Functional variation at an expressed MHC class IIß locus associates with Ranavirus infection intensity in larval anuran populations.

Infectious diseases are causing catastrophic losses to global biodiversity. Iridoviruses in the genus Ranavirus are among the leading causes of amphibian disease-related mortality. Polymorphisms in major histocompatibility complex (MHC) genes are significantly associated with variation in amphibian pathogen susceptibility. MHC genes encode two classes of polymorphic cell-surface molecules that can recognize and bind to diverse pathogen peptides. While MHC class I genes are the classic mediators of viral-acquired immunity, larval amphibians do not express them. Consequently, MHC class II gene diversity may be an important predictor of Ranavirus susceptibility in larval amphibians, the life stage most susceptible to Ranavirus. We surveyed natural populations of larval wood frogs (Rana sylvatica), which are highly susceptible to Ranavirus, across 17 ponds and 2 years in Maryland, USA. We sequenced the peptide-binding region of an expressed MHC class IIß locus and assessed allelic and genetic diversity. We converted alleles to functional supertypes and determined if supertypes or alleles influenced host responses to Ranavirus. Among 381 sampled individuals, 26% were infected with Ranavirus. We recovered 20 unique MHC class IIß alleles that fell into two deeply diverged clades and seven supertypes. MHC genotypes were associated with Ranavirus infection intensity, but not prevalence. Specifically, MHC heterozygotes and supertype ST1/ST7 had significantly lower Ranavirus infection intensity compared to homozygotes and other supertypes. We conclude that MHC class IIß functional genetic variation is an important component of Ranavirus susceptibility. Identifying immunogenetic signatures linked to variation in disease susceptibility can inform mitigation strategies for combatting global amphibian declines.

Complete genome sequence of a divergent strain of Tibetan frog hepatitis B virus associated with a concave-eared torrent frog (Odorrana tormota).

Viruses of the family Hepadnaviridae are characterized by partially dsDNA circular genomes of approximately 3.2 kb, which are reverse transcribed from RNA intermediates. Hepadnaviruses have a broad host range, which includes humans (hepatitis B virus), other mammals (genus Orthohepadnavirus), and birds (genus Avihepadnavirus). The known host specificity of hepadnaviruses has been expanded by reports of new viruses infecting fish, amphibians, and reptiles. Tibetan frog hepatitis B virus (TFHBV) was recently discovered in a member of the species Nanorana parkeri (family Dicroglossidae) from Tibet. To increase our understanding of hepadnaviruses that infect amphibian hosts, we identified the full-length genome of a divergent strain, TFHBV-Ot, associated with a concave-eared torrent frog (Odorrana tormota, family Ranidae) from China by searching deep-sequencing data. TFHBV-Ot shared a genomic organization and 76.6% overall genome sequence nucleotide identity with the prototype TFHBV associated with N. parkeri (TFHBV-Np). The pairwise amino acid sequence identity between the predicted gene products of TFHBV-Ot and TFHBV-Np ranged between 63.9% and 77.9%. Multiple tissue/organ-specific RNAseq datasets suggested a broad tropism of TFHBV, including muscle, gonads and brain. In addition, we provide information about putative virus-derived small RNAs from an amphibian hepadnavirus. The results presented here expand the known genetic diversity and host range of TFHBV to Ranidae frogs, and warrant an investigation of hepadnaviral infection of amphibian brains.

Divergent transcriptomic responses underlying the ranaviruses-amphibian interaction processes on interspecies infection of Chinese giant salamander.

BACKGROUND: Ranaviruses (family Iridoviridae, nucleocytoplasmic large DNA viruses) have been reported as promiscuous pathogens of cold-blooded vertebrates. Rana grylio virus (RGV, a ranavirus), from diseased frog Rana grylio with a genome of 105.79 kb and Andrias davidianus ranavirus (ADRV), from diseased Chinese giant salamander (CGS) with a genome of 106.73 kb, contains 99% homologous genes. RESULTS: To uncover the differences in virus replication and host responses under interspecies infection, we analyzed transcriptomes of CGS challenged with RGV and ADRV in different time points (1d, 7d) for the first time. A total of 128,533 unigenes were obtained from 820,858,128 clean reads. Transcriptome analysis revealed stronger gene expression of RGV than ADRV at 1 d post infection (dpi), which was supported by infection in vitro. RGV replicated faster and had higher titers than ADRV in cultured CGS cell line. RT-qPCR revealed the RGV genes including the immediate early gene (RGV-89R) had higher expression level than that of ADRV at 1 dpi. It further verified the acute infection of RGV in interspecies infection. The number of differentially expressed genes and enriched pathways from RGV were lower than that from ADRV, which reflected the variant host responses at transcriptional level. No obvious changes of key components in pathway "Antigen processing and presentation" were detected for RGV at 1 dpi. Contrarily, ADRV infection down-regulated the expression levels of MHC I and CD8. The divergent host immune responses revealed the differences between interspecies and natural infection, which may resulted in different fates of the two viruses. Altogether, these results revealed the differences in transcriptome responses among ranavirus interspecies infection of amphibian and new insights in DNA virus-host interactions in interspecies infection. CONCLUSION: The DNA virus (RGV) not only expressed self-genes and replicated quickly after entry into host under interspecies infection, but also avoided the over-activation of host responses. The strategy could gain time for the survival of interspecies pathogen, and may provide opportunity for its adaptive evolution and interspecies transmission.

FV3-like ranavirus infection outbreak in black-spotted pond frogs (Rana nigromaculata) in China.

In April 2016, an outbreak emerged in a cultured population of black-spotted pond frog tadpoles in Shuangliu County, China, whereas tadpoles were suffering from substantial mortality (90%). Principal clinical signs of diseased tadpoles were comprised haemorrhage on their body surface, swollen abdomen with yellow ascites, congestion and swelling of the liver. The diseased tadpole's homogenates tissue were inoculated into epithelioma papulosum cyprini (EPC) cells at 25 °C for 4 days which caused typical cytopathic effect, and the viral titer TCID50 reached 107/0.1 mL. In pathogenicity tests, tadpoles were immersed in 2‰ virus fluid for 8 h, the clinical signs were observed similar to those recognized in naturally infected tadpoles and mortality rate were reached up to 80%, which affirms that the virus was the main cause for this disease. In addition, transmission electron microscopy of EPC cells infected with isolated virus reflected that the virus was in a regular hexagon way (shape) with capsule like structure. The diagonal diameter was recorded 135 ±â€¯8 nm, wherever virus particles were arrayed in crystalline manner in the cytoplasm. The electrophoresis of MCP gene PCR-product showed that the samples of diseased tadpoles, aquaculture water source and isolated virus were all positive. The sequence of the isolate revealed more than 99% similarities to ranavirus based on homology and genetic evolution analysis of the whole MCP gene, and the isolate belongs to FV3-like virus group. This study confirmed that ranavirus was the causative agent of this outbreak, and named the virus as Rana nigromaculata ranavirus (RNRV).

Pathogen richness and abundance predict patterns of adaptive major histocompatibility complex variation in insular amphibians.

The identification of the factors responsible for genetic variation and differentiation at adaptive loci can provide important insights into the evolutionary process and is crucial for the effective management of threatened species. We studied the impact of environmental viral richness and abundance on functional diversity and differentiation of the MHC class Ia locus in populations of the black-spotted pond frog (Pelophylax nigromaculatus), an IUCN-listed species, on 24 land-bridge islands of the Zhoushan Archipelago and three nearby mainland sites. We found a high proportion of private MHC alleles in mainland and insular populations, corresponding to 32 distinct functional supertypes, and strong positive selection on MHC antigen-binding sites in all populations. Viral pathogen diversity and abundance were reduced at island sites relative to the mainland, and islands housed distinctive viral communities. Standardized MHC diversity at island sites exceeded that found at neutral microsatellites, and the representation of key functional supertypes was positively correlated with the abundance of specific viruses in the environment (Frog virus 3 and Ambystoma tigrinum virus). These results indicate that pathogen-driven diversifying selection can play an important role in maintaining functionally important MHC variation following island isolation, highlighting the importance of considering functionally important genetic variation and host-pathogen associations in conservation planning and management.

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.

Pathogenesis of Frog Virus 3 ( Ranavirus, Iridoviridae) Infection in Wood Frogs ( Rana sylvatica).

Wood frogs ( Rana sylvatica) are highly susceptible to infection with Frog virus 3 (FV3, Ranavirus, Iridoviridae), a cause of mass mortality in wild populations. To elucidate the pathogenesis of FV3 infection in wood frogs, 40 wild-caught adults were acclimated to captivity, inoculated orally with a fatal dose of 104.43 pfu/frog, and euthanized at 0.25, 0.5, 1, 2, 4, 9, and 14 days postinfection (dpi). Mild lesions occurred sporadically in the skin (petechiae) and bone marrow (necrosis) during the first 2 dpi. Severe lesions occurred 1 to 2 weeks postinfection and consisted of necrosis of medullary and extramedullary hematopoietic tissue, lymphoid tissue in spleen and throughout the body, and epithelium of skin, mucosae, and renal tubules. Viral DNA was first detected (polymerase chain reaction) in liver at 4 dpi; by dpi 9 and 14, all viscera tested (liver, kidney, and spleen), skin, and feces were positive. Immunohistochemistry (IHC) first detected viral antigen in small areas devoid of histologic lesions in the oral mucosa, lung, and colon at 4 dpi; by 9 and 14 dpi, IHC labeling of viral antigen associated with necrosis was found in multiple tissues. Based on IHC staining intensity and lesion severity, the skin, oral, and gastrointestinal epithelium and renal tubular epithelium were important sites of viral replication and shedding, suggesting that direct contact (skin) and fecal-oral contamination are effective routes of transmission and that skin tissue, oral, and cloacal swabs may be appropriate antemortem diagnostic samples in late stages of disease (>1 week postinfection) but poor samples to detect infection in clinically healthy frogs.

Clinical signs, pathology and dose-dependent survival of adult wood frogs, Rana sylvatica, inoculated orally with frog virus 3 Ranavirus sp., Iridoviridae.

Amphibian populations suffer massive mortalities from infection with frog virus 3 FV3, genus Ranavirus, family Iridoviridae, a pathogen also involved in mortalities of fish and reptiles. Experimental oral infection with FV3 in captive-raised adult wood frogs, Rana sylvatica Lithobates sylvaticus, was performed as the first step in establishing a native North American animal model of ranaviral disease to study pathogenesis and host response. Oral dosing was successful LD50 was 10(2.93 2.423.44) p.f.u. for frogs averaging 35mm in length. Onset of clinical signs occurred 614days post-infection p.i. median 11 days p.i. and time to death was 1014 days p.i. median 12 days p.i.. Each tenfold increase in virus dose increased the odds of dying by 23-fold and accelerated onset of clinical signs and death by approximately 15. Ranavirus DNA was demonstrated in skin and liver of all frogs that died or were euthanized because of severe clinical signs. Shedding of virus occurred in faeces 710 days p.i. 34.5days before death and skin sheds 10 days p.i. 01.5days before death of some frogs dead from infection. Most common lesions were dermal erosion and haemorrhages haematopoietic necrosis in bone marrow, kidney, spleen and liver and necrosis in renal glomeruli, tongue, gastrointestinal tract and urinary bladder mucosa. Presence of ranavirus in lesions was confirmed by immunohistochemistry. Intracytoplasmic inclusion bodies probably viral were present in the bone marrow and the epithelia of the oral cavity, gastrointestinal tract, renal tubules and urinary bladder. Our work describes a ranaviruswood frog model and provides estimates that can be incorporated into ranavirus disease ecology models.

High susceptibility of the endangered dusky gopher frog to ranavirus.

Amphibians are one of the most imperiled vertebrate groups, with pathogens playing a role in the decline of some species. Rare species are particularly vulnerable to extinction because populations are often isolated and exist at low abundance. The potential impact of pathogens on rare amphibian species has seldom been investigated. The dusky gopher frog Lithobates sevosus is one of the most endangered amphibian species in North America, with 100-200 individuals remaining in the wild. Our goal was to determine whether adult L. sevosus were susceptible to ranavirus, a pathogen responsible for amphibian die-offs worldwide. We tested the relative susceptibility of adult L. sevosus to ranavirus (103 plaque-forming units) isolated from a morbid bullfrog via 3 routes of exposure: intra-coelomic (IC) injection, oral (OR) inoculation, and water bath (WB) exposure. We observed 100% mortality of adult L. sevosus in the IC and WB treatments after 10 and 19 d, respectively. Ninety-five percent mortality occurred in the OR treatment over the 28 d evaluation period. No mortality was observed in the control treatment after 28 d. Our results indicate that L. sevosus is susceptible to ranavirus, and if adults in the wild are exposed to this pathogen, significant mortality could occur. Additionally, our study demonstrates that some adult amphibian species can be very susceptible to ranavirus, which has been often overlooked in North American studies. We recommend that conservation planners consider testing the susceptibility of rare amphibian species to ranavirus and that the adult age class is included in future challenge experiments.

Infection and co-infection by the amphibian chytrid fungus and ranavirus in wild Costa Rican frogs.

Amphibian populations are globally threatened by emerging infectious diseases, and 2 pathogens in particular are recognized as major threats: the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) and ranaviruses. Here, we evaluated the prevalence of infection by Bd and ranavirus in an assemblage of frogs from a lowland wet forest in Costa Rica. We found an overall prevalence of 21.3% for Bd and 16.6% for ranavirus, and detected both pathogens widely among our 20 sampled species. We found a positive association between ranavirus and Bd infection in one of our 4 most commonly sampled species. We also found a positive but non-significant association between infection by ranavirus and infection by Bd among species overall. Our study is among the first detailed evaluations of ranavirus prevalence in the American tropics, and to our knowledge is the first to detect a positive association between Bd and ranavirus in any species. Considerable research attention has focused on the ecology of Bd in tropical regions, yet we argue that greater research focus is necessary to understand the ecology and conservation impact of ranaviruses on amphibian populations already decimated by the emergence of Bd.

Co-infection by alveolate parasites and frog virus 3-like ranavirus during an amphibian larval mortality event in Florida, USA.

A multispecies amphibian larval mortality event, primarily affecting American bullfrogs Lithobates catesbeianus, was investigated during April 2011 at the Mike Roess Gold Head Branch State Park, Clay County, Florida, USA. Freshly dead and moribund tadpoles had hemorrhagic lesions around the vent and on the ventral body surface, with some exhibiting a swollen abdomen. Bullfrogs (100%), southern leopard frogs L. sphenocephalus (33.3%), and gopher frogs L. capito (100%) were infected by alveolate parasites. The intensity of infection in bullfrog livers was high. Tadpoles were evaluated for frog virus 3 (FV3) by histology and PCR. For those southern leopard frog tadpoles (n = 2) whose livers had not been obscured by alveolate spore infection, neither a pathologic response nor intracytoplasmic inclusions typically associated with clinical infections of FV3-like ranavirus were noted. Sequencing of a portion (496 bp) of the viral major capsid protein gene confirmed FV3-like virus in bullfrogs (n = 1, plus n = 6 pooled) and southern leopard frogs (n = 1, plus n = 4 pooled). In July 2011, young-of-the-year bullfrog tadpoles (n = 7) were negative for alveolate parasites, but 1 gopher frog tadpole was positive. To our knowledge, this is the first confirmed mortality event for amphibians in Florida associated with FV3-like virus, but the extent to which the virus played a primary role is uncertain. Larval mortality was most likely caused by a combination of alveolate parasite infections, FV3-like ranavirus, and undetermined etiological factors.

Sequencing and analysis of the complete genome of Rana grylio virus (RGV).

Infection with Rana grylio virus (RGV), an iridovirus isolated in China in 1995, resulted in a high mortality rate in frogs. The complete genome sequence of RGV was determined and analyzed. The genomic DNA was 105,791 bp long, with 106 open reading frames (ORFs). Dot plot analysis showed that the gene order of RGV shared colinearity with three completely sequenced ranaviruses. A phylogenetic tree was constructed based on concatenated sequences of iridovirus 26 core-gene-encoded proteins, and the result showed high bootstrap support for RGV being a member of the genus Ranavirus and that iridoviruses of other genera also clustered closely. A microRNA (miRNA) prediction revealed that RGV could encode 18 mature miRNAs, many of which were located near genes associated with virus replication. Thirty-three repeated sequences were found in the RGV genome. These results provide insight into the genetic nature of RGV and are useful for laboratory diagnosis for vertebrate iridoviruses.

Induction of antimicrobial peptides from Rana dybowskii under Rana grylio virus stress, and bioactivity analysis.

The skin glands of Ranidae are a rich source of antimicrobial peptides. In this study, the genomic RNA of Rana dybowskii was extracted from its skin while under Rana grylio virus stress. Five new cDNA sequences encoding 5 mature peptides, Ranatuerin-2YJ (GLMDIFKVAVNKLLAAGMNKPRCKAAHC), Dybowskin-YJb (IIPLPLGYFAKKP), Dybowskin-YJa (IIPLPLGYFAKKKKKKDPVPLDQ), Temperin-YJa (VLPLLETCSMTCWENNQTFGK), and Temperin-YJb (VLPLVGNLLNDLLGK), were obtained by reverse transcription polymerase chain reaction with a pair of degenerate primers designed according to the conserved terminal sequences of cDNA encoding antimicrobial peptide precursors of genus Rana. The antimicrobial activities of the peptides were analyzed, and the results demonstrated that all these peptides showed a significant anti-Rana grylio virus activity, and the virus was gradually cleared with the increase in gene expression. Among the 5 peptides obtained in this work, Ranatuerin-2YJ also showed a broad-spectrum anti-Gram-positive bacteria and anti-Gram-negative bacteria activity with a minimal inhibitory concentration of 22.5 µg/mL and 7.64% hemolysis activity, both of which were significantly lower (p < 0.05) than that of the other peptides. Moreover, Ranatuerin-2YJ was widely distributed in the skin, liver, spleen, and blood of R. dybowskii, while the other 4 peptides could only be cloned from the skin, indicating that the Ranatuerin-2YJ in vivo plays an important role in the protection against pathogen invasion.

Prevalence of Ranavirus Infection in Three Anuran Species across South Korea.

To cope with amphibian die-offs caused by ranavirus, it is important to know the underlying ranavirus prevalence in a region. We studied the ranavirus prevalence in tadpoles of two native and one introduced anuran species inhabiting agricultural and surrounding areas at 49 locations across eight provinces of South Korea by applying qPCR. The local ranavirus prevalence and the individual infection rates at infected locations were 32.6% and 16.1%, respectively, for Dryophytes japonicus (Japanese tree frog); 25.6% and 26.1% for Pelophylax nigromaculatus (Black-spotted pond frog); and 30.5% and 50.0% for Lithobates catesbeianus (American bullfrog). The individual infection rate of L. catesbeianus was significantly greater than that of D. japonicus. The individual infection rate of P. nigromaculatus was related to the site-specific precipitation and air temperature. The individual infection rate gradually increased from Gosner development stage 39, and intermittent infection was confirmed in the early and middle developmental stages. Our results show that ranavirus is widespread among wild amphibians living in agricultural areas of South Korea, and mass die-offs by ranavirus could occur at any time.

Sustained Ranavirus Outbreak Causes Mass Mortality and Morbidity of Imperiled Amphibians in Florida.

A persistent 2-month long outbreak of Ranavirus in a natural community of amphibians contributed to a mass die-off of gopher frog tadpoles (Lithobates capito) and severe disease in striped newts (Notophthalmus perstriatus) in Florida. Ongoing mortality in L. capito and disease signs in N. perstriatus continued for 5 weeks after the first observation. Hemorrhagic disease and necrosis were diagnosed from pathological examination of L. capito tadpoles. We confirmed detection of a frog virus 3 (FV3)-like Ranavirus via quantitative PCR in all species. Our findings highlight the susceptibility of these species to Rv and the need for long-term disease surveillance during epizootics.

Effects of two amphibian pathogens on the developmental stability of green frogs.

Developmental instability, measured as fluctuating asymmetry (FA), is often used as a tool to measure stress and the overall quality of organisms. Under FA, it is assumed that control of symmetry during development is costly and that under stress the trajectory of development is disturbed, resulting in asymmetric morphologies. Amphibian emergent infectious diseases (EIDs), such as Ranavirus and chytrid fungus, have been involved in several mortality events, which makes them stressors and allows for the study of FA. We analyzed nine populations of green frogs (Rana clamitans) for the presence or absence of Ranavirus and chytrid fungus. Individuals were measured to determine levels of FA in seven traits under the hypothesis that FA is more likely to be observed in individuals infected by the pathogens. Significantly higher levels of FA were found in individuals with Ranavirus compared with uninfected individuals among all populations and all traits. We did not observe FA in individuals infected with chytrid fungus for any of the traits measured. Additionally, we observed a significant association between Ranavirus infection and levels of FA in both males and females, which may indicate this viral disease is likely to affect both sexes during development. Altogether, our results indicate that some EIDs may have far-reaching and nonlethal effects on individual development and populations harboring such diseases and that FA can be used as a conservation tool to identify populations subject to such a stress.

Cloning, expression and subcellular distribution of a Rana grylio virus late gene encoding ERV1 homologue.

An essential for respiration and viability (ERV1) homologue, 88R, was cloned and characterized from Rana grylio virus (RGV). Database searches found its homologues in all sequenced iridoviruses, and sequence alignment revealed a highly conserved motif shared by all ERV1 family proteins: Cys-X-X-Cys. RT-PCR and western blot analysis revealed that 88R begins to transcribe and translate at 6 h postinfection (p.i.) and remains detectable at 48 h p.i. during RGV infection course. Furthermore, using drug inhibition analysis by a de novo protein synthesis inhibitor and a viral DNA replication inhibitor, RGV 88R was classified as a late (L) viral gene during the in vitro infection. 88R-EGFP fusion protein was observed in both the cytoplasm and nucleus of pEGFP-N3-88R transfected EPC cells. Although result of immunofluorescence is similar, 88R protein was not detected in viromatrix. Moreover, function of RGV 88R on virus replication were evaluated by RNAi assay. Nevertheless, effect of knockdown of RGV 88R expression on virus replication was not detected in cultured fish cell lines. Collectively, current data indicate that RGV 88R was a late gene of iridovirus encoding protein that distributed both the cytoplasm and nucleus.

Ranavirus in wild edible frogs Pelophylax kl. esculentus in Denmark.

A survey for the amphibian pathogens ranavirus and Batrachochytrium dendrobatidis (Bd) was conducted in Denmark during August and September 2008. The public was encouraged via the media to register unusual mortalities in a web-based survey. All members of the public that registered cases were interviewed by phone and 10 cases were examined on suspicion of disease-induced mortality. All samples were negative for Bd. Ranavirus was isolated from 2 samples of recently dead frogs collected during a mass mortality event in an artificial pond near Slagelse, Denmark. The identity of the virus was confirmed by immunofluorescent antibody test. Sequencing of the major capsid protein gene showed the isolate had more than 97.3% nucleotide homology to 6 other ranaviruses.

Pathologic findings in larval and juvenile anurans inhabiting farm ponds in Tennessee, USA.

Amphibian populations are declining globally, yet general pathologic surveys for free-ranging amphibians are uncommon. Pathologic surveys are necessary to provide insight into the impacts of humans on emergence of pathogens in amphibian populations. During 2005, 104 American bullfrog (Rana catesbeiana) and 80 green frog (Rana clamitans) larvae and 40 green frog juveniles were collected from farm ponds in Tennessee, and complete necropsies were performed. Diagnostic testing included bacterial culture, virus testing, fecal parasite analysis, and histologic examination. Gross and histologic examination revealed that all individuals, except one bullfrog tadpole, could be classified as clinically normal. The clinically abnormal tadpole had swollen erythemic legs, and was positive for Aeromonas hydrophila but negative for Ranavirus. Parasites were common (43%) among specimens, with myxosporidium and trematodes most often noted. Commensal and opportunistic microorganisms were cultured from the tissues. Ranavirus was detected in 29% of individuals but generally not associated with significant histopathologic changes. Myxosporidia and Ranavirus coinfections occurred in 7 and 26% of green and bullfrog tadpoles, respectively, with the highest coinfection rate (83%) in bullfrog tadpoles during winter. Protozoans were most common in fecal examination. These data can serve as a baseline to evaluate the presence of clinical disease in larval and juvenile amphibians.

Pathogen Dynamics in an Invasive Frog Compared to Native Species.

Emerging infectious diseases threaten the survival of wildlife populations and species around the world. In particular, amphibians are experiencing population declines and species extinctions primarily in response to two pathogens, the fungus Batrachochytrium dendrobatidis (Bd) and the iridovirus Ranavirus (Rv). Here, we use field surveys and quantitative (q)PCR to compare infection intensity and prevalence of Bd and Rv across species and seasons on Jekyll Island, a barrier island off the coast of Georgia, USA. We collected oral and skin swabs for 1 year from four anuran species and three families, including two native hylids (Hyla cinerea and Hyla squirella), a native ranid (Rana sphenocephala), and the invasive rain frog Eleutherodactylus planirostris. Bd infection dynamics did not vary significantly over sampling months, but Rv prevalence and intensity were significantly higher in fall 2014 compared to spring 2015. Additionally, Rv prevalence and intensity were significantly higher in E. planirostris than in the other three species. Our study highlights the potential role of invasive amphibians as drivers of disease dynamics and demonstrates the importance of pathogen surveillance across multiple time periods and species to accurately capture the infectious disease landscape.