We have been monitoring amphibian communities located in the Picos de Europa National Park (PNPE) since 2005, when we first recorded ranavirus infection, disease, and mass mortality. We began demographic surveys in 2007, making annual counts of target species at 15 sites across the park ( Figure 1 ). This predates the first published records of lethal ranavirus infections (occurring in September 2007) in the PNPE in two of our study species []. Ranaviruses are large, double-stranded DNA viruses of the family Iridoviridae, which are emerging pathogens with broad geographical and host ranges. They infect and cause disease in fish and reptiles but are noted for their ability to cause lethal disease in amphibians in the Americas, Europe, Asia, and Australia []. Despite Ranavirus's broad geographic distribution and documented multihost epizootics in amphibian communities [], quantitative evidence for amphibian demographic decline due to ranavirosis has previously been reported for only a single amphibian host species [].

Map shows park boundary and sites where amphibian communities have been monitored since 2005. Sites that experienced observed amphibian mass mortality events are expanded to display the amphibian community present, and affected hosts are bordered in red. Hosts with confirmed CMTV infection are denoted by a blue virus particle in bottom left corner of their image. Disease and mass mortality associated with CMTV infection at Igüedri (IGU) was reported by Balseiro et al. (2009) []. See also Figure S1 and Table S1

Pathology, isolation and molecular characterisation of a ranavirus from the common midwife toad Alytes obstetricans on the Iberian Peninsula.

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

Pathology, isolation and molecular characterisation of a ranavirus from the common midwife toad Alytes obstetricans on the Iberian Peninsula.

Outbreak of common midwife toad virus in alpine newts (Mesotriton alpestris cyreni) and common midwife toads (Alytes obstetricans) in northern Spain: a comparative pathological study of an emerging ranavirus.

Pathology, isolation and molecular characterisation of a ranavirus from the common midwife toad Alytes obstetricans on the Iberian Peninsula.

We successfully amplified a suite of ranavirus loci (six partial open reading frames and an intergenic region) from three of the host species sampled at locations in the PNPE and all three species sampled in Galicia. Sequences from amplification products of all loci were aligned against whole genomes covering known global amphibian-like ranavirus diversity and joined to form a final concatenated alignment 2,274 bp in length. Phylogenetic assessment of the concatenated sequences derived from seven diseased animals show that the ranaviruses associated with disease and mortality in northern Spain were all related or near identical to the genome of CMTV. This virus was originally isolated from a diseased animal sampled in 2008 in the PNPE and was associated with mass mortality and the same distinctive signs of disease ( Figure 1 ) []. Sequences amplified from all hosts sampled within the PNPE at sites of mass mortality exhibited 99.96% homology (varying at a single base) with the sequence of the type CMTV irrespective of what host species was sampled. Sequences from hosts sampled at the Galician site were highly similar to each other (two were identical and were 99.96% identical to the third, with variation again confined to a single base). They formed the sister clade to the CMTV cluster and are hereafter referred to as Bosca’s newt virus (BNV; Figure 2 ). In contrast, an isolate from a common midwife toad at Ándara Lake, where ranavirosis and mass mortality have not been observed—Ándaran Alytes obstetricans virus (AAOV)—grouped with “FV3-like” viruses. We also screened animals from three other sites in the PNPE where there has been no observed disease or mass mortality, and we found no evidence of ranavirus infection. These observations support the assumption that disease and mass mortality can be used as a reliable indicator of CMTV incidence.

Ándaran Alytes obstetricans virus (AAOV) is a member of the “FV3-like” viruses, which form a monophyletic group; Bosca’s newt virus (BNV) and Common midwife toad virus (CMTV) are considered “CMTV-like.” The tree was constructed from seven concatenated multiple sequence alignments (details of loci are included in Experimental Procedures ). Node support values are annotated on the best maximum-likelihood tree and were calculated using maximum-likelihood (bootstraps, black) and Bayesian inference (posterior probabilities, red) under a GTR model of molecular evolution. Scale of branch lengths is in nucleotide substitutions per site. Additional sequences included are Frog virus 3 (FV3, GenBank accession number AY548484 ), Tiger frog virus (TFV, AF389451 ), Ambystoma tigrinum virus (ATV, AY150217 ), Epizootic hematopoietic necrosis virus (EHNV, FJ433873 ), Soft-shelled turtle iridovirus (STIV, NC012637 ), Rana grylio virus (RGV, JQ654586 ), and European sheatfish virus (ESV, JQ724856 ).

Outbreak of common midwife toad virus in alpine newts (Mesotriton alpestris cyreni) and common midwife toads (Alytes obstetricans) in northern Spain: a comparative pathological study of an emerging ranavirus.

Molecular diagnostics have confirmed that infection with Ranavirus is associated with disease at all five of these locations ( Table S1 ). We also screened for Batrachochytrium dendrobatidis (Bd), a fungal pathogen commonly associated with amphibian die-offs in Iberia [], at eight locations in the PNPE. Bd was present at two sites (25%; Table S1 B), occurring in the absence of observed disease and mortality at La Güelga (LAG, a ranavirus-negative site, Figure 1 ) and at low prevalence in 2005 only at an artificial pond (near Áliva refuge). A more widespread survey for bacteria and viruses was also undertaken as part of the first published account of ranavirus infection and mass mortality in the PNPE [] (at Igüedri [IGU], Figure 1 ). That mass mortality event affected some of our study species, which exhibited the same suite of lesions observed in this study. There were no other pathogens found besides the Ranavirus Common midwife toad virus (CMTV). We consider it unlikely that other environmental factors, such as pollution, are contributing to the observed mass mortality events, given the pristine nature of the PNPE and the heterogeneity among study sites in terms of the type and situation of water bodies.

Pathology, isolation and molecular characterisation of a ranavirus from the common midwife toad Alytes obstetricans on the Iberian Peninsula.

Evidence of a chytrid fungus infection involved in the decline of the common midwife toad (Alytes obstetricans) in protected areas of central Spain.

We have continued to record mass mortality events consistent with ranavirosis affecting amphibian communities at four locations in the PNPE (Áliva, Ercina, Lloroza, and Moñetas [ALIVA, ERC, LLOR, and MON], Figure 1 ). During annual field surveys, we encountered numerous dead and dying adult, juvenile, and larval caudate and anuran amphibians, including all six common species inhabiting the park. Ranavirus infections may be subclinical [] but are more typically associated with overt, distinctive disease in the form of systemic or ulcerative syndromes [] and accompanied by host mass mortality. Sick and dead animals in the PNPE exhibited superficial and ulcerating skin lesions, internal hemorrhages, and severe limb necrosis, all gross signs typical of lethal ranavirosis (see Figure S1 A available online). Additionally, since 2010 we have been observing mortality associated with signs of ranavirosis at a location 200 km west of the PNPE in Galicia, affecting two caudate amphibian species that are rare or absent in the PNPE and one squamate reptile ( Figure S1 B).

Outbreak of common midwife toad virus in alpine newts (Mesotriton alpestris cyreni) and common midwife toads (Alytes obstetricans) in northern Spain: a comparative pathological study of an emerging ranavirus.

The worst-affected species was the common midwife toad, Alytes obstetricans. Midwife toads experienced steep decline at both diseased sites where we monitored population trends of this host species but did not decline at two other locations where signs of ranavirosis were never detected ( Figure 3 Table S1 A). Alpine newts (Mesotriton alpestris) and common toads (Bufo bufo) also experienced significant declines at diseased sites ( Figure 3 ) but did not show this dynamic at locations where we did not observe amphibians exhibiting signs of ranavirosis, with one exception (B. bufo at Soto-Covadonga Road [SCR], Figure 1 ). Amphibian species that were enumerated at disease-free locations did not generally decline from 2007 to 2012 and sometimes appeared to be increasing in numbers during the course of our surveillance ( Table S1 A). When all monitored host populations were considered independently, infection with CMTV was significantly associated with host declines (Fisher’s exact test, p = 0.0093; raw data summarized in Figure S2 ). The same was true when we accounted for potential nonindependence of multiple species at the same site by using site data rather than host species data (Fisher’s exact test, p = 0.022; Figure S2 ).

Populations of host species in diseased communities in the PNPE have consistently experienced statistically significant, persistent, and in some cases catastrophic population declines, which was not the case at sites where disease had not been observed ( Figure 3 Table S1 A). The onset of decline for all species experiencing ranavirosis was contemporary with our first records of disease in the PNPE, but there is insufficient resolution of disease records to match decline and disease onset at individual locations. Our first records of mass mortality and ranavirosis in the region were at an artificial pond adjacent to Lloroza (LLOR, Figure 1 ; subsequently drained for other reasons) and a roadside pool at Áliva mine (ALIVA, Figure 1 ), both sampled in 2005.

Common midwife toad ranavirus (CMTV) infections have been confirmed within amphibian communities at Moñetas, Lloroza, and Ercina Lake (black lines). Ranavirus infection and disease were first observed in the Picos de Europa National Park in 2005 (see Multispecies Host Declines ). Trends are also shown for all other sites where monitoring of Alytes obstetricans, Mesotriton alpestris, and Bufo bufo has been ongoing but CMTV is assumed to be absent due to molecular screening and/or the absence of disease and mass mortality events (gray lines). Circles denote counts for adults or tadpoles; Squares denote counts for egg masses. See also Table S1

Our findings indicate that CMTV-like ranaviruses recently emerged in northern Spain and are responsible for mortality of every amphibian species that we have sampled. Additionally, an isolate with 99.96% sequence identity to viruses isolated from amphibians in Galicia, and closely related to CMTV, was generated from esophageal tissue taken from a viperine snake (Natrix maura) found dead in the process of ingesting diseased amphibians [] and with ulcerating lesions along its gullet. The ability of FV3-like ranaviruses (the sister clade to CMTV-likes; Figure 2 ]) to be transmitted among both closely related and highly diverged cold-blooded vertebrate taxa is well documented []. However, recurrent epizootics caused by FV3-like viruses in the Americas, the United Kingdom (UK), and Southeast Asia have only been linked to host population decline in UK common frogs (Rana temporaria), and UK FV3-like viruses appear limited in their ability to cause disease and significant mortality in other native UK amphibian species []. We have also detected FV3-like ranaviruses cocirculating in the PNPE (at Ándara Lake [AND], Figure 2 ) that caused morbidity without lesions in A. obstetricans but with no evidence of mass mortality or population declines. Reports of amphibian mass mortality events associated with ranavirosis are rapidly accruing across Europe, and CMTV-like forms are implicated: a partial sequence of the major capsid protein gene of the virus isolate responsible for the death of thousands of pool frogs (Pelophylax lessonae) and smooth newts (Lissotriton vulgaris) in the Netherlands reported by Kik et al. exhibited 100% sequence similarity to CMTV [], as did sequences from the same locus derived from a Ranavirus isolated from North American bullfrog larvae sampled from invasive populations in Belgium []. The accumulating body of evidence indicates that CMTV-like ranaviruses may be in the process of emerging in amphibian host communities across Europe, with the capacity to infect and cause significant disease and death in a wide range of hosts.

Community-Level Effects

27 Johnson P.T.J.

Preston D.L.

Hoverman J.T.

Richgels K.L.D. Biodiversity decreases disease through predictable changes in host community competence. 28 Wood C.L.

Lafferty K.D.

DeLeo G.

Young H.S.

Hudson P.J.

Kuris A.M. Does biodiversity protect humans against infectious disease?. 29 Parrish C.R.

Holmes E.C.

Morens D.M.

Park E.-C.

Burke D.S.

Calisher C.H.

Laughlin C.A.

Saif L.J.

Daszak P. Cross-species virus transmission and the emergence of new epidemic diseases. 30 Kassen R. The experimental evolution of specialists, generalists, and the maintenance of diversity. 31 Benmayor R.

Hodgson D.J.

Perron G.G.

Buckling A. Host mixing and disease emergence. 32 Woolhouse M.E.J.

Taylor L.H.

Haydon D.T. Population biology of multihost pathogens. 33 Bitter W.

Gerrits H.

Kieft R.

Borst P. The role of transferrin-receptor variation in the host range of Trypanosoma brucei. 1 LaDeau S.L.

Kilpatrick A.M.

Marra P.P. West Nile virus emergence and large-scale declines of North American bird populations. 2 Fisher M.C.

Garner T.W.J.

Walker S.F. Global emergence of Batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time, and host. 4 Frick W.F.

Pollock J.F.

Hicks A.C.

Langwig K.E.

Reynolds D.S.

Turner G.G.

Butchkoski C.M.

Kunz T.H. An emerging disease causes regional population collapse of a common North American bat species. Infection and disease frequency are both known to decrease with increasing host community species richness (see e.g. []), even if the relationship is not always straightforward []. This is because diverse host communities present a range of barriers to infection, and universal pathogen strategies for overcoming host barriers are rare []. Accordingly, pathogens typically exhibit significant variation in their ability to infect and cause disease across host species and must evolve novel traits to exploit a broader host range []. It is therefore unusual for an emerging pathogen to exploit a broad range of host species and extremely rare for multiple host species to suffer synchronous mass mortality and decline when infection emerges. For the rare exceptions, the results for hosts can be notably catastrophic (e.g., West Nile virus emergence in North America [], Batrachochytrium dendrobatidis emergence in the Neotropics and Australia [], and white-nose syndrome in North American bats []).

We see no evidence of increased host species diversity hampering the ability of a novel pathogen to exploit a host community in northern Spain. At the most species-rich site in the PNPE, all six amphibian species are experiencing mortality associated with signs of disease and have done so since disease was first observed. Instead of being inhibited by host species diversity, it appears that a single strain of CMTV has the capacity to exploit multiple host species de novo. This hypothesis is supported by an almost complete lack of variation at seven loci located across the type CMTV genome and recovered at multiple points in space, time, and host species—spanning the width of the PNPE, several years, and diverse hosts. The synchrony of host declines across sites, beginning shortly after disease was first detected in the park, provides further support. Taken together with the obstacles to rapid amphibian dispersal presented by the rugged, mountainous terrain of the PNPE, all of the evidence points to a single introduction of CMTV at multiple locations within the park and affecting all amphibian species. Such an introduction could have occurred via human translocations of infectious materials along with equipment or livestock.