of the skin, subcutaneous and intramuscular edema, swollen and pale livers with multifocal hemorrhage, and distended fluid-filled intestines with areas of hemorrhage. Light microscopy revealed intracytoplasmic inclusions, suggestive of a viral infection, in a variety of organs. Electron microscopy of ultra thin sections of the same tissues revealed iridovirus-like particles within the inclusions. These viruses were isolated from a variety of organs, indicating a systemic infection. Representative viral isolates from the three mortality events were characterized using molecular assays. Characterization confirmed that the viral isolates were iridoviruses and that the two tiger salamander isolates were similar and could be distinguished from the spotted salamander isolate. The spotted salamander isolate was similar to frog virus 3, the type species of the genus Ranavirus, while the tiger salamander isolates were not. These data indicate that different species of salamanders can become infected and die in association with different iridoviruses. Challenge assays are required to determine the fish and amphibian host range of these isolates and to assess the susceptibility of tiger and spotted salamanders to heterologous virus isolates.     

Frog virus 3-like infections in aquatic amphibian communities

Frog virus 3 (FV3) and FV3-like viruses, are members of the genus Ranavirus (family Iridoviridae), and they have been associated with infectious diseases that may be contributing to amphibian population declines. We examined the mode of transmission of an FV3-like virus, and potential hosts and reservoirs of the virus in a local amphibian community. Using the polymerase chain reaction to detect infected animals, we found an FV3-like virus in south-central Ontario, Canada, amphibian communities, where it infects sympatric amphibian species, including ranid and hylid tadpoles (Rana sylvatica, Hyla versicolor, and Pseudacris spp.), larval salamanders (Ambystoma spp.), and adult eastern-spotted newts (Notophthalmus viridescens). The high prevalence of FV3-like infections in caudate larvae suggests that salamanders are likely to be both hosts and reservoirs. In laboratory FV3 challenges of R. sylvatica, the rate of infection was dependent on the amount of virus to which the animals were exposed. In addition, although vertical transmission was suspected, horizontal transmission through exposure to infected pond water is the most likely route of infection in tadpoles. Based on our observations, a simple model of FV3/FV3-like virus transmission postulates that, in aquatic amphibian communities, transmission of the virus occurs between anuran and urodele species, with ambystomatid salamanders the most likely reservoir for the ranavirus in our study.     

Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina

A large icosahedral virus was isolated from diseased grouper Epinephelus tauvina. The virus grew well in several cultured fish cell lines, with stable and high infectivity after serial passages in grouper cell line (GP). The virus was sensitive to both acid and heat treatments. Virus replication was inhibited by 5-iodo-2-deoxyuridine (IUDR), indicative of a DNA-containing genome. The virus infectivity was reduced with ether treatment, suggesting that the virus was lipid-enveloped. Electron micrographs showed abundant cytoplasmic icosahedral virons in the virus-infected GP cells. The size of the intracellular nucleocapsid was 154 nm between the opposite sides, or 176 nm between the opposite vertices with an inner electron-dense core of 93 nm. Virus particles were released through budding from plasma membranes with a size of 200 nm in diameter. SDS-PAGE of purified virus revealed 20 structural protein bands and a major capsid protein (MCP) of 49 kDa. A DNA fragment of approximately 500 nucleotides was successfully amplified by polymerase chain reaction (PCR) using the primers from conserved regions of the MCP gene of frog virus 3 (FV3), the type species of Ranavirus. Subsequent multiple alignment and phylogenetic analysis showed that the newly isolated grouper virus was closely related to largemouth bass virus (LMBV), FV3 and Regina ranavirus (RRV). Our data suggests that the virus isolate is a novel member of genus Ranavirus, family Iridoviridae. We tentatively name the virus as Singapore grouper iridovirus (SGIV). SGIV was able to cause serious systemic disease capable of killing 96% of grouper fry.     

Influence of larval stage and virus inoculum on virus yield in insect host Neodiprion abietis (Hymenoptera: Diprionidae)

Virus yield produced by dead larvae of balsam fir sawfly, Neodiprion abietis (Harris) (Hymenoptera: Diprionidae), that had been infected at four different larval stages (second, third, fourth, or fifth instar) with two virus concentrations (10(5) polyhedral inclusion bodies (PIB) /ml or 10(7) PIB/ml), were analyzed and compared to determine the effects of instar and amount of virus inoculum on virus production. The results indicate that both larval stage and inoculation dosage significantly affect virus yield. On average, each dead larva produced 1.36-12.21 x 10(7) PIB, depending upon larval age and virus concentration of inoculation. Although each dead larva produced more PIB when it was inoculated in the fourth or fifth stage, inoculation of these larvae did not result in the highest virus yield because of low larval mortality. In terms of net virus return, third instars would maximize virus yield when they are inoculated with a virus concentration that can cause 95-100% larval mortality.     

First case of ranavirus-associated mass mortality in a natural population of the Huanren frog (Rana huanrenensis) tadpoles in South Korea

Globally, ranavirus is often responsible for the mass mortality of a variety of captive and wild amphibians. In Asia, several mass mortality cases of captive amphibians by ranavirus are known, but one mass mortality case in the wild has been reported in a non-endemic larval bullfrog population in Japan. In order to verify factors involved in mass mortality of Rana huanrenensis tadpoles (> 200 tadpoles) in a mountain stream in South Korea, we investigated possible infections by ranavirus, chytrid fungus, and lethal bacteria by conducting PCR assays of pathogens with specific primers. We found that all R. huanrenensis tadpoles collected (two alive and ten carcasses) showed positive PCR results for two different ranavirus primer sets targeting partial genes of a major capsid protein (MCP). The identified MCP sequence was more closely related to Rana catesbeiana virus JP MCP, isolated from invasive bullfrog tadpoles in Japan. We could not detect any lethal bacteria or chytrid fungus in the specimens. Our finding is the first report in Asia that ranavirus is involved in the mass mortality of endemic wild amphibians.     

Identification and characterization of a ross river virus variant that grows persistently in macrophages, shows altered disease kinetics in a mouse model, and exhibits resistance to type I interferon

Alphaviruses, such as chikungunya virus, o'nyong-nyong virus, and Ross River virus (RRV), cause outbreaks of human rheumatic disease worldwide. RRV is a positive-sense single-stranded RNA virus endemic to Australia and Papua New Guinea. In this study, we sought to establish an in vitro model of RRV evolution in response to cellular antiviral defense mechanisms. RRV was able to establish persistent infection in activated macrophages, and a small-plaque variant (RRV(PERS)) was isolated after several weeks of culture. Nucleotide sequence analysis of RRV(PERS) found several nucleotide differences in the nonstructural protein (nsP) region of the RRV(PERS) genome. A point mutation was also detected in the E2 gene. Compared to the parent virus (RRV-T48), RRV(PERS) showed significantly enhanced resistance to beta interferon (IFN-β)-stimulated antiviral activity. RRV(PERS) infection of RAW 264.7 macrophages induced lower levels of IFN-β expression and production than infection with RRV-T48. RRV(PERS) was also able to inhibit type I IFN signaling. Mice infected with RRV(PERS) exhibited significantly enhanced disease severity and mortality compared to mice infected with RRV-T48. These results provide strong evidence that the cellular antiviral response can direct selective pressure for viral sequence evolution that impacts on virus fitness and sensitivity to alpha/beta IFN (IFN-α/β).     

Patterns of habitat and invertebrate diet overlap between tiger salamanders and ducks in prairie potholes

During the breeding season, migratory waterfowl are attracted to wetlands characterized by high macroinvertebrate availability. Many of these prairie potholes are fishless and this apparent void is filled, at least partially, by tiger salamanders. Based on gut contents from 98 tiger salamanders and published diet data from over 1500 ducks, we show that there is general overlap in diet between both larval and adult tiger salamanders and 10 duck species. Furthermore, when the ducks were split into foraging guilds and compared with tiger salamanders, prey type overlap was 1.7 times higher and prey size was 1.8 times higher with dabbling ducks than diving ducks. Field surveys show that tiger salamander density is more highly correlated with diving duck density across potholes than dabbling duck density. Tiger salamanders have higher diet overlap with dabbling ducks than diving ducks whereas tiger salamanders have higher spatial overlap with diving ducks than dabbling ducks suggesting that these consumers coarsely partition diet and habitat resources. It has been reported that tiger salamanders have specialized diets that are associated with foraging preferences for benthic habitats. This view is too narrow: in southwestern Manitoba, Canada, tiger salamanders are more general consumers with diets more like dabbling ducks that forage mostly in planktonic and littoral habitats. Our results suggest that dabbling and diving ducks are, to different extents, liable to the effects of indirect interactions, specifically competition for common prey, with tiger salamanders.     

Amphibian malformations and inbreeding

Inbreeding may lead to morphological malformations in a wide variety of taxa. We used genetic markers to evaluate whether malformed urodeles were more inbred and/or had less genetic diversity than normal salamanders. We captured 687 adult and 1259 larval tiger salamanders (Ambystoma tigrinum tigrinum), assessed each individual for gross malformations, and surveyed genetic variation among malformed and normal individuals using both cytoplasmic and nuclear markers. The most common malformations in both adults and larvae were brachydactyly, ectrodactyly and polyphalangy. The overall frequency of adults with malformations was 0.078 compared to 0.081 in larval samples. Genetic diversity was high in both normal and malformed salamanders, and there were no significant difference in measures of inbreeding (f and F), allele frequencies, mean individual heterozygosity or mean internal relatedness. Environmental contaminants or other extrinsic factors may lead to genome alternations that ultimately cause malformations, but our data indicate that inbreeding is not a causal mechanism.     

Pathogen Host Switching in Commercial Trade with Management Recommendations

Global wildlife trade exacerbates the spread of nonindigenous species. Pathogens also move with hosts through trade and often are released into naïve populations with unpredictable outcomes. Amphibians are moved commercially for pets, food, bait, and biomedicine, and are an excellent model for studying how wildlife trade relates to pathogen pollution. Ranaviruses are amphibian pathogens associated with annual population die-offs; multiple strains of tiger salamander ranaviruses move through the bait trade in the western United States. Ranaviruses infect amphibians, reptiles, and fish and are of additional concern because they can switch hosts. Tiger salamanders are used as live bait for freshwater fishing and are a potential source for ranaviruses switching hosts from amphibians to fish. We experimentally injected largemouth bass with a bait trade tiger salamander ranavirus. Largemouth bass became infected but exhibited no signs of disease or mortality. Amphibian bait ranaviruses have the potential to switch hosts to infect fish, but fish may act as dead-end hosts or nonsymptomatic carriers, potentially spreading infection as a result of trade.     

Five amphibian mortality events associated with ranavirus infection in south central Ontario, Canada

Using field, molecular and histological methods, an epizootic, systemic disease causing death within wood frog Rana sylvatica tadpoles and leopard frog Rana pipiens metamorphs at 3 different locations within Southern Ontario, Canada, has been investigated. Our results demonstrated that the probable cause of this disease was a ranavirus. Affected amphibians were found to exhibit necrosis within the hematopoietic cells. Liver tissue samples were found positive for the virus by PCR amplification of the ranavirus (Family: Iridoviridae) major capsid protein (MCP). Positive samples were confirmed by sequence analysis. Clinically normal, laboratory-raised wood frog egg broods were also found to test weakly positive for ranavirus. The population effects of disease on these amphibian communities have not yet been conclusively associated with population declines, but warrant more focused consideration.     

Dose and host characteristics influence virulence of ranavirus infections

Parasites play a prominent role in the ecology, evolution, and more recently, conservation of many organisms. For example, emerging infectious diseases, including a group of lethal ranaviruses, are associated with the declines and extinctions of amphibians around the world. An increasingly important basic and applied question is: what controls parasite virulence? We used a dose-response experiment with three laboratory-bred clutches of tiger salamander larvae (Ambystoma tigrinum) to test how the size of inoculum and host genetic factors influence the dynamics and outcome of ranavirus infections. We found that infection rates increased with dose and were strongly affected by clutch identity and host life history stage. Case mortality increased with dose of inoculum, but was unaffected by host characteristics. Average survival time decreased with dose and differed among clutches, but this was largely due to differences in the time to onset of symptoms. Overall, our results suggest that dose of inoculum and host characteristics (life history stage and genetic background) influence the establishment and early virus replication, and therefore the virulence of ranavirus infections.     

Functional design of the feeding mechanism in lower vertebrates: unidirectional and bidirectional flow systems in the tiger salamander

There are two basic designs of the aquatic feeding mechanism in lower vertebrates: unidirectional and bidirectional flow systems. Larval salamanders and most fishes posses a unidirectional flow design in which water drawn into the mouth with the prey passes over the gills and exits posteriorly. Metamorphosed salamanders and all other aquatic vertebrates possess a bidirectional system in which water flows into and out of the mouth during a single feeding cycle. We investigated the functional consequences of these two feeding designs in larval and metamorphosed tiger salamanders ( Ambystoma tigrinum ) feeding in the water. Buccal cavity pressures were measured during feeding and 11 variables measured from the pressure traces. Significant differences were found between the larval and metamorphosed salamanders in eight variables. Larval salamanders generate significantly greater negative pressures than do metamorphosed individuals and a principal components analysis of the 11 pressure variables completely separates larval from metamorphosed salamanders. Larval individuals are significantly better at capturing elusive prey than are metamorphosed salamanders, apparently because of changes in the structure of the feeding mechanism and the concomitant functional modifications.     

Helminth and leech community structure in tadpoles and caudatan larvae of two amphibian species from Western Nebraska

Currently no comparative studies exist on helminth and leech community structure among sympatric anuran tadpoles and salamander larvae. During June-August 2007-2009, we examined 50 bullfrog tadpoles, Rana catesbeiana , 50 barred tiger salamander larvae, Ambystoma mavortium , and 3 species of snails from Nevens Pond, Keith County, Nebraska for helminth and leech infections. The helminth and leech compound community of this larval amphibian assemblage consisted of at least 7 species, 4 in bullfrog tadpoles and 4 in barred tiger salamander larvae. Bullfrog tadpoles were infected with 2 species of nematodes ( Gyrinicola batrachiensis and Spiroxys sp.) and 2 types of metacercariae ( Telorchis sp. and echinostomatids), whereas barred tiger salamander larva were infected with 1 species of leech ( Placobdella picta ), 2 species of adult trematodes ( Telorchis corti and Halipegus sp.), and 1 species of an unidentified metacercaria. The component community of bullfrog tadpoles was dominated by helminths acquired through active penetration, or incidentally ingested through respiratory currents, or both, whereas the component community of larval salamanders was dominated by helminths acquired through ingestion of intermediate hosts (χ2 = 3,455.00, P < 0.00001). Differences in amphibian larval developmental time (2-3 yr for bullfrog tadpoles versus 2-5 mo for salamander larvae), the ephemeral nature of intermediate hosts in Nevens Pond, and the ability of bullfrog tadpole to eliminate echinostome infections had significant effects on mean helminth species richness among amphibian species and years (t = 12.31, P < 0.0001; t = 2.09, P = 0.04). Differences in herbivorous and carnivorous diet and time to metamorphosis among bullfrog tadpoles and barred tiger salamander larvae were important factors in structuring helminth communities among the larval stages of these 2 sympatric amphibian species, whereas size was important in structuring helminth and leech communities in larval salamanders, but not in bullfrog tadpoles.