J. Comp. Path. 2012, Vol. 146, 269e273 Available online at www.sciencedirect.com

www.elsevier.com/locate/jcpa

DISEASE IN WILDLIFE OR EXOTIC SPECIES

Systemic Herpesvirus and MorbillivirusCo-Infection in a Striped Dolphin (Stenella

coeruleoalba)

Cor

002

doi

S. Soto*,†, B. Gonz�alez‡, K. Willoughbyx, M. Maleyx, A. Olvera†,k,S. Kennedy{, A. Marco* and M. Domingo*,†

*Departament de Sanitat i d’Anatomia Animals, Facultat de Veterin�aria, Universitat Aut�onoma de Barcelona (UAB),†Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Aut�onoma de Barcelona,‡CRAM (Fundaci�o per la Conservaci�o y Recuperaci�o d’Animals Marins), Cam�ı Ral 239, 08330 Premi�a de Mar,

Barcelona, Spain,xMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK, kDepartament de

Gen�etica i Microbiologia, Facultat de Bioci�encies, Universitat Aut�onoma de Barcelona (UAB), 08193 Bellaterra,

Barcelona, Spain and {Agri-Food and Biosciences Institute, AFBI Newforge, BT9 5PX Belfast, UK

resp

1-99

:10.1

Summary

During 2007 a dolphin morbillivirus epizootic affected the westernMediterranean and several striped dolphins(Stenella coeruleoalba) stranded on the Catalonian coasts. One of those animals had severe lymphoid depletion,necrosis and syncytial formation in lymph nodes and spleen, with large basophilic nuclear inclusions compat-ible with herpesvirus detected by immunohistochemical and ultrastructural examination. Non-suppurative en-cephalitis with associated morbillivirus antigen and morbillivirus antigen within alveolar macrophages werealso observed. A pan-herpesvirus nested polymerase chain reaction amplified a sequence virtually identicalto two cetacean herpesvirus sequences previously identified in systemic infections in an Atlantic Cuvier’sbeaked whale (Ziphius cavirostris) and in a Mediterranean striped dolphin. The herpesviral infection was prob-ably secondary to the immunosuppression caused by the morbillivirus. To our knowledge, this is the first reportof a cetacean co-infected by dolphin morbillivirus and herpesvirus with evidence of lesions attributable to bothviruses.

� 2011 Elsevier Ltd. All rights reserved.

Keywords: herpesvirus; morbillivirus; Stenella coeruleoalba; striped dolphin

Two dolphin morbillivirus (DMV) epizootics havecaused mass mortalities of Mediterranean striped dol-phins (Stenella coeruleoalba) in 1990e1992 (Domingoet al., 1992) and, with less severity, in 2006e2007(Fern�andez et al., 2008; Raga et al., 2008). Affecteddolphins suffered a systemic disease characterized bybronchiolointerstitial pneumonia, lymphoid depletionand non-suppurative encephalitis. Multinucleate syn-cytia and cytoplasmic and nuclear eosinophilic inclu-sions were detected in lung, lymphoid organs andbrain (Domingo et al., 1992; Raga et al., 2008).

ondence to: S. Soto (e-mail: sara.soto@uab.es).

75/$ - see front matter

016/j.jcpa.2011.04.002

Few cases of herpesvirus infection have been re-ported in cetaceans. Alphaherpesviruses have beenassociated with fatal systemic infections in bottlenosedolphins (Tursiops truncatus) (Blanchard et al., 2001)and in a Cuvier’s beaked whale (Ziphius cavirostris)(Arbelo et al., 2010), and also with cutaneous lesionsin bottlenose dolphins (Smolarek Benson et al.,2006). Gammaherpesviruses have been identified inmucosal lesions in bottlenose dolphins, Risso’s dol-phins (Grampus griseus), dwarf sperm whales (Kogiasima) and Blainville’s beaked whales (Mesoplodon den-

sirostris) (Saliki et al., 2006; Smolarek Bensonet al., 2006; van Elk et al., 2009). In addition,non-purulent encephalitis associated with herpesvirus

� 2011 Elsevier Ltd. All rights reserved.

Fig. 1. Lung-associated lymph node showing severe diffuse lym-phoid depletion. HE. Bar, 250 mm. Inset: mononuclearand syncytial lymph node cells showing large basophilicnuclear inclusions. HE. Bar, 15 mm.

270 S. Soto et al.

infection has been described in a harbour porpoise(Phocoena phocoena) (Kennedy et al., 1992). In the firstreport of herpesvirus infection in Mediterranean Seacetaceans, eight different herpesvirus sequences werefound in five of eight striped dolphins, which werealso infected with DMV (Belli�ere et al., 2010). In thesedolphins no lesions were found that could be attrib-uted to herpesvirus infection and the pathogenic con-tribution of these herpesviruses remains unclear. Inthis report we document a case of a striped dolphinco-infected by herpesvirus and DMV, with histologi-cal and ultrastructural evidence of pathogenicity forthis host.

A juvenile, 148 cm long, male striped dolphin wasfound stranded on the Catalonian Mediterraneancoast (Sant Pere Pescador, Girona) on August 13th,2007, the peak of the second DMV epizootic reportedin this area. The dolphin died 2 h after stranding withtetanic episodes. It was transported to the VeterinarySchool of Barcelona,where a complete necropsy exam-ination was performed. The dolphin was in poorbodily condition (26 kg body weight) and abundantsubcutaneous Phyllobothrium delphini cysts were theonly relevant gross findings. Samples from the lung,lung-associated lymph node, prescapular lymphnode, mesenteric lymph node, spleen, intestine, liver,kidney, heart, brain and anterior cervical spinal cordwere fixed in 10% neutral buffered formalin and em-bedded in paraffin wax. Five different locations weresampled from the brain, including the cerebral cortex(two sections), cerebellum, diencephalon and brain-stem. Sections (5 mm)were stained with haematoxylinand eosin (HE). Immunohistochemistry (IHC) witha mouse monoclonal antibody specific for the caninedistemper virus nucleoprotein (VMRD, Pullman,Washington, USA) was performed (Raga et al.,2008) on the samples of lung, lung-associated lymphnode, prescapular lymph node, mesenteric lymphnode, kidney, liver, spleen, brain and spinal cord.

Microscopically, the most prominent lesions wereobserved in lymphoid organs. All lymph nodes andthe spleen showed severe lymphocytic depletion,with cellular pyknosis and karyorrhexis and the pres-ence of multinucleate syncytial cells. The nucleiof many leucocytes and syncytial cells showed mar-gination of the peripheral chromatin with a largebasophilic nuclear inclusion (Fig. 1). Scattered hae-morrhages were present in the lymph nodes. Thelung showed mild multifocal interstitial pneumonia,with mononuclear leucocyte infiltration and syncytialformation. Similar viral inclusions were detected inthe nuclei of these cells. The liver displayed mild por-tal fibrosis and biliary hyperplasia, with occasionalmononuclear cells and syncytia with similar nuclearinclusion bodies in the portal areas. The renal pelvic

epithelium showed ballooning degeneration, withsome groups of degenerate or necrotic sloughed epi-thelial cells. A low number of mononuclear inflamma-tory cells and syncytial cells were observed in theunderlying interstitium. These cells and the transi-tional epithelial cells also had nuclear viral inclusions.Scant multifocal glial nodules with neuronal satellito-sis were observed in the brainstem, diencephalon andcerebral cortex.

Although depletion of lymphoid organs and bron-chiolointerstitial pneumonia are hallmarks of DMVinfection in dolphins (Domingo et al., 1992), the find-ing of basophilic nuclear inclusions in many mononu-clear cells and multinucleate syncytia was notindicative of DMV infection. No intranuclear inclu-sions were observed in the brain.

IHC for morbillivirus confirmed this hypothesis, asviral antigen could not be found in lymphoid organsin spite of the evident lesions. In fact, only lowamounts of morbillivirus antigen were found ina few alveolar macrophages, mostly not associatedwith the pneumonic lesions, and in the body and den-drites of occasional neurons in the brain (Fig. 2).Mul-tinucleate syncytial cells were negative in all tissues,but these are normally strongly labelled when in-fected by DMV (Domingo et al., 1992). Therefore, al-though infected by DMV, another virus wasassociated with the lymphoid depletion with syncy-tium formation. Some herpesviruses in cetaceans areassociated with syncytial cells, nuclear inclusionsand lesions in lymphoid organs (Blanchard et al.,2001; Arbelo et al., 2010).

To elucidate the possible participation of a herpes-virus as the causative agent of these lesions, immuno-histochemical, ultrastructural and molecular studies

Fig. 2. Morbillivirus antigen in a neuronal body and, with a punc-tiform distribution, in neuronal dendrites. Glial cells andmononuclear leucocytes surround the affected neuron.IHC. Bar, 50 mm.

Fig. 3. Splenic mononuclear cell nucleus showing numeroushexagonal virions. Transmission electron microscopy.Bar, 100 nm.

Virus Co-infection in a Striped Dolphin 271

were performed. IHC with a non-commercial poly-clonal anti-porcine herpesvirus-1 was performed(Kennedy et al., 1992) for the detection of herpesvirusantigen in lung, lung-associated lymph node, kidney,liver, spleen and brain. Brain sections from a case ofAujeszky’s disease in a pig were used as positive con-trol material. Positive labelling was observed associ-ated with the nuclear inclusion bodies in the lung,lymph nodes and spleen, and also in the cytoplasmof leucocytes in the lung and lymph node. The restof the tissues were negative.

Ultrastructural studies were performed onformalin-fixed samples of the spleen. Numerous hex-agonal 90e100 nm virions, compatible with herpesvi-rus, were found inside the inclusions in the nucleus ofsplenic cells (Fig. 3).

DNA was extracted from sections (10 mm) from theparaffin wax-embedded lung and lung-associatedlymph node (RecoverAll� total nucleic acid isolationkit for FFPE[Ambion]; Applied Biosystems, Warring-ton, Cheshire, UK), and a pan-herpesvirus nestedpolymerase chain reaction (PCR) was performed us-ing degenerate deoxyinosine-substituted primers spe-cific for the herpesvirus DNA polymerase gene(Ehlers et al., 1999). As a positive control for the nestedPCR reaction, DNA extracted from the buffy coat ofan ovine herpesvirus-2 positive cow using the DNeasyBlood and Tissue Kit (Qiagen Ltd., Crawley, WestSussex, UK) was used. Pan-herpesvirus nested PCRamplified a 190 base pair (bp) product, which was se-quenced and comparedwith other sequences availableinGenbank using the BLAST search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). This analysis showed thatthe amplified sequence was virtually identical to two

cetacean herpesvirus sequences: GU066291 (froma Cuvier’s beaked whale; Arbelo et al., 2010) andGU068981 (from a striped dolphin; Belli�ere et al.,2010), with 99% and 98% sequence identity, respec-tively. The sequence amplified in this study was classi-fied within the cetacean alphaherpesvirus group andsubmitted to GenBank (accession numberHQ214675). More distant relationships were studiedby phylogenetic analysis. Sequence HQ214675 wasalignedwith the cetacean alphaviruses present inGen-Bank (06-01-2010) using MUSCLE (BMC bioinfor-matics; 19th August, 2004) and the alignmentcurated using GBlocks (www.phylogeny.fr). A maxi-mum likelihood tree with 1000 bp was constructed us-ing MEGA4 (Tamura et al., 2007). As expected,sequence HQ214675 was closely related to the previ-ously mentioned sequences isolated in Spain between2007 and 2008 (Fig. 4). These sequences were groupedin a monophyletic branch with sequences obtainedfrom different cetacean species between 1999 and2009 without a clear geographical clustering. Thehigh homology with the other two alphaherpesvirussequences from cetaceans in the area suggests thatthis alphaherpesvirus is latently maintained in ceta-ceans in Mediterranean waters.

Morbillivirus infection in dolphins is strongly im-munosuppressive and secondary pathogens such asToxoplasma gondii and Aspergillus spp. are known tohave contributed to the fatal consequences of theDMV epizootic of 1990 (Domingo et al., 1992) and2007 (unpublished data). In fact, no single case oftoxoplasmosis or systemic mycosis has been observedin our caseload in the inter-epizootic period, during

Fig. 4. Maximum likelihood tree of cetacean alphaherpesvirus. The Tamura-Nei substitution model and nearest-neighbour-interchangeML heuristic method were used for tree reconstruction and 1,000 bootstraps for tree evaluation. Bootstrap values below 50 are notindicated. The isolate from the present study (GenBank accession numberHQ214675) showed greatest homology (99% and 98%,respectively) with GenBank sequences GU066291 and GU068981.

272 S. Soto et al.

which more than 110 striped dolphins have been sub-jected to gross necropsy and microscopical examina-tions (data not shown). This is despitea seroprevalence of 11% for Toxoplasma exposuredemonstrated for this species in the area (Cabez�onet al., 2004). Therefore, the herpesvirus infection inthis dolphin could be considered a secondary infectionthat had potentially been reactivated following im-munocompromise by the DMV infection. The highnumber of DMV-infected dolphins also positive forherpesvirus by PCR (five of eight; Belli�ere et al.,2010) is in line with this interpretation.

The capacity of herpesvirus to cause disease in ceta-ceans is unclear and may show variations dependingon different individual conditions and within species.In a series of 128 samples of skin and mucosal lesionsfrom 12 different cetacean species, as many as 11 dif-ferent herpesvirus sequences were amplified (ninefrom 32 mucosal lesions and two from a total of 88skin lesions; Smolarek Benson et al., 2006), showingan apparently high association of herpesvirus infec-

tion with mucosal and skin lesions. However, systemicdisease or internal lesions attributable to herpesvirushave been described only sporadically in cetaceans(Blanchard et al., 2001 in two bottlenose dolphins;Arbelo et al., 2010 in a Cuvier’s beaked whale;Kennedy et al., 1992 in a harbour porpoise and inthe present report).

The present case shows similarities and differencesto previous reports of fatal cetacean herpesvirus infec-tion. The main common feature of these cases is theformation of nuclear inclusions in many mononuclearcells. Extensive damage of the lymphoid system wasfound in one of the bottlenose dolphins reported byBlanchard et al. (2001), the Cuvier’s beaked whale(Arbelo et al., 2010) and the striped dolphin describedin this paper. In all of these cases the herpesviruses de-tected belonged to the Alphaherpesvirus subfamily,contrary to what might be predicted for a herpesviruswith lymphoid tropism, a typical feature of Gamma-herspesvirus (Roizmann et al., 1992). Syncytial cellformation appears to be frequent, although it was

Virus Co-infection in a Striped Dolphin 273

not reported in the Cuvier’s beaked whale (Arbeloet al., 2010) or in the harbour porpoise (Kennedyet al., 1992). Additionally, necrotizing vasculitis andthrombosis, described in lymphoid organs in thebeaked whale (Arbelo et al., 2010), were not seen instriped dolphins or in bottlenose dolphins.

At least in the striped dolphin it would appear thatherpesvirus multiplication and/or disease occurrenceis triggered or exacerbated by immunosuppressiveconditions, such as morbillivirus infection (Belli�ereet al., 2010), but more observations are needed in or-der to understand the role of herpesvirus in diseasecausation in this cetacean species. The interesting fea-ture of the present case is that the dolphin sufferedfrom two systemic viral infections (DMV and analphaherpesvirus). To our knowledge, this is the firstreport of such co-infection with evidence of lesionsattributable to both viruses.

Acknowledgements

The authors thank Dr. M. C. Horzinek (Spes Nostra,The Netherlands) and R.W. Nordhausen (CaliforniaAnimal Health and Food Safety Laboratory, Davis,United States) for their help in the analysis of ultra-structural images. We also thank M. P�erez (CReSA,Centre de Recerca en Sanitat Animal), B. P�erez(Departament de Sanitat i d’Anatomia Animals,Facultat de Veterin�aria, Universitat Aut�onoma deBarcelona) and Mr S. Ellison (Agri-Food and Biosci-ences Institute) for their technical support. The Mor-edun Research Institute is funded by the ScottishGovernment.

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eceived, September 9th, 2010

ccepted, April 5th, 2011

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