Frog Virus Research Articles

Developmental exposure to chemicals associated with unconventional oil and gas extraction alters immune homeostasis and viral immunity of the amphibian Xenopus

Although aquatic vertebrates and humans are increasingly exposed to water pollutants associated with unconventional oil and gas extraction (UOG), the long-term effects of these pollutants on immunity remains unclear. We have established the amphibian Xenopus laevis and the ranavirus Frog Virus 3 (FV3) as a reliable and sensitive model for evaluating the effects of waterborne pollutants. X. laevis tadpoles were exposed to a mixture of equimass amount of UOG chemicals with endocrine disrupting activity (0.1 and 1.0 μg/L) for 3 weeks, and then long-term effects on immune function at steady state and following viral (FV3) infection was assessed after metamorphosis. Notably, developmental exposure to the mixture of UOG chemicals at the tadpole stage affected metamorphic development and fitness by significantly decreasing body mass after metamorphosis completion. Furthermore, developmental exposure to UOGs resulted in perturbation of immune homeostasis in adult frogs, as indicated by significantly decreased number of splenic innate leukocytes, B and T lymphocytes; and a weakened antiviral immune response leading to increased viral load during infection by the ranavirus FV3. These findings suggest that mixture of UOG-associated waterborne endocrine disruptors at low but environmentally–relevant levels have the potential to induce long-lasting alterations of immune function and antiviral immunity in aquatic vertebrates and ultimately human populations.

Localization of Frog Virus 3 Conserved Viral Proteins 88R, 91R, and 94L.

The characterization of the function of conserved viral genes is central to developing a greater understanding of important aspects of viral replication or pathogenesis. A comparative genomic analysis of the iridoviral genomes identified 26 core genes conserved across the family Iridoviridae. Three of those conserved genes have no defined function; these include the homologs of frog virus 3 (FV3) open reading frames (ORFs) 88R, 91R, and 94L. Conserved viral genes that have been previously identified are known to participate in a number of viral activities including: transcriptional regulation, DNA replication/repair/modification/processing, protein modification, and viral structural proteins. To begin to characterize the conserved FV3 ORFs 88R, 91R, and 94L, we cloned the genes and determined their intracellular localization. We demonstrated that 88R localizes to the cytoplasm of the cell while 91R localizes to the nucleus and 94L localizes to the endoplasmic reticulum (ER).

First report of ranavirus mortality in a common snapping turtle Chelydra serpentina.

An adult male snapping turtle with marked palpebral edema and multifocal skin ulceration was found alive in a marsh in southern Ontario in summer 2017. The turtle was transported to a rehabilitation facility and died 4 d after arrival. The carcass was submitted to the Canadian Wildlife Health Cooperative for post-mortem examination. Gross lesions included ulcerative conjunctivitis, necrotizing stomatitis, and splenomegaly. Microscopically, this corresponded to multisystemic fibrinonecrotizing vasculitis and severe fibrinous splenic necrosis. Liver tissue tested positive for frog virus 3-like ranavirus and negative for herpesvirus via polymerase chain reaction. The gross and microscopic lesions were consistent with previous reports of ranavirus infection in turtles and were severe enough to have been the cause of death in this case. This is the first report of morbidity and mortality in a common snapping turtle with a ranavirus infection, and the first reported case of ranavirus infection in a reptile in Canada. Ranaviruses are considered to be an emerging infectious disease in chelonians as they are increasing in distribution, prevalence, and host range.

Class A Scavenger Receptors Are Used by Frog Virus 3 During Its Cellular Entry.

Frog virus 3 (FV3) is the type species of the genus Ranavirus (family Iridoviridae). FV3 and FV3-like viruses are globally distributed infectious agents with the capacity to replicate in three vertebrate classes (teleosts, amphibians, and reptiles). At the cellular level, FV3 and FV3-like viruses can infect cells from virtually all vertebrate classes. To date, the cellular receptors that are involved in the FV3 entry process are unknown. Class A scavenger receptors (SR-As) are a family of evolutionarily conserved cell-surface receptors that bind a wide range of chemically distinct polyanionic ligands and can function as cellular receptors for other DNA viruses, including vaccinia virus and herpes simplex virus. The present study aimed to determine whether SR-As are involved in FV3 cellular entry. By using well-defined SR-A competitive and non-competitive ligand-blocking assays and absolute qPCR, we demonstrated that the SR-A competitive ligands drastically reduced the quantities of cell-associated viral loads in frog cells. Moreover, inducing the expression of a human SR-AI in an SR-A null cell line significantly increased FV3–cell association. Together, our results indicate that SR-As are utilized by FV3 during the cellular entry process.

Structural transformation-mediated dimerization of caspase recruitment domain revealed by the crystal structure of CARD-only protein in frog virus 3

Caspase recruitment domain (CARD)-only proteins (COPs), regulate apoptosis, inflammation, and innate immunity. They inhibit the assembly of NOD-like receptor complexes such as the inflammasome and NODosome, which are molecular complexes critical for caspase-1 activation. COPs are known to interact with either caspase-1 CARD or RIP2 CARD via a CARD-CARD interaction, and inhibit caspase-1 activation or further downstream signaling. In addition to the human COPs, Pseudo-ICE, INCA, and ICEBERG, several viruses also contain viral COPs that help them escape the host immune system. To elucidate the molecular mechanism of host immunity inhibition by viral COPs, we solved the structure of a viral COP for the first time. Our structure showed that viral COP forms a structural transformation-mediated dimer, which is unique and has not been reported in any structural study of a CARD domain. Based on the current structure, and the previously solved structures of other death domain superfamily members, we propose that structural transformation-mediated dimerization might be a new strategy for dimer assembly in the death domain superfamily.

Pathogen Risk Analysis for Wild Amphibian Populations Following the First Report of a Ranavirus Outbreak in Farmed American Bullfrogs (Lithobates catesbeianus) from Northern Mexico.

Ranaviruses are the second deadliest pathogens for amphibian populations throughout the world. Despite their wide distribution in America, these viruses have never been reported in Mexico, the country with the fifth highest amphibian diversity in the world. This paper is the first to address an outbreak of ranavirus in captive American bullfrogs (Lithobates catesbeianus) from Sinaloa, Mexico. The farm experienced high mortality in an undetermined number of juveniles and sub-adult bullfrogs. Affected animals displayed clinical signs and gross lesions such as lethargy, edema, skin ulcers, and hemorrhages consistent with ranavirus infection. The main microscopic lesions included mild renal tubular necrosis and moderate congestion in several organs. Immunohistochemical analyses revealed scant infected hepatocytes and renal tubular epithelial cells. Phylogenetic analysis of five partial ranavirus genes showed that the causative agent clustered within the Frog virus 3 clade. Risk assessment with the Pandora+ protocol demonstrated a high risk for the pathogen to affect amphibians from neighboring regions (overall Pandora risk score: 0.619). Given the risk of American bullfrogs escaping and spreading the disease to wild amphibians, efforts should focus on implementing effective containment strategies and surveillance programs for ranavirus at facilities undertaking intensive farming of amphibians.

Co-exposure to multiple ranavirus types enhances viral infectivity and replication in a larval amphibian system.

Multiple pathogens commonly co-occur in animal populations, yet few studies demonstrate how co-exposure of individual hosts scales up to affect transmission. Although viruses in the genus Ranavirus are globally widespread, and multiple virus species or strains likely co-occur in nature, no studies have examined how co-exposure affects infection dynamics in larval amphibians. We exposed individual northern red-legged frog Rana aurora larvae to 2 species of ranavirus, namely Ambystoma tigrinum virus (ATV), frog virus 3 (FV3), or an FV3-like strain isolated from a frog-culturing facility in Georgia, USA (RCV-Z2). We compared single-virus to pairwise co-exposures while experimentally accounting for dosage. Co-exposure to ATV and FV3-like strains resulted in almost twice as many infected individuals compared to single-virus exposures, suggesting an effect of co-exposure on viral infectivity. The viral load in infected individuals exposed to ATV and FV3 was also higher than the single-dose FV3 treatment, suggesting an effect of co-exposure on viral replication. In a follow-up experiment, we examined how the co-occurrence of ATV and FV3 affected epizootics in mesocosm populations of larval western chorus frogs Pseudacris triseriata. Although ATV did not generally establish within host populations (<4% prevalence), when ATV and FV3 were both present, this co-exposure resulted in a larger epizootic of FV3. Our results emphasize the importance of multi-pathogen interactions in epizootic dynamics and have management implications for natural and commercial amphibian populations.

Unique Composition of Intronless and Intron-Containing Type I IFNs in the Tibetan Frog Nanorana parkeri Provides New Evidence To Support Independent Retroposition Hypothesis for Type I IFN Genes in Amphibians.

In vertebrates, intron-containing and intronless type I IFN genes have recently been reported in amphibian model species Xenopus tropicalis and X. laevis. However, whether intronless type I IFNs in amphibians are the ancestral genes of type I IFNs in amniotes or just represent the independent divergence in amphibians is unknown or even uninvestigated. In this study, both intron-containing and intronless type I IFN genes, as well as their receptor genes, were identified in the Tibetan frog Nanorana parkeri The evidence obtained from homology, synteny, phylogeny, and divergence time showed that intronless type I IFN genes in N. parkeri and in Xenopus might have arisen from two independent retroposition events occurred in these two lineages, and the retrotransposition causing the generation of intronless type I IFN genes in amniotes is another independent event beyond the two in amphibians. It can then be proposed that intronless type I IFNs in N. parkeri and Xenopus may not be the ancestral genes of intronless type I IFNs in amniotes but may just represent two independent bifurcations in the amphibian lineage. Furthermore, both intronless and intron-containing type I IFNs in N. parkeri showed strong ability in inducing the expression of IFN-stimulated genes and the strong antiviral activity against frog virus 3. The present study thus provides the evolutionary evidence to support the independent retroposition hypothesis for the occurrence of intronless type I IFN genes in amphibians and contributes to a functional understanding of type I IFNs in this group of vertebrates.

Environmental Drivers of Ranavirus in Free-Living Amphibians in Constructed Ponds

Amphibian ranaviruses occur globally, but we are only beginning to understand mechanisms for emergence. Ranaviruses are aquatic pathogens which can cause > 90% mortality in larvae of many aquatic-breeding amphibians, making them important focal host taxa. Host susceptibilities and virulence of ranaviruses have been studied extensively in controlled laboratory settings, but research is needed to identify drivers of infection in natural environments. Constructed ponds, essential components of wetland restoration, have been associated with higher ranavirus prevalence than natural ponds, posing a conundrum for conservation efforts, and emphasizing the need to understand potential drivers. In this study, we analyzed 4 years of Frog virus 3 prevalence and associated environmental parameters in populations of wood frogs (Lithobates sylvaticus) and green frogs (Lithobates clamitans) in a constructed pond system. High prevalence was best predicted by low temperature, high host density, low zooplankton concentrations, and Gosner stages approaching metamorphosis. This study identified important variables to measure in assessments of ranaviral infection risk in newly constructed ponds, including effects of zooplankton, which have not been previously quantified in natural settings. Examining factors mediating diseases in natural environments, particularly in managed conservation settings, is important to both validate laboratory findings in situ, and to inform future conservation planning, particularly in the context of adaptive management.

Amphibian (Xenopus laevis) Tadpoles and Adult Frogs Differ in Their Use of Expanded Repertoires of Type I and Type III Interferon Cytokines.

While amphibians around the globe are facing catastrophic declines, in part because of infections with pathogens such as the Frog Virus 3 (FV3) ranavirus; the mechanisms governing amphibian susceptibility and resistance to such pathogens remain poorly understood. The type I and type III interferon (IFN) cytokines represent a cornerstone of vertebrate antiviral immunity, while our recent work indicates that tadpoles and adult frogs of the amphibian Xenopus laevis may differ in their IFN responses to FV3. In this respect, it is notable that anuran (frogs and toads) tadpoles are significantly more susceptible to FV3 than adult frogs, and thus, gaining greater insight into the differences in the tadpole and adult frog antiviral immunity would be invaluable. Accordingly, we examined the FV3-elicited expression of a panel of type I and type III IFN genes in the skin (site of FV3 infection) and kidney (principal FV3 target) tissues and isolated cells of X. laevis tadpoles and adult frogs. We also examined the consequence of tadpole and adult frog skin and kidney cell stimulation with hallmark pathogen-associated molecular patterns (PAMPs) on the IFN responses of these cells. Together, our findings indicate that tadpoles and adult frogs mount drastically distinct IFN responses to FV3 as well as to viral and non-viral PAMPs, while these expression differences do not appear to be the result of a distinct pattern recognition receptor expression by tadpoles and adults.

Water Contaminants Associated With Unconventional Oil and Gas Extraction Cause Immunotoxicity to Amphibian Tadpoles.

Chemicals associated with unconventional oil and gas (UOG) operations have been shown to contaminate surface and ground water with a variety of endocrine disrupting compounds (EDCs) inducing multiple developmental alteration in mice. However, little is known about the impacts of UOG-associated contaminants on amphibian health and resistance to an emerging ranavirus infectious disease caused by viruses in the genus Ranavirus, especially at the vulnerable tadpole stage. Here we used tadpoles of the amphibian Xenopus laevis and the ranavirus Frog virus 3 (FV3) as a model relevant to aquatic environment conservation research for investigating the immunotoxic effects of exposure to a mixture of 23 UOG-associated chemicals with EDC activity. Xenopus tadpoles were exposed to an equimass mixture of 23 UOG-associated chemicals (range from 0.1 to 10 µg/l) for 3 weeks prior to infection with FV3. Our data show that exposure to the UOG chemical mixture is toxic for tadpoles at ecological doses of 5 to 10 µg/l. Lower doses significantly altered homeostatic expression of myeloid lineage genes and compromised tadpole responses to FV3 through expression of TNF-α, IL-1β, and Type I IFN genes, correlating with an increase in viral load. Exposure to a subset of 6 UOG chemicals was still sufficient to perturb the antiviral gene expression response. These findings suggest that UOG-associated water pollutants at low but environmentally relevant doses have the potential to induce acute alterations of immune function and antiviral immunity.

Adoptive Transfer of Fluorescently Labeled Immune Cells in Xenopus.

Adoptive cell transfer from inbred adult Xenopus to inbred tadpoles is a useful way to study the dissemination of immune cells or pathogen-infected immune cells in tadpoles. For example, Xenopus peritoneal leukocytes (PLs) can be readily infected by pathogens such as Frog virus 3 (FV3) and Mycobacterium marinum (M. marinum). By transferring fluorescently labeled, FV3-infected PLs into tadpoles, we observed infiltration of these cells into the tadpole's brain, which indicates that FV3-infected PLs can cross blood brain barrier. Taking advantage of tadpoles' transparency, fluorescently labeled immune cells can be tracked in real time using fluorescence microscopy.

Optimizing, validating, and field testing a multiplex qPCR for the detection of amphibian pathogens.

Amphibian populations worldwide are facing numerous threats, including the emergence and spread of infectious diseases. In the past 2 decades, Batrachochytrium dendrobatidis (Bd), a parasitic fungus, and a group of viruses comprising the genus Ranavirus have become widespread and resulted in mass mortality events and extirpations worldwide. In 2013, another novel fungus, B. salamandrivorans (Bsal), was attributed to dramatic declines in populations of fire salamander Salamandra salamandra in the Netherlands. Experimental infections demonstrated that Bsal is highly pathogenic to numerous salamander genera. In an effort to prevent the introduction of Bsal to North America, the US Fish and Wildlife Service (USFWS) listed 201 salamander species as injurious wildlife under the Lacey Act. To determine infection status and accurately assess amphibian health, the development of a sensitive and specific diagnostic assay was needed. We describe the optimization and validation of a multiplex quantitative polymerase chain reaction (qPCR) protocol for the simultaneous detection of Bd, Bsal, and frog virus 3-like ranaviruses. A synthetic genome template (gBlock®) containing the target genes from all 3 pathogens served as the positive control and allowed accurate quantification of pathogen genes. The assay was validated in the field using an established non-lethal swabbing technique to survey local amphibian populations throughout a range of habitats. This multiplex qPCR demonstrates high reproducibility, sensitivity, and was capable of detecting both Bd and ranavirus in numerous locations, species, and life stages. Bsal was not detected at any point during these sampling efforts.

EXPERIMENTAL TRANSMISSION OF FROG VIRUS 3-LIKE RANAVIRUS IN JUVENILE CHELONIANS AT TWO TEMPERATURES.

The pathogenicity of frog virus 3 (FV3)-like ranavirus varies in adult chelonian species at different environmental temperatures, but differences in pathogenicity at different temperatures has yet to be determined in juveniles. Our objective was to determine the susceptibility to FV3-like ranavirus in four species of juvenile chelonians: red-eared sliders (RES; Trachemys scripta elegans), Mississippi map turtles ( Graptemys pseudogeographica kohnii), false map turtles (FMT; Graptemys pseudogeographica), and eastern river cooters ( Pseudemys concinna concinna) at two environmental temperatures. Two simultaneous trials ( n=8 treatment and n=4 controls of each species) were conducted in separate temperature-controlled rooms with animals maintained at 22 C or 27 C. All of the inoculated animals of each species at each temperature died, but no mortality was observed in control animals. Median survival times varied between 8 d and 11 d, based on species and temperature, with RES in the 27 C trial surviving the shortest time and the FMT in the 22 C trial surviving the longest. Combining all species, turtles in the 27 C trial survived for fewer days than those housed at 22 C, despite all turtles in both trials having similar viral copies detected in postmortem tissues. Lesions in inoculated turtles resembled those noted in natural and experimental FV3-like ranavirus infections and included vasculitis, thrombosis, hemorrhage in multiple organs, renal tubular necrosis, and hepatic necrosis. Myositis was not present in any juvenile, infected turtles in this study. This study confirmed that juvenile chelonians have a high susceptibility to ranaviral disease.

Protein A from orange-spotted grouper nervous necrosis virus triggers type I interferon production in fish cell

Family Nodaviridae consists of two genera: Alphanodavirus and Betanodavirus, and the latter is classified into four genotypes, including red-spotted grouper nervous necrosis virus, tiger puffer nervous necrosis virus, striped jack nervous necrosis virus, and barfin flounder nervous necrosis virus. Type I interferons (IFNs) play a central role in the innate immune system and antiviral responses, and the interactions between IFN and NNV have been investigated in this study. We have found that the RNA-dependent RNA polymerase (RdRp) from orange-spotted nervous necrosis virus (OGNNV), named protein A, was capable of activating IFN promoter in fathead minnow (FHM) cells. Transient expression of protein A was found to induce IFN expression and secretion, endowing FHM cells with anti-tiger frog virus ability. Protein A from SJNNV can also induce IFN expression in FHM cells but that from Flock House virus (FHV), a well-studied representative species of genus Alphanodavirus, cannot. RdRp activity and mitochondrial localization were shown to be required for protein A to induce IFN expression by means of activating IRF3 but not NFκB. Furthermore, DsRNA synthesized in vitro transcription and poly I:C activated IFN promoter activity when transfected into FHM cells, and dsRNA were also detected in NNV-infected cells. We postulated that dsRNA, a PAMP, was produced by protein A, leading to activation of innate immune response. These results suggest that protein As from NNV are the agonists of innate immune response. This is the first work to demonstrate the interaction between NNV protein A and innate immune system, and may help to understand pathogenesis of NNV.

Protein-protein interactions among the structural proteins of Chilo iridescent virus.

Chilo iridescent virus (CIV), officially named invertebrate iridescent virus 6 (IIV6), is a nucleocytoplasmic virus with a ~212-kb linear dsDNA genome that encodes 215 putative open reading frames (ORFs). Proteomic analysis has revealed that the CIV virion consists of 54 virally encoded proteins. In this study, we identified the interactions between the structural proteins using the yeast two-hybrid system. We cloned 47 structural genes into both bait and prey vectors, and then analysed the interactions in Saccharomyces cerevisiae strain AH109. A total of 159 protein-protein interactions were detected between the CIV structural proteins. Only ORF 179R showed a self-association. Four structural proteins that have homologues in iridoviruses (118L, 142R, 274L and 295L) showed indirect interactions with each other. Seven proteins (138R, 142R, 361L, 378R, 395R, 415R and 453R) interacted with the major capsid protein 274L. The putative membrane protein 118L, a homologue of the frog virus 3/Ranagrylio virus 53R protein, showed direct interactions with nine other proteins (117L, 229L, 307L, 355R, 366R, 374R, 378R, 415R and 422L). The interaction between 118L and 415R was confirmed by a GST pull-down assay. These data indicate that 415R is a potential matrix protein connecting the envelope protein 118L with the major capsid protein 274L.

Differentiation-dependent antiviral capacities of amphibian (Xenopus laevis) macrophages

Infections by ranaviruses such as Frog virus 3 (Fv3), are significantly contributing to worldwide amphibian population declines. Notably, amphibian macrophages (Mφs) are important to both the Fv3 infection strategies and the immune defense against this pathogen. However, the mechanisms underlying amphibian Mφ Fv3 susceptibility and resistance remain unknown. Mφ differentiation is mediated by signaling through the colony-stimulating factor-1 receptor (CSF-1R) which is now known to be bound not only by CSF-1, but also by the unrelated interleukin-34 (IL-34) cytokine. Pertinently, amphibian (Xenopus laevis) Mφs differentiated by CSF-1 and IL-34 are highly susceptible and resistant to Fv3, respectively. Accordingly, in the present work, we elucidate the facets of this Mφ Fv3 susceptibility and resistance. Because cellular resistance to viral replication is marked by expression of antiviral restriction factors, it was intuitive to find that IL-34-Mφs possess significantly greater mRNA levels of select restriction factor genes than CSF-1-Mφs. Xenopodinae amphibians have highly expanded repertoires of antiviral interferon (IFN) cytokine gene families, and our results indicated that in comparison with the X. laevis CSF-1-Mφs, the IL-34-Mφs express substantially greater transcripts of representative IFN genes, belonging to distinct gene family clades, as well as their cognate receptor genes. Finally, we demonstrate that IL-34-Mφ-conditioned supernatants confer IFN-mediated anti-Fv3 protection to the virally susceptible X. laevis kidney (A6) cell line. Together, this work underlines the differentiation pathways leading to Fv3-susceptible and -resistant amphibian Mφ populations and defines the molecular mechanisms responsible for these differences.

Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses

Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ranaviruses. Here we report the development and partial validation of a TaqMan real-time qPCR assay. The primers and TaqMan probe targeted a conserved region of the major capsid protein (MCP) gene. A series of experiments using a 10-fold dilution series of Frog virus 3 (FV3) MCP plasmid DNA revealed linearity over a range of 7 orders of magnitude (107-101), a mean correlation coefficient (R2) of >0.99, and a mean efficiency of 96%. The coefficient of variation of intra- and inter-assay variability ranged from <0.1-3.5% and from 1.1-2.3%, respectively. The analytical sensitivity was determined to be 10 plasmid copies of FV3 DNA. The qPCR assay detected a panel of 33 different ranaviral isolates originating from fish, amphibian, and reptile hosts from all continents excluding Africa and Antarctica, thereby representing the global diversity of ranaviruses. The assay did not amplify highly divergent ranaviruses, members of other iridovirus genera, or members of the alloherpesvirus genus Cyprinivirus. DNA from fish tissue homogenates previously determined to be positive or negative for the ranavirus Epizootic hematopoietic necrosis virus by virus isolation demonstrated a diagnostic sensitivity of 95% and a diagnostic specificity of 100%. The reported qPCR assay provides an improved expedient diagnostic tool and can be used to elucidate important aspects of ranaviral pathogenesis and epidemiology in clinically and sublinically affected fish, amphibians, and reptiles.

DETECTION OF RANAVIRUS USING BONE MARROW HARVESTED FROM MORTALITY EVENTS IN EASTERN BOX TURTLES ( TERRAPENE CAROLINA CAROLINA).

The causes of free-living chelonian mortality events are often unknown because of infrequent recovery of remains and rapid postmortem decomposition. This study describes a technique to harvest bone marrow and detect frog virus 3-like ranavirus (FV3) using quantitative polymerase chain reaction in skeletonized eastern box turtles ( Terrapene carolina carolina) ( N = 87), and assesses agreement with concurrent perimortem samples ( N = 14). FV3 was detected in bone marrow samples from 12 turtle shells (14%). Three of 14 turtles had detectable FV3 loads in both bone marrow and perimortem samples, two turtles had detectable FV3 in bone marrow only, and nine turtles tested FV3 negative in both bone marrow and concurrent perimortem samples. There was substantial agreement between FV3 testing of bone marrow and other tissues ( κ = 0.658). Harvesting bone marrow from shells is easily performed and can serve as a means for biologists and wildlife veterinarians to improve postmortem surveillance for systemically distributed pathogens, including FV3.

Effect of imidacloprid on the survival of Xenopus tadpoles challenged with wild type frog virus 3

The sensitivity of amphibians to Ranavirus may be increased by exposure to other environmental stressors, including chemical contaminants. Neonicotinoid insecticides comprise 27% of the global insecticide market and have been detected in wetlands and other aquatic habitats. The present study focused on the effects of exposure of pre-metamorphic Xenopus laevis to the neonicotinoid, imidacloprid (IMI) on sensitivity to frog virus 3 (FV3) infection. It was hypothesized that exposure of tadpoles to IMI at sublethal concentrations of 1 and 500μgL−1 would increase FV3 related mortalities relative to tadpole mortalities in a control treatment with only the virus. However, contrary to the predicted outcome, IMI reduced the rates of mortality following viral challenge, although the total mortalities by the 25th day after infection did not differ among the treatments. These results should not be interpreted as an indication that neonicotinoid insecticides are beneficial to aquatic ecosystems, since these insecticides cause toxic responses at low concentrations to other non-target aquatic organisms.