Investigation of amphibian mortality events in wildlife reveals an on-going ranavirus epidemic in the North of the Netherlands

In the four years following the first detection of ranavirus (genus Ranavirus, family Iridoviridae) infection in Dutch wildlife in 2010, amphibian mortality events were investigated nationwide to detect, characterize and map ranaviruses in amphibians over time, and to establish the affected host species and the clinico-pathological presentation of the disease in these hosts. The ultimate goal was to obtain more insight into ranavirus disease emergence and ecological risk. In total 155 dead amphibians from 52 sites were submitted between 2011 and 2014, and examined using histopathology, immunohistochemistry, virus isolation and molecular genetic characterization. Ranavirus-associated amphibian mortality events occurred at 18 sites (35%), initially only in proximity of the 2010 index site. Specimens belonging to approximately half of the native amphibian species were infected, including the threatened Pelobates fuscus (spadefoot toad). Clustered massive outbreaks involving dead adult specimens and ranavirus genomic identity indicated that one common midwife toad virus (CMTV)-like ranavirus strain is emerging in provinces in the north of the Netherlands. Modelling based on the spatiotemporal pattern of spread showed a high probability that this emerging virus will continue to be detected at new sites (the discrete reproductive power of this outbreak is 0.35). Phylogenetically distinct CMTV-like ranaviruses were found in the south of the Netherlands more recently. In addition to showing that CMTV-like ranaviruses threaten wild amphibian populations not only in Spain but also in the Netherlands, the current spread and risk of establishment reiterate that understanding the underlying causes of CMTV-like ranavirus emergence requires international attention

Investigation of Amphibian Mortality Events in Wildlife Reveals an On-Going Ranavirus Epidemic in the North of the Netherlands

In the four years following the first detection of ranavirus (genus Ranavirus, family Iridoviridae) infection in Dutch wildlife in 2010, amphibian mortality events were investigated nationwide to detect, characterize and map ranaviruses in amphibians over time, and to establish the affected host species and the clinico-pathological presentation of the disease in these hosts. The ultimate goal was to obtain more insight into ranavirus disease emergence and ecological risk. In total 155 dead amphibians from 52 sites were submitted between 2011 and 2014, and examined using histopathology, immunohistochemistry, virus isolation and molecular genetic characterization. Ranavirus-associated amphibian mortality events occurred at 18 sites (35%), initially only in proximity of the 2010 index site. Specimens belonging to approximately half of the native amphibian species were infected, including the threatened Pelobates fuscus (spadefoot toad). Clustered massive outbreaks involving dead adult specimens and ranavirus genomic identity indicated that one common midwife toad virus (CMTV)-like ranavirus strain is emerging in provinces in the north of the Netherlands. Modelling based on the spatiotemporal pattern of spread showed a high probability that this emerging virus will continue to be detected at new sites (the discrete reproductive power of this outbreak is 0.35). Phylogenetically distinct CMTV-like ranaviruses were found in the south of the Netherlands more recently. In addition to showing that CMTV-like ranaviruses threaten wild amphibian populations not only in Spain but also in the Netherlands, the current spread and risk of establishment reiterate that understanding the underlying causes of CMTV-like ranavirus emergence requires international attention

Severity and frequency of lesions per species at sites with or without ranavirus.

<p>Severity and frequency of lesions per species at sites with or without ranavirus.</p

Spatiotemporal distribution of ranavirus associated amphibian mortality events, the Netherlands, 2011–2014.

<p>(A) Country overview, 2011–2012. (B) Country overview, 2013–2014. (C) Close-up of area around index site, 2011–2014. The green surface contains all sites with confirmed ranavirus-associated mortality events in 2011 (dark green shade), extending southwards in 2012 (light green shade). The yellow surface is the area to the north of the index site where additional events occurred ≤ 20 km from the index site in 2013 (dark shade) and 2014 (light shade). The numbers correspond to the site numbers for sites with confirmed ranavirus presence. ND = Not determined.</p

Phylogeny of the fully sequenced ranavirus isolate associated with amphibian mortality at site no. 35.

<p>Maximum-likelihood phylogeny based on the 26 iridovirus core proteins of the fully sequenced ranavirus from site no. 35 and other publically available ranavirus genomes. The grouper iridoviruses were used as an outgroup (not shown). The bootstrap support is shown at the nodes.</p

Characteristics of ranavirus associated mortality events, grouped by distance from the index site (no. 0).

<p>ND = Not determined. NA = Not applicable. All = No, or virtually no, live specimens remain in the water body in the immediate aftermath of the initial mortality event.</p

Geographical coordinates of sites (WGS 84), with longitude in decimal degrees East (x), and latitude in decimal degrees North (y).

<p>Geographical coordinates of sites (WGS 84), with longitude in decimal degrees East (x), and latitude in decimal degrees North (y).</p

Macroscopic lesions in amphibians naturally infected with CMTV-like ranavirus from the Netherlands.

<p>(A) Adult <i>Pelophylax</i> kl. <i>esculentus</i> with mild erythema of the skin from the inguinal region (black arrow head). (B) Internal inspection of animal from Fig 2A with enlarged pale liver showing marked hepatic necrosis (black arrow head) and areas of hemorrhage in the coelomic cavity (black arrow). (C) Neotenic adult <i>Lissotriton vulgaris</i> presenting with focal area of hemorrhage in the cloaca (black arrow). (D) Smooth newt larvae presenting with hemorrhages in the limbs (black arrows).</p

Phylogeny of the partially sequenced ranaviruses associated with amphibian mortality events in the Dutch wildlife.

<p>Maximum-likelihood phylogeny of ranaviruses based on concatenated alignments of seven partial gene sequences. The ranavirus samples from the Netherlands cluster in two distinct phylogenetically related groups (NL group I and NL group II) within the CMTV-like ranavirus group, clearly distinct from CMTV from Spain (CMTV-E) and Bosca’s newt virus from Spain (BNV-E). The fully sequenced ranavirus from site no. 35 is indicated with an asterisk (*). The bootstrap support is shown at the nodes. Only values >50% are shown.</p