Stephen J. Price

Collapse of Amphibian Communities Due to an Introduced Ranavirus

The emergence of infectious diseases with a broad host range can have a dramatic impact on entire communities and has become one of the main threats to biodiversity. Here, we report the simultaneous exploitation of entire communities of potential hosts with associated severe declines following invasion by a novel viral pathogen. We found two phylogenetically related, highly virulent viruses (genus Ranavirus, family Iridoviridae) causing mass mortality in multiple, diverse amphibian hosts in northern Spain, as well as a third, relatively avirulent virus. We document host declines in multiple species at multiple sites in the region. Our work reveals a group of pathogens that seem to have preexisting capacity to infect and evade immunity in multiple diverse and novel hosts, and that are exerting massive impacts on host communities. This report provides an exceptional record of host population trends being tracked in real time following emergence of a wildlife disease and a striking example of a novel, generalist pathogen repeatedly crossing the species barrier with catastrophic consequences at the level of host communities.

A de novo Assembly of the Common Frog (Rana temporaria) Transcriptome and Comparison of Transcription Following Exposure to Ranavirus and Batrachochytrium dendrobatidis

Amphibians are experiencing global declines and extinctions, with infectious diseases representing a major factor. In this study we examined the transcriptional response of metamorphic hosts (common frog, Rana temporaria) to the two most important amphibian pathogens: Batrachochytrium dendrobatidis (Bd) and Ranavirus. We found strong up-regulation of a gene involved in the adaptive immune response (AP4S1) at four days post-exposure to both pathogens. We detected a significant transcriptional response to Bd, covering the immune response (innate and adaptive immunity, complement activation, and general inflammatory responses), but relatively little transcriptional response to Ranavirus. This may reflect the higher mortality rates found in wild common frogs infected with Ranavirus as opposed to Bd. These data provide a valuable genomic resource for the amphibians, contribute insight into gene expression changes after pathogen exposure, and suggest potential candidate genes for future host-pathogen research.

Reconstructing the emergence of a lethal infectious disease of wildlife supports a key role for spread through translocations by humans

There have been few reconstructions of wildlife disease emergences, despite their extensive impact on biodiversity and human health. This is in large part attributable to the lack of structured and robust spatio-temporal datasets. We overcame logistical problems of obtaining suitable information by using data from a citizen science project and formulating spatio-temporal models of the spread of a wildlife pathogen (genus Ranavirus, infecting amphibians). We evaluated three main hypotheses for the rapid increase in disease reports in the UK: that outbreaks were being reported more frequently, that climate change had altered the interaction between hosts and a previously widespread pathogen, and that disease was emerging due to spatial spread of a novel pathogen. Our analysis characterized localized spread from nearby ponds, consistent with amphibian dispersal, but also revealed a highly significant trend for elevated rates of additional outbreaks in localities with higher human population density—pointing to human activities in also spreading the virus. Phylogenetic analyses of pathogen genomes support the inference of at least two independent introductions into the UK. Together these results point strongly to humans repeatedly translocating ranaviruses into the UK from other countries and between UK ponds, and therefore suggest potential control measures.

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

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

Xenopus laevis and Emerging Amphibian Pathogens in Chile

Amphibians face an extinction crisis with no precedence. Two emerging infectious diseases, ranaviral disease caused by viruses within the genus Ranavirus and chytridiomycosis due to Batrachochytrium dendrobatidis (Bd), have been linked with amphibian mass mortalities and population declines in many regions of the globe. The African clawed frog (Xenopus laevis) has been indicated as a vector for the spread of these pathogens. Since the 1970s, this species has been invasive in central Chile. We collected X. laevis and dead native amphibians in Chile between 2011 and 2013. We conducted post-mortem examinations and molecular tests for Ranavirus and Bd. Eight of 187 individuals (4.3xa0%) tested positive for Ranavirus: seven X. laevis and a giant Chilean frog (Calyptocephallela gayi). All positive cases were from the original area of X. laevis invasion. Bd was found to be more prevalent (14.4xa0%) and widespread than Ranavirus, and all X. laevis Bd-positive animals presented low to moderate levels of infection. Sequencing of a partial Ranavirus gene revealed 100xa0% sequence identity with Frog Virus 3. This is the first report of Ranavirus in Chile, and these preliminary results are consistent with a role for X. laevis as an infection reservoir for both Ranavirus and Bd.

Detection and reporting of ranavirus in amphibians: Evaluation of the roles of the world organisation for animal health and the published literature

Abstract Pathogens of wildlife can have direct impacts on human and livestock health as well as on biodiversity, as causative factors in population declines and extinctions. The World Organization for Animal Health (OIE) seeks to facilitate rapid sharing of information about animal diseases to enable up-to-date risk assessments of translocations of animals and animal products. The OIE also produces manuals of recommended methods to standardize diagnostic testing. Ranaviruses are important amphibian pathogens that may have spread through international trade, and infections became notifiable to OIE in 2009. We surveyed and reviewed published literature for data on sampling, diagnostic testing, and reporting of ranavirus during 2009–14. We also investigated attitudes and awareness of the OIE and its recommendations for best practice. We found that sampling effort is uneven and concentrated in the northern hemisphere. We also identified citizen science projects that have the potential to improve the quantity and quality of data on the incidence of ranavirus infection and the circumstances surrounding disease outbreaks. We found reporting of infection to be inconsistent: reporting was split between the published literature (where it was subject to a 2-yr lag) and the OIE with little overlap, results of negative diagnostic tests were underreported, and scientific researchers lacked awareness of the role of the OIE. Approaches to diagnostic screening were poorly harmonized and heavily reliant on molecular methods. These flaws in the mechanisms of ranavirus detection and reporting hamper the construction of a comprehensive disease information database.

Temperature is a key driver of a wildlife epidemic and future warming will increase impacts

Increasing environmental temperatures are predicted to have increasingly severe and deleterious effects on biodiversity. For the most part, the impacts of a warming environment are presumed to be direct, however some predict increasingly severe disease epidemics, primarily from vector-borne pathogens, that will have the capacity to deplete host populations. Data to support this hypothesis are lacking. Here we describe increasing severity of ranavirosis driven by increasing temperature affecting a widely distributed amphibian host. Both in vitro and in vivo experiments showed that increasing environmental temperature leads to increased propagation of ranavirus and, in the latter, increased incidence of host infection and mortality. Also, temperature was shown to be a key determinant of disease dynamics in wild amphibians, raising the odds and severity of disease incidents. The direction of this effect was highly consistent in the context of other interacting variables such as shading around ponds. Projections based on future climate indicate that changes in seasonal weather in the UK will result in the increased incidence of severe cases of ranavirosis in amphibian populations that could affect recruitment. These complementary lines of evidence present a clear case of direct environmental modulation of a host-pathogen interaction and provide information for proposing mitigation actions.

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 10u2009µg/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 10u2009µg/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.

A novel approach to wildlife transcriptomics provides evidence of disease-mediated differential expression and changes to the microbiome of amphibian populations.

Ranaviruses are responsible for a lethal, emerging infectious disease in amphibians and threaten their populations throughout the world. Despite this, little is known about how amphibian populations respond to ranaviral infection. In the United Kingdom, ranaviruses impact the common frog (Rana temporaria). Extensive public engagement in the study of ranaviruses in the UK has led to the formation of a unique system of field sites containing frog populations of known ranaviral disease history. Within this unique natural field system, we used RNA sequencing (RNA‐Seq) to compare the gene expression profiles of R. temporaria populations with a history of ranaviral disease and those without. We have applied a RNA read‐filtering protocol that incorporates Bloom filters, previously used in clinical settings, to limit the potential for contamination that comes with the use of RNA‐Seq in nonlaboratory systems. We have identified a suite of 407 transcripts that are differentially expressed between populations of different ranaviral disease history. This suite contains genes with functions related to immunity, development, protein transport and olfactory reception among others. A large proportion of potential noncoding RNA transcripts present in our differentially expressed set provide first evidence of a possible role for long noncoding RNA (lncRNA) in amphibian response to viruses. Our read‐filtering approach also removed significantly more bacterial reads from libraries generated from positive disease history populations. Subsequent analysis revealed these bacterial read sets to represent distinct communities of bacterial species, which is suggestive of an interaction between ranavirus and the host microbiome in the wild.

Host Microbiome Richness Predicts Resistance to Disturbance by Pathogenic Infection in a Vertebrate Host

Environmental heterogeneity is known to modulate the interactions between pathogens and hosts. However, the impact of environmental heterogeneity on the structure of host-associated microbial communities, and how these communities respond to pathogenic exposure remain poorly understood. Here we use an experimental framework to probe the links between environmental heterogeneity, skin microbiome structure and infection by the emerging pathogen Ranavirus in a vertebrate host, the European common frog (Rana temporaria). We provide evidence that environmental complexity directly influences the diversity and structure of the host skin microbiome, and that more diverse microbiomes are more resistant to perturbation associated with exposure to Ranavirus. Our data also indicate that host microbiome diversity covaries with survival following exposure to Ranavirus. Our study highlights the importance of extrinsic factors in driving host-pathogen dynamics in vertebrate hosts, and suggests that environment-mediated variation in the structure of the host microbiome may covary with observed differences in host susceptibility to disease in the wild.