JMM Case Reports (2014)
Case Report
DOI 10.1099/jmmcr.0.001198
Epidemic ranaviral disease in imported captive
frogs (Dendrobates and Phyllobates spp.), Japan,
2012: a first report
Yumi Une,1 Tomoo Kudo,1 Ken-ichi Tamukai2 and Masaru Murakami3
Correspondence
1
Yumi Une
[email protected]
Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17-71
Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan
2
Den-enchofu Animal Hospital, 2-1-3 Denenchofu, Ota-ku, Tokyo 145-0071, Japan
3
Laboratory of Molecular Biology, School of Veterinary Medicine, Azabu University, 1-17-71
Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan
Introduction: Ranavirus infection is associated with mass die-off and population decline in
amphibians worldwide, and is listed by the World Organization for Animal Health (OIE) as a
notifiable disease under the Aquatic Animal Health Code. Work is ongoing to control the spread
of ranavirus, because this agent is considered an emerging pathogen of amphibians. In Japan,
ranaviral diseases have been detected at each episode of die-off of wild bullfrog juveniles since
2008. However, ranaviral disease has not been found in captive anurans.
Case presentation: Epidemic and lethal ranaviral disease in captive Dendrobates and
Phyllobates species imported from the Netherlands is reported. Poison dart frogs of three genera
were imported from the Netherlands in March 2012. Disease and death were noted
approximately 10 days after importation. Fifty-three adults of five species died in about 1 month,
including frogs that had been kept previously. Microscopic examination revealed necrosis and
intracytoplasmic basophilic inclusion bodies in the parenchyma of multiple organs. Electron
microscopy showed cytoplasmic ranavirus-like particles within haematopoietic cells of the kidney.
These particles were icosahedral, with a diameter of approximately 144 nm. Thirty-three of
41 dead frogs showed positive PCR results for the major capsid protein gene of Ranavirus. The
sequence obtained from five frogs was identical and did not match the registered sequence of
any ranavirus.
Received 12 December 2013
Accepted 24 June 2014
Conclusion: This is the first report, to our knowledge, of ranaviral disease in imported anurans of
the family Dendrobatidae and genera Phyllobates and Dendrobates in Japan.
Keywords: frog; infectious diseases; ranavirus.
Introduction
Ranavirus infection is associated with mass die-off and
population decline in amphibians worldwide, and is listed
by the World Organization for Animal Health (OIE) as a
notifiable disease under the Aquatic Animal Health Code.
Work is ongoing to control the spread of ranavirus,
because this agent is considered an emerging pathogen
(OIE, 2012; Schloegel et al., 2010). In Japan, ranaviral
diseases have been detected at each episode of die-off of
wild bullfrog juveniles (Une et al., 2009) since 2008.
Although ranaviral disease has not been found in captive
anurans, we discovered epidemic and lethal ranaviral
disease in anurans imported from the Netherlands. In the
Netherlands, ranaviral diseases have been discovered in
free-range frogs (Pelophylax spp.), common newts
Abbreviation: MCP, major capsid protein.
(Lissotriton vulgaris) (Kik et al., 2011), and imported redtailed knobby newts (Tylototriton kweichowensis) (Pasmans
et al., 2008). Further, ranaviral disease has recently been
confirmed in breeding poison dart frogs (Kik et al.,
2012).We describe here the first occurrence to our
knowledge of ranaviral disease and mortality in captive
Dendrobates and Phyllobates species in Japan.
Case report
Thirty poison dart frogs of three species (Dendrobates
auratus, Dendrobates azureus, Dendrobates tinctorius) were
imported from the Netherlands in mid-March 2012. Onset
of disease and death occurred approximately 10 days after
importation. Subsequently, four species of frog (D.
auratus, Dendrobates leucomelas, D. tinctorius, Phyllobates
terribilis) that were bred from a previous stock of frogs also
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Y. Une and others
began to die. Before this, there was indirect contact
between the imported frogs and frogs that had been kept
previously in the same container at the importer’s facility.
Symptoms were nonspecific – sloughing failure, difficulty
moving, lethargy and sudden death. Surviving frogs were
treated in medicated baths containing an antimycotic drug
(itraconazole) for chytrid fungus; however, death of frogs
continued. In total, 53 adults of 5 species died in about
1 month (Table 1).
3 (FJ459783). The neighbour-joining tree based on the
MCP gene sequences confirmed that the ranavirus
obtained in this study clustered with Frog virus 3 with a
64 % bootstrap value (Fig. 1). Nested PCR examination
for Batrachochytrium dendrobatidis was performed using
samples obtained by swabbing the skin of frogs with cotton
swabs and using a primer for the BdITS gene region, as
described by Goka et al. (2009).
Of 33 frogs positive for ranavirus on PCR (Table 1), the
livers were blackish and friable in 22 and splenomegaly was
observed in 17. Tissue samples (skin, liver, spleen, kidney
and digestive tract) from 20 dead frogs were fixed in 10 %
phosphate-buffered formalin, embedded in paraffin, sectioned at 3–4 mm, and stained with haematoxylin and
eosin. Histological lesions common to all cases included:
necrosis and degeneration in varying degrees in the
glomeruli and tubules of the kidney and in the hepatocytes;
basophilic or amphophilic intracytoplasmic inclusion
bodies present in interstitial haematopoietic cells, hepatocytes, renal tubular epithelial cells and some glomerular
cells; and apoptosis in the haematopoietic cells of the liver
and kidney (Fig. 2). No lesions associated with chytridiomycosis or chytrid fungus were found in the skin. In
addition, no bacterial infection was seen. For ultrastructural examination, a portion of formalin-fixed kidney
tissue was refixed with 2.5 % glutaraldehyde and 1 %
osmium tetroxide, and embedded in epoxy resin. Ultrathin sections (90–150 nm) were stained with uranyl acetate
and lead citrate. Electron microscopy showed cytoplasmic
ranavirus-like particles within haematopoietic cells of the
kidney. These particles were icosahedral, with a diameter of
¡144 nm.
Examinations were performed on 52 (41 dead and 11
surviving) of the frogs. There were no skin lesions except
adhesion of sloughed skin. Total DNA was extracted from
fresh kidneys by using the DNeasy Tissue kit (Qiagen)
according to the manufacturer’s protocol. We used three
primer sets [1, RanaM68F (59-GCACCACCTCTACTCTTATG-39) and BIV MCP 154 (59-CCATCGAGCCGTTCATGATG-39), 230 bp; 2, FV3MCP4F (59-GACTTGGCCACTTATGAC-39) and FV3MCP5R (59-GTCTCTGGAGAAGAAGAA-39), 530 bp (Mao et al., 1997); and 3, RanaJP556F
(59-GGTTCTTCCCCTCCCATTCTTCTT-39) and RanaJP772R
(59-GGTCATGTAGACGTTGGCCTCGAC-39),
217 bp] and amplified a Ranavirus-specific gene encoding
major capsid protein (MCP) from the 52 frog specimens. If
two or more of these primer sets were positive, that sample
was determined to be positive. As a consequence, 33 of the
41 dead frogs and 3 of the 11 surviving frogs tested
positive. The nucleotide sequences obtained were aligned
using the Clustal W method of the program MEGA v. 5.2.
The phylogenetic tree was constructed using the neighbour-joining algorithm with the Tamura three-parameter
model. To determine the robustness of the tree, a bootstrap
test of 2 000 replicates was applied. The corresponding
European catfish virus (FJ358608, FJ358609) and shortfinned eel ranavirus (FJ358612) were used as an outgroup.
The MCP gene sequence (1392 bp) was separately
determined from five frogs. Sequence alignment showed
no variation in the sequence from these frogs. The
comparison of the sequence obtained from the poison
dart frogs with previously published data in GenBank/
DDBJ/EMBL databases using the BLAST system showed that
the sequence obtained in this study had a 99 % identity
(1386/1392 bp) with the MCP gene sequence of Frog virus
Discussion
Considering the setting of the epidemic together with
characteristic microscopic lesions and molecular investigations, we decided that a ranaviral disease caused the
outbreak in the imported poison dart frogs of this
report. As far as we know, this is the first documented
case of significant morbidity and mortality associated
with a ranavirus in captive anurans in Japan. There are
Table 1. Profile of the frogs
Dead
Scientific name
Total number
n
PCR+/dead*
n
PCR+/surviving*
D. auratus
D. azureus
D. tinctorius
D. leucomelas
P. terribilis
Total
26
12
21
10
10
79
16
7
14
9
7
53
13/15
2/5
11/14
3/3
4/4
33/41
10
5
7
1
3
26
2/4
0/3
1/4
nd
nd
3/11
*Number examined by the PCR method.
2
Surviving
ND,
Not done
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JMM Case Reports
Epidemic ranaviral disease in imported captive frogs
Fig. 1. Phylogenetic tree based on MCP sequences of Ranavirus. The comparison of the obtained sequence from the poison
dart frogs with previously published data in GenBank/DDBJ/EMBL databases using the BLAST system showed that the
sequence obtained in this study had a 99 % identity (1386/1392 bp) with the MCP gene sequence of Frog virus 3
(FJ459783). The neighbour-joining tree based on the MCP gene sequences confirmed the ranavirus obtained in this study
clustered with Frog virus 3 with a 64 % bootstrap value.
two reports of concurrent infection with ranavirus and
B. dendrobatidis in the poison dart frog (Kik et al., 2012;
Miller et al., 2008). In these reports, it was difficult to
determine which of the present pathogens contributed
more to the deaths of the frogs, because either agent has
(b)
(a)
http://jmmcr.sgmjournals.org
(c)
the pathogenic potential to kill amphibians. This is the
first report that shows clearly that ranavirus itself has
lethal pathogenicity and the potential to induce an
outbreak of epidemic proportions in the poison dart
frog.
Fig. 2. Kidney. (a) Glomerular necrosis and
degeneration of renal tubular epithelium.
Apoptosis of interstitial haematopoietic cells
is abundant. Inset shows an intracytoplasmic
inclusion body (arrowhead) in a renal tubular
epithelial cell. H&E stain. ID no.12065X (D.
auratus that was imported and died in March
2012). (b) Ranavirus-like particles in the
cytoplasm of haematopoietic cells. Bar, 3 mm;
electron microscopy. (c) The particles are
icosahedral, with a diameter of 144 nm. Bar,
0.5 mm; electron microscopy. ID no.12067X
(D. auratus that was imported and died in
March 2012).
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Y. Une and others
Cunningham et al. (1996) identified two main disease
syndromes: one characterized by skin ulceration (ulcerative
syndrome) and one characterized by systemic haemorrhages (haemorrhagic syndrome). Among affected common frogs (Rana temporaria) in Britain, individuals were
found with lesions common to both of these syndromes.
Ulceration of the skin is a common lesion in infected
amphibians (Cunningham et al., 1996; Une et al., 2009).
The ulcer is an important lesion, and can lead to secondary
infection such as a bacterial infection in aquatic animals.
However, we examined 52 frogs and did not find a distinct
skin ulcer. Type and severity of lesion will vary depending
upon the species and condition of the host, and the strain
of the virus. In addition, lesion formation is related to the
length of the clinical course, which is affected by the
sensitivity of the species of frog to the specific virus. A
possible explanation for the absence of skin ulcers is that
the frog species in this report had high susceptibility to the
ranavirus, and the clinical course was short. Therefore,
diagnosis should be based on synthetic methods, which are
not influenced by the presence or absence of a specific
lesion such as the skin ulcer. In ranaviral disease of the
poison dart frog in this case, degeneration and necrosis of
parenchymal organs with intracytoplasmic inclusion
bodies were found to be characteristic, which concurs
with common findings in previously reported ranaviral
disease (Miller et al., 2011; Une et al., 2009).
Course of onset and detection of the same sequence of the
MCP portion in all Ranavirus sequences suggest horizontal
transmission at the facility in Japan; a ranavirus outbreak
as in this case has never previously been confirmed in
Japan. In The Netherlands, the common midwife toad
virus (CMTV), or CMTV-like virus, and Frog virus 3 have
been identified in both captive frogs and free-range frogs
(Kik et al., 2011, 2012; Pasmans et al., 2008), and the virus
in this report is closely related to Frog virus 3. The importer
went to the Netherlands and brought the frogs directly
back to Japan himself. Further, the importer had not
experienced previous outbreaks, and this epidemic started
after the introduction of these frogs. Therefore, the frogs
imported from the Netherlands might have represented the
original route of entry into the facility. A spill-over of
ranavirus present in a captive population of frogs is a
serious threat to wild amphibian populations. Considering
the worldwide trade in amphibians, the risk of introducing
this infection into wild populations with intentionally
released animals is high (Picco & Collins, 2008). In
conclusion, this is, we believe, the first report of ranavirus
in imported animals of the Dendrobatidae in Japan.
4
Acknowledgements
This study was partially supported by a research project grant
awarded by Azabu University and JSPS KAKENHI, grant number
B23310169. According to the guidelines and practices for ethical
animal experiments of Azabu University, all animals were handled
correctly.
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