Effects of historic and projected climate change on the range and impacts of an emerging wildlife disease.

The global trend of increasing environmental temperatures is often predicted to result in more severe disease epidemics. However, unambiguous evidence that temperature is a driver of epidemics is largely lacking, because it is demanding to demonstrate its role among the complex interactions between hosts, pathogens, and their shared environment. Here, we apply a three-pronged approach to understand the effects of temperature on ranavirus epidemics in UK common frogs, combining in vitro, in vivo, and field studies. Each approach suggests that higher temperatures drive increasing severity of epidemics. In wild populations, ranavirosis incidents were more frequent and more severe at higher temperatures, and their frequency increased through a period of historic warming in the 1990s. Laboratory experiments using cell culture and whole animal models showed that higher temperature increased ranavirus propagation, disease incidence, and mortality rate. These results, combined with climate projections, predict severe ranavirosis outbreaks will occur over wider areas and an extended season, possibly affecting larval recruitment. Since ranaviruses affect a variety of ectothermic hosts (amphibians, reptiles, and fish), wider ecological damage could occur. Our three complementary lines of evidence present a clear case for direct environmental modulation of these epidemics and suggest management options to protect species from disease.

Simulating contact networks for livestock disease epidemiology: a systematic review.

Contact structure among livestock populations influences the transmission of infectious agents among them. Models simulating realistic contact networks therefore have important applications for generating insights relevant to livestock diseases. This systematic review identifies and compares such models, their applications, data sources and how their validity was assessed. From 52 publications, 37 models were identified comprising seven model frameworks. These included mathematical models (n = 8; including generalized random graphs, scale-free, Watts-Strogatz and spatial models), agent-based models (n = 8), radiation models (n = 1) (collectively, considered 'mechanistic'), gravity models (n = 4), exponential random graph models (n = 9), other forms of statistical model (n = 6) (statistical) and random forests (n = 1) (machine learning). Overall, nearly half of the models were used as inputs for network-based epidemiological models. In all models, edges represented livestock movements, sometimes alongside other forms of contact. Statistical models were often applied to infer factors associated with network formation (n = 12). Mechanistic models were commonly applied to assess the interaction between network structure and disease dissemination (n = 6). Mechanistic, statistical and machine learning models were all applied to generate networks given limited data (n = 13). There was considerable variation in the approaches used for model validation. Finally, we discuss the relative strengths and weaknesses of model frameworks in different use cases.

Social contact patterns relevant for infectious disease transmission in Cambodia.

Social mixing patterns are key determinants of infectious disease transmission. Mathematical models parameterised with empirical data from contact pattern surveys have played an important role in understanding epidemic dynamics and informing control strategies, including for SARS-CoV-2. However, there is a paucity of data on social mixing patterns in many settings. We conducted a community-based survey in Cambodia in 2012 to characterise mixing patterns and generate setting-specific contact matrices according to age and urban/rural populations. Data were collected using a diary-based approach from 2016 participants, selected by stratified random sampling. Contact patterns were highly age-assortative, with clear intergenerational mixing between household members. Both home and school were high-intensity contact settings, with 27.7% of contacts occurring at home with non-household members. Social mixing patterns differed between rural and urban residents; rural participants tended to have more intergenerational mixing, and a higher number of contacts outside of home, work or school. Participants had low spatial mobility, with 88% of contacts occurring within 1 km of the participants' homes. These data broaden the evidence-base on social mixing patterns in low and middle-income countries and Southeast Asia, and highlight within-country heterogeneities which may be important to consider when modelling the dynamics of pathogens transmitted via close contact.

Is Xenopus laevis introduction linked with Ranavirus incursion, persistence and spread in Chile?

Ranaviruses have been associated with amphibian, fish and reptile mortality events worldwide and with amphibian population declines in parts of Europe. Xenopus laevis is a widespread invasive amphibian species in Chile. Recently, Frog virus 3 (FV3), the type species of the Ranavirus genus, was detected in two wild populations of this frog near Santiago in Chile, however, the extent of ranavirus infection in this country remains unknown. To obtain more information about the origin of ranavirus in Chile, its distribution, species affected, and the role of invasive amphibians and freshwater fish in the epidemiology of ranavirus, a surveillance study comprising wild and farmed amphibians and wild fish over a large latitudinal gradient (2,500 km) was carried out in 2015-2017. In total, 1,752 amphibians and 496 fish were tested using a ranavirus-specific qPCR assay, and positive samples were analyzed for virus characterization through whole genome sequencing of viral DNA obtained from infected tissue. Ranavirus was detected at low viral loads in nine of 1,011 X. laevis from four populations in central Chile. No other amphibian or fish species tested were positive for ranavirus, suggesting ranavirus is not threatening native Chilean species yet. Phylogenetic analysis of partial ranavirus sequences showed 100% similarity with FV3. Our results show a restricted range of ranavirus infection in central Chile, coinciding with X. laevis presence, and suggest that FV3 may have entered the country through infected X. laevis, which appears to act as a competent reservoir host, and may contribute to the spread the virus locally as it invades new areas, and globally through the pet trade.

High pathogen prevalence in an amphibian and reptile assemblage at a site with risk factors for dispersal in Galicia, Spain.

Ranaviruses are agents of disease, mortality and population declines in ectothermic vertebrates and emergences have been repeatedly linked to human activities. Ranaviruses in the common midwife toad ranavirus lineage are emerging in Europe. They are known to be severe multi-host pathogens of amphibians and can also cause disease in reptiles. Recurrent outbreaks of ranavirus disease and mortality affecting three species have occurred at a small reservoir in north-west Spain but no data were available on occurrence of the pathogen in the other amphibian and reptile species present or at adjacent sites. We sampled nine species of amphibians and reptiles at the reservoir and nearby sites and screened for ranavirus presence using molecular methods. Our results show infection with ranavirus in all nine species, including first reports for Hyla molleri, Pelophylax perezi, Rana iberica, and Podarcis bocagei. We detected ranavirus in all four local sites and confirmed mass mortality incidents involving Lissotriton boscai and Triturus marmoratus were ongoing. The reservoir regularly hosts water sports tournaments and the risks of ranavirus dispersal through the translocation of contaminated equipment are discussed.

Diversity-Stability Dynamics of the Amphibian Skin Microbiome and Susceptibility to a Lethal Viral Pathogen.

Variation among animals in their host-associated microbial communities is increasingly recognized as a key determinant of important life history traits including growth, metabolism, and resistance to disease. Quantitative estimates of the factors shaping the stability of host microbiomes over time at the individual level in non-model organisms are scarce. Addressing this gap in our knowledge is important, as variation among individuals in microbiome stability may represent temporal gain or loss of key microbial species and functions linked to host health and/or fitness. Here we use controlled experiments to investigate how both heterogeneity in microbial species richness of the environment and exposure to the emerging pathogen Ranavirus influence the structure and temporal dynamics of the skin microbiome in a vertebrate host, the European common frog (Rana temporaria). Our evidence suggests that altering the bacterial species richness of the environment drives divergent temporal microbiome dynamics of the amphibian skin. Exposure to ranavirus effects changes in skin microbiome structure irrespective of total microbial diversity, but individuals with higher pre-exposure skin microbiome diversity appeared to exhibit higher survival. Higher diversity skin microbiomes also appear less stable over time compared to lower diversity microbiomes, but stability of the 100 most abundant ("core") community members was similar irrespective of microbiome richness. Our study highlights the importance of extrinsic factors in determining the stability of host microbiomes over time, which may in turn have important consequences for the stability of host-microbe interactions and microbiome-fitness correlations.

Screening of a long-term sample set reveals two Ranavirus lineages in British herpetofauna.

Reports of severe disease outbreaks in amphibian communities in mainland Europe due to strains of the common midwife toad virus (CMTV)-like clade of Ranavirus are increasing and have created concern due to their considerable population impacts. In Great Britain, viruses in another clade of Ranavirus-frog virus 3 (FV3)-like-have caused marked declines of common frog (Rana temporaria) populations following likely recent virus introductions. The British public has been reporting mortality incidents to a citizen science project since 1992, with carcasses submitted for post-mortem examination, resulting in a long-term tissue archive spanning 25 years. We screened this archive for ranavirus (458 individuals from 228 incidents) using molecular methods and undertook preliminary genotyping of the ranaviruses detected. In total, ranavirus was detected in 90 individuals from 41 incidents focused in the north and south of England. The majority of detections involved common frogs (90%) but also another anuran, a caudate and a reptile. Most incidents were associated with FV3-like viruses but two, separated by 300 km and 16 years, involved CMTV-like viruses. These British CMTV-like viruses were more closely related to ranaviruses from mainland Europe than to each other and were estimated to have diverged at least 458 years ago. This evidence of a CMTV-like virus in Great Britain in 1995 represents the earliest confirmed case of a CMTV associated with amphibians and raises important questions about the history of ranavirus in Great Britain and the epidemiology of CMTV-like viruses. Despite biases present in the opportunistic sample used, this study also demonstrates the role of citizen science projects in generating resources for research and the value of maintaining long-term wildlife tissue archives.

A quantitative-PCR based method to estimate ranavirus viral load following normalisation by reference to an ultraconserved vertebrate target.

Ranaviruses are important pathogens of amphibians, reptiles and fish. To meet the need for an analytical method for generating normalised and comparable infection data for these diverse host species, two standard-curve based quantitative-PCR (qPCR) assays were developed enabling viral load estimation across these host groups. A viral qPCR targeting the major capsid protein (MCP) gene was developed which was specific to amphibian-associated ranaviruses with high analytical sensitivity (lower limit of detection: 4.23 plasmid standard copies per reaction) and high reproducibility across a wide dynamic range (coefficient of variation below 3.82% from 3 to 3×108 standard copies per reaction). The comparative sensitivity of the viral qPCR was 100% (n=78) based on agreement with an established end-point PCR. Comparative specificity with the end-point PCR was also 100% (n=94) using samples from sites with no history of ranavirus infection. To normalise viral quantities, a host qPCR was developed which targeted a single-copy, ultra-conserved non-coding element (UCNE) of vertebrates. Viral and host qPCRs were applied to track ranavirus growth in culture. The two assays offer a robust approach to viral load estimation and the host qPCR can be paired with assays targeting other pathogens to study infection burdens.