Abstract Book of EAVLD2012 - eavld congress 2012

S3 - P - 11
DOLPHIN MORBILLIVIRUS INFECTION IN A CAPTIVE HARBOR SEAL (PHOCA VITULINA)
Sandro Mazzariol 1 , Francesco Grande 2 , Cristina Pilenga 2 , Paola Modesto 3 , Cristina Biolatti 3 , Giovanni Di
Guardo 4 , Alessandra Mondin 5 , Simone Peletto 3 , Pier Luigi Acutis 3
1 Department of Comparative Biomedicine and Nutrition (BCA), University of Padua, Legnaro, Italy
2 Zoomarine Italia, Rome, Italy
3 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Turin, Italy
4 Department of Comparative Biomedical Sciences, University of Teramo, Teramo, Italy
5
Department of Animal Medicine and Health, University of Padua, Legnaro, Italy
morbillivirus, DMV, harbor seal, transmission, phylogeny
Introduction
During the last 25 years morbilliviral infections have caused
dramatic mortalities among several aquatic mammal species and
populations worldwide (1). In particular, Dolphin Morbillivirus
(DMV) represents a major biological threat for free-ranging
cetaceans. In the Mediterranean Sea, DMV has been responsible
for two epidemics, the first of which caused a dramatic mortality
among striped dolphins (Stenella coeruleoalba) between 1990
and 1992, while the second one was firstly reported in the
western Mediterranean in 2006-2008, moving thereafter eastward
along the French and the Italian coastlines. In June 2011, one
bottlenose dolphin (Tursiops truncatus) and one striped dolphin
stranded along the Tyrrhenian coastline, close to Rome. Both
animals were found molecularly (RT-PCR) positive to
Morbillivirus. Personnel from a marine park helped the stranding
network with the bottlenose dolphin, which stranded alive and
died after one day of rehabilitation. Standard quarantine protocols
were applied, in order to avoid any contact with cetaceans kept in
the park. On July 25 th , 2011, an 8-years-old male harbor seal
(Phoca vitulina) hosted inside the aforementioned park was
submitted for post-mortem examination after a short history of
anorexia, tremors, abdominal contractions, and polyuria with
hypothermia and vomit in the last phases.
Materials & methods
A complete post-mortem investigation was carried out the day
after death. Samples from main organs (brain, lungs, heart, liver,
kidneys, GI tract, spleen, lymph nodes) were fixed in 10% neutral
buffered formalin, paraffin-embedded and routinely processed for
histopathology. Samples from intestine and kidneys were
submitted for routine microbiological exams while cerebral,
hepatic, cardiac, splenic, pulmonary and lymph node tissues
were preserved frozen for molecular analyses.
Molecular investigations were carried out to look for the presence
of Canine Distemper Virus (CDV), Phocine Distemper Virus
(PDV) and Cetacean Morbillivirus (CeMV) nucleic acids. Multiple
sequence alignment was performed with the software BioEdit
using CLUSTAL W. An identity matrix was created to compare
sequenced products with morbillivirus sequences deposited in
GenBank. Phylogenetic analysis was performed using MEGA 5.0
software. The tree topology was inferred by the maximum
likelihood method with Kimura 2-parameter model. The reliability
of the topology was tested by bootstrapping 1,000 replicates.
Results
Necropsy revealed a mild gastro-enteritis and splenitis due to
Aeromonas hydrophyla, along with hepatic and renal necrosis
presumably due to endotoxemia. Besides these pathological
findings, a generalized and diffuse lymph node enlargement was
observed, along with a severe meningeal hyperemia and
choroidal edema. Histologically, a severe and diffuse reactive
lymphadenopathy was apparent, with lesions being characterized
by lymphoid cell depletion, hyalinosis, and several multinucleate
syncytia. A mild, multifocal, chronic choriomeningitis with white
matter spongiosis and demyelination were also apparent, with
eosinophilic inclusion bodies being also found in the cytoplasm of
brain neurons and glial cells. Furthermore, the lung showed a
mild, chronic bronchointerstitial pneumonia, with simultaneous
evidence of bronchiolar epithelial cell hyperplasia/hypertrophy.
Pathological findings suggested a morbilliviral infection, which
was confirmed by RT-PCR, with CeMV-specific genome
sequences being subsequently amplified from the brain of this
seal. CDV and PDV molecular analyses were negative. Similarity
was 97.6-99.5% with known DMV sequences and 85.9-89.6%
with other CeMV members (Porpoise Morbillivirus and the
tentatively named Pilot Whale Morbillivirus). Phylogenetic
analysis confirmed clustering of the harbor seal CeMV sequence
with previously reported DMV sequences from cetacean species
(Figure 1).
53
75
DMV AJ608288
95 DMV AY586536
DMV Z36978
DMV AJ224705
94
DMV Sc/2007 HQ829973
75 DMV Gme/2007 HQ829972
Harbor seal
PWMV 20E FJ842382
90
PMV IRL88 FJ648457
PMV 2990 AY586537
MeV UK140H94 U29285
69
RPV LA96 JN234010
PPRV ICV89 EU267273
PDV AF479277
CDV EU716337
SeV X56131
0.2
Figure 1: Maximum Likelihood dendrogram, based on the Kimura
2-parameter model, illustrating haemagglutinin gene (H)
nucleotide sequence relationships of morbilliviruses.
Discussion & conclusions
During the last epidemic outbreak, morbilliviral infection was
reported in several species besides striped dolphins, namely pilot
whales (Globicephala melas), bottlenose dolphins, Risso’s
dolphins (Grampus griseus), and fin whales (Balaenoptera
physalus). Although the susceptibility of these cetacean species
was already known, no reports other than the present one have
ever described DMV transmission to harbor seals. Indeed,
infection of pinniped species with morbilliviruses of cetacean
origin was previously speculated on the basis of serological
cross-reactivity and sequence homology (2). On a global scale,
mortality events in both this and in other pinniped species such
as grey seals (Halichoerus grypus), Bajkal seals (Pusa siberica),
and Caspian seals (P. caspica) have been respectively linked to
PDV and to CDV, with the former agent being more closely
related to CDV than to any other known morbillivirus, while DMV
appears to be more closely related to Rinderpest Virus (RPV).
Apart from their diverging phylogeny and their phylogenetic
distances, DMV and CDV could share similar epidemiologic
features. This hypothesis is strongly supported by the
simultaneous evidence of morbilliviral infection in the two freeranging
dolphins found beached ashore near Rome before this
captive seal died. Therefore, a possible route of virus (DMV)
entry into the facility could have been represented by either the
personnel and/or the instruments used for the rehabilitation of the
Morbillivirus-infected bottlenose dolphin that was found stranded
alive one month before. In conclusion, this report emphasizes
that, apart from cetaceans, also pinniped species such as harbor
seals are susceptible to DMV infection and related mortality.
Therefore, strict quarantine procedures should be enforced also
in structures where pinnipeds are kept when injured or stranded
cetaceans are hospitalized within these facilities.
References
1. Di Guardo G, Mazzariol S, Fernández A. (2011). Biologically threatened
dolphins and whales. Environmental Microbiology, 13(11), 2833-4.
2. Van de Bildt MWG, Martina BEE, Vedder EJ et al. (2000). Identification
of morbilliviruses of probable cetacean origin in carcases of Mediterranean
monk seals (Monachus monachus). Veterinary Record, 10, 691-4.