This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
Author's personal copy
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
Ana Balseiroa,*, Kevin P. Daltonb, Ana del Cerroa, Isabel Márqueza, Francisco Parrab,
José M. Prietoa, R. Casaisa
aSERIDA, Servicio Regional de Investigación y Desarrollo Agroalimentario, Laboratorio de Sanidad Animal, 33299 Jove, Gijón, Spain
bDepartamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain
a r t i c l ei n f o
Accepted 31 July 2009
Common midwife toad virus
Mesotriton alpestris cyreni
a b s t r a c t
This report describes the isolation and characterisation of the common midwife toad virus (CMTV) from
juvenile alpine newts (Mesotriton alpestris cyreni) and common midwife toad (CMT) tadpoles (Alytes
obstetricans) in the Picos de Europa National Park in Northern Spain in August 2008. A comparative path-
ological and immunohistochemical study was carried out using anti-CMTV polyclonal serum. In the kid-
neys, glomeruli had the most severe histological lesions in CMT tadpoles, while both glomeruli and renal
tubular epithelial cells exhibited foci of necrosis in juvenile alpine newts. Viral antigens were detected by
immunohistochemical labelling mainly in the kidneys of CMT tadpoles and in ganglia of juvenile alpine
newts. This is the first report of ranavirus infection in the alpine newt, the second known species to be
affected by CMTV in the past 2 years.
? 2009 Elsevier Ltd. All rights reserved.
In recent years, chytridiomycosis and ranavirus infections have
caused outbreaks of high mortality in amphibians and appear to be
linked to amphibian population declines (Daszak et al., 1999). The
number of reported ranavirus outbreaks has increased greatly
since the 1990s, with recurring epidemics occurring in native
amphibian populations in the United Kingdom (Cunningham
et al., 1996; Teacher et al., 2009).
The common midwife toad virus (CMTV) is a ranavirus origi-
nally isolated from common midwife toad (CMT) tadpoles (Alytes
obstetricans) from the Picos de Europa National Park in Northern
Spain in September 2007 (Balseiro et al., 2009). This was the first
description of a ranavirus disease in Spain. No further cases of
the disease were detected until August 2008, when high mortality
was observed in CMT tadpoles and juvenile alpine newts (Mesotri-
ton alpestris cyreni) in a pond approximately 1 km from the perma-
nent water trough where the first outbreak occurred. In this study
we report the isolation of CMTV from juvenile alpine newts, along
with CMT tadpoles at the same location, and perform a compara-
tive pathological and immunohistochemical study. No further
cases of CMTV infection were detected in the permanent water
trough, which had been disinfected after the outbreak in 2007.
Macroscopically, CMT tadpoles exhibited systemic haemorrhag-
es. Juvenile alpine newts had haemorrhages on the ventral surface
(Fig. 1), but not in internal organs. Systemic haemorrhages are
common in ranavirus infections (Fox et al., 2006; Cunningham
et al., 2008). However, in the United Kingdom, ranavirus infection
can present with cutaneous ulceration but no internal gross lesions
(Cunningham et al., 1996, 2008). Microscopic lesions in CMT tad-
poles and juvenile alpine newts in the present outbreak were sim-
ilar to those described for the systemic haemorrhagic form of
ranavirus disease in CMT tadpoles previously (Balseiro et al., 2009).
Three whole CMT tadpoles and three juvenile alpine newts
were fixed in 10% neutral buffered formalin immediately after
death, dehydrated in graded ethanol solutions, embedded in paraf-
fin wax, sectioned at 4 lm thickness and stained with haematoxy-
linandeosin (H&E). On
intracytoplasmic inclusion bodies (Fig. 2) were associated with
small foci of necrosis in the skin, liver, kidney, pancreas and gastro-
intestinal tract. Pycnotic cell nuclei were also observed in these or-
gans. Vesicles and focal thickening were observed in the epidermis
in both species. In the kidneys, glomeruli were the structures most
affected in CMT tadpoles, while both glomeruli and tubular epithe-
lial cells exhibited foci of necrosis (Fig. 2C) in juvenile alpine newts.
Another ranavirus, FV3, exhibits trophism for the kidney, specifi-
cally for the proximal renal tubular epithelium (Robert et al.,
2005). Necrosis of neuroepithelial tissue, previously described in
salamanders (Docherty et al., 2003), was not found in infected
CMT tadpoles or juvenile alpine newts.
Samples of kidneys from CMT tadpoles and juvenile alpine
newts were fixed in 2.5% glutaraldehyde, embedded in resin and
ultrathin sections were stained with uranyl acetate and lead citrate
1090-0233/$ - see front matter ? 2009 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +34 985 308470; fax: +34 985 327811.
E-mail address: email@example.com (A. Balseiro).
The Veterinary Journal 186 (2010) 256–258
Contents lists available at ScienceDirect
The Veterinary Journal
journal homepage: www.elsevier.com/locate/tvjl
Author's personal copy
for transmission electron microscopy (TEM) using a JEOL JEM-1010
microscope. Virions were detected in the kidneys of both CMT tad-
poles and juvenile alpine newts examined by TEM (Fig. 3B and C).
Most virions appeared to be extracellular and were located in areas
of necrosis found on histological examination. Virus particles ap-
peared to be more abundant in CMT tadpoles than juvenile alpine
Epithelioma papulosum cyprini (EPC) cells were inoculated
with tissue homogenates of lung, liver, kidney and gastrointestinal
tract from four diseased CMT tadpoles and four diseased juvenile
alpine newts for virus isolation (Balseiro et al., 2009). Cytopathic
effects were evident after incubation for 5 days at 15 ?C. Virions
were purified from EPC-infected cells (Balseiro et al., 2009) and
negatively stained with 2% phosphotungstic acid (pH 6.2). Electron
approximately 160–180 nm in diameter with hexagonal nucleo-
capsid morphology (Fig. 3A).
DNA was extracted from tissue homogenates of diseased ani-
mals, using the illustra tissue and cells genomicPrep Mini Spin
Kit (GE Healthcare) and amplified by PCR using as forward primer
50-GACTTGGCCACTTATGAC-30and reverse primer 50-GTCTCTGGA-
GAAGAAGAA-30within the FV3 major capsid protein (MCP) gene
(Mao et al., 1997). The DNA products (530 nucleotides, including
the primer sequences) were gel-purified using the TaKaRa RECO-
CHIP and sequenced using the ABI prism BigDye terminator v3.1
kit (Applied BioSystems) and the Applied BioSystems 3100 Genetic
Analyser. The nucleotide sequences obtained for the viruses iso-
lated from CMT tadpoles and juvenile adult newts in the August
2008 outbreak were identical to each other and to that of the CMTV
isolated in September 2007 (GenBank FM213466; Balseiro et al.,
2009). These results suggest that the viruses isolated from CMT
tadpoles and juvenile alpine newts in the 2008 outbreak are most
likely to be CMTV.
Immunohistochemistry was performed using the peroxidase
anti-peroxidase method. Sections were incubated with specific
rabbit anti-serum raised against purified virions of the 2007 isolate
CMTV and diluted 1/1000 (Balseiro et al., 2009). In CMT tadpoles,
the strongest staining on immunohistochemistry was observed in
renal glomeruli (Fig. 2B). However, in juvenile alpine newts,
Fig. 2. Comparative analysis of histopathological features of the kidney, liver and ganglia from a CMTV-infected CMT tadpole and a CMTV-infected juvenile alpine newt using
H&E and immunohistochemistry (PAP). (A) Viral inclusions (arrows) and necrotic cells in renal glomeruli. Bar = 20 lm. (B) Renal glomerulus with intense immunolabelling.
Bar = 20 lm. (C) Necrotic tubular epithelial cells with viral inclusions (arrow) in a renal tubule. Bar = 20 lm. (D) Renal glomerulus showing weak immunolabelling.
Bar = 20 lm. (E) Areas of focal necrosis with hepatocytes containing viral inclusions (arrows). Bar = 20 lm. (F) Hepatocytes showing intense CMTV-specific immunolabelling.
Bar = 20 lm. (G) Areas of necrosis with hepatocytes containing viral inclusions (arrows). Bar = 50 lm. (H) Hepatocytes showing weak immunolabelling. Bar = 20 lm. (I)
Ganglion with absence of microscopic lesions. Bar = 100 lm. (J) Low level immunolabelling within the ganglion. Bar = 50 lm. (K) Ganglion with absence of microscopic
lesions. Bar = 100 lm. (L) Strong CMTV-specific immunolabelling. Bar = 50 lm. Original magnifications 40? (I), 100? (K), 200? (G, J, L), 400? (A, B, D, E, F, H) and 1000? (C).
Fig. 1. Two juvenile alpine newts (Mesotriton alpestris cyreni), about 5 cm in length,
infected with CMTV, showing skin haemorrhages on their ventral body surfaces
A. Balseiro et al./The Veterinary Journal 186 (2010) 256–258
Author's personal copy Download full-text
ganglia located in the muscle were most strongly immunolabelled
(Fig. 2L), although necrosis or viral inclusions were not observed at
these sites (Fig. 2K). In both species, there was focal immunolabel-
ling of CMTV antigen in the epidermis and dermis. CMTV antigen
was also detected in the intestinal mucosa and occasionally in
endothelial cells in the submucosa. Antigen labelling within the
pancreas was detected in the exocrine glandular cells.
This study shows that the alpine newt is susceptible to CMTV
infection. Ranavirus transmission could be occurring between the
CMT and alpine newts in the North of Spain. It is not known
whether other amphibians, fish or reptiles are susceptible to CMTV
infection, but other ranaviruses, such as FV3, can infect a variety of
amphibian and fish species (Duffus et al., 2008). Subclinical infec-
tions with ranaviruses have been documented in free-ranging
amphibians and clinical signs in late stage tadpoles sometimes re-
solve (Fox et al., 2006). Adult frogs may serve as reservoirs of the
virus (Robert et al., 2005; Fox et al., 2006). In both outbreaks of
CMTV in Spain, mortality was observed in larvae or juvenile
amphibians, but not in adults. It is not known if CMTV has an intra-
specific reservoir similar to Ambystoma tigrinum (Brunner et al.,
This is the first report of a ranavirus infection in the alpine newt
and the second species known to be infected with CMTV in the past
2 years. The alpine newt is considered to be vulnerable in Spain
(Nores et al., 2007) and it is important to monitor CMTV in this
species to prevent its extinction due to this infection.
Conflict of interest statement
None of the authors of this paper has a financial or personal
relationship with other people or organisations that could inappro-
priately influence or bias the content of the paper.
The authors wish to thank the Veterinary Services of the Picos
de Europa National Park, the Electron Microscopy Area of the Uni-
versity of León and P. Solano for helping with the processing of
samples. A.B. is a recipient of a Contrato de Investigación para
Doctores from the Instituto Nacional de Investigación Agraria y
Agroalimentaria (INIA) and R.C. is recipient of a Ramón y Cajal con-
tract from the Spanish Ministerio de Educación y Ciencia co-fi-
nanced by Fondo Social Europeo.
Balseiro, A., Dalton, K.P., Del Cerro, A., Márquez, I., Cunningham, A.A., Parra, F.,
Prieto, J.M., Casais, R., 2009. Pathology, isolation and molecular characterization
of a ranavirus from the common midwife toad (Alytes obstetricans) on the
Iberian Peninsula. Diseases of Aquatic Organisms 84, 95–104.
Brunner, J.L., Schock, D.M., Davidson, E.W., Collins, J.P., 2004. Intraspecific
reservoirs: complex life history and the persistence of a lethal ranavirus.
Ecology 85, 560–566.
Cunningham, A.A., Langton, T.E.S., Bennett, P.M.B., Lewin, J.F., Drury, S.E.N., Gough,
R.E., Macgregor, S.K., 1996. Pathological and microbiological findings from
incidents of unusual mortality of the common frog (Rana temporaria).
Philosophical Transactions of the Royal Society of London: Biological Sciences
Cunningham,A.A., Tems,C.A., Russell,
demonstration of ranavirus antigen in the tissues of infected frogs (Rana
temporaria) with systemic haemorrhagic or cutaneous ulcerative disease.
Journal of Comparative Pathology 138, 3–11.
Daszak, P., Berger, L., Cunningham, A.A., Hyatt, A.D., Green, D.E., Speare, R., 1999.
Emerging infectious diseases and amphibian population declines. Emerging
Infectious Diseases 5, 735–748.
Duffus, A.L.J., Pauli, B.D., Wozney, K., Brunetti, C.R., Berrill, M., 2008. Frog virus 3-like
infections in aquatic amphibian communities. Journal of Wildlife Diseases 44,
Docherty, D.E., Meteyer, C.U., Wang, J., Mao, J., Case, S.T., Chinchar, V.G., 2003.
Diagnostic and molecular evaluation of three iridovirus-associated salamander
mortality events. Journal of Wildlife Diseases 39, 556–566.
Fox, S.F., Greer, A.L., Torres-Cervantes, R., Collins, J.P., 2006. First case of ranavirus-
associated morbidity and mortality in natural populations of the South
American frog Atelognathus patagonicus. Diseases of Aquatic Organisms 72,
Mao, J., Hedrick, R.P., Chinchar, V.G., 1997. Molecular characterization, sequence
analysis, and taxonomic position of newly isolated fish iridoviruses. Virology
Nores, C., García Rovés, P., Segura, A., 2007. Anfibios. In: Nores, C., García Rovés, P.
(Eds.), Libro Rojo de la Fauna del Principado de Asturias. Imprastur, Asturias,
Spain, pp. 162–163.
Robert, J., Morales, H., Back, W., Cohen, N., Marr, S., Gantress, J., 2005. Adaptive
immunity and histopathology in frog virus-3 infected Xenopus. Virology 332,
Teacher, A.G.F., Garner, T.W.J., Nichols, R.A., 2009. Evidence for directional selection
at a novel major histocompatibility class I marker in wild common frogs (Rana
temporaria) exposed to a viral pathogen (ranavirus). Public Library of Science 4,
Fig. 3. Electron micrographs of CMTV. (A) CMTV isolated from inoculated EPC cells. Negatively stained 2% phosphotungstic acid pH 6.2. (B) Inclusion body (asterisk) and
CMTV virions (arrows) in a necrotic cell in the kidney of a juvenile alpine newt. (C) CMTV virions (arrows) in the renal glomerulus of a CMT tadpole. B and C stained with
uranyl acetate and lead citrate. Scale bars = 50 nm. Original magnifications 200,000? (A), 25,000? (B), 50,000? (C).
A. Balseiro et al./The Veterinary Journal 186 (2010) 256–258