Abstract
Over the last 25 years, morbilliviral infections have caused dramatic mortalities among aquatic mammal species and populations worldwide. Dolphin morbillivirus (DMV) poses a major threat for free-ranging cetaceans, having been responsible for two epidemics in the Mediterranean Sea, the second of which (2006–2011) involved several other species besides striped dolphins (Stenella coeruleoalba).1,2,4,6,9
During the 2006–2011 morbilliviral epidemic, DMV genome sequences were molecularly detected in the brain tissue from one live-stranded bottlenose dolphin (Tursiops truncatus), which died after one day of medical care. Field operations were supported by personnel of a zoo, in which standard quarantine protocols were strictly applied. Despite these precautions, 28 days after the bottlenose dolphin stranding event, an adult male harbor seal (Phoca vitulina) hosted inside the zoo died. A detailed necropsy was carried out within 24 hours after death; tissue samples were fixed in 10% neutral buffered formalin for histopathology, refrigerated for microbiology and parasitology, and frozen for biomolecular investigations.
Postmortem investigations identified as the likely cause of death an acute endotoxic/septicemic shock due to Aeromonas hydrophyla. Furthermore, there was a generalized and diffuse lymphadenopathy, characterized by lymphoid cell depletion, hyalinosis in germinal centers, and several multinucleate syncytia (Figure 1A), accompanied by a mild, multifocal, chronic choriomeningitis with rare mononuclear cell perivascular cuffs (Figure 1B). Furthermore, a mild, multifocal white matter spongiosis and demyelination were apparent in the seal's brain, where intracytoplasmic eosinophilic inclusion bodies could be also observed in glial cells (Figure 1C), as well as in epithelial cells lining the urinary bladder and palatine tonsils. Pathological findings were suggestive of morbilliviral infection, which was subsequently confirmed by RT-PCR,3 with DMV sequences being amplified from the lungs, inguinal lymph nodes, and brain5. The resulting phylogenetic tree obtained combining the N1, F, and H gene regions showed a well-separated cluster corresponding to the available DMV sequences (Figure 2). Even though the analysis indicated that the overall genetic diversity of the known circulating DMV strains was low, our multigene approach allowed us to discriminate a distinct sub-clade, including the DMV sequences obtained from the harbor seal and two dolphins stranded in 2011, strongly supporting the hypothesis of DMV transmission from these stranded dolphins to the captive seal.
These two cetaceans, particularly the live-stranded bottlenose dolphin, were hypothesized as the likely source of DMV infection for the captive seal on the basis of the overlap of biomolecular characterization results; nevertheless, the exact route by which the virus entered the zoo remains unknown, due to the quarantine the facility administrators had imposed following the two wild dolphins' strandings.
In conclusion, the present case of DMV infection in a captive harbor seal, which occurred without any direct contact with two DMV-infected dolphins, confirm the possible DMV interspecies transmission from cetaceans to pinnipeds.10 Furthermore, it calls for new guidelines to enforce and extend quarantine protocols for zoological parks housing marine mammals and rehabilitating animals, considering the biologic features of the DMV isolates reported herein and the potential for interspecies viral transmission.7,8
| Figure 1. Microscopic findings in the harbor seal with DMV infection | 
Note the lymphoid cell depletion and multinucleate syncytia in the prescapular lymph nodes (arrows) in Figure 1A (HE, 10x, bar = 20 µm), and the mononuclear cell perivascular cuffing around a small capillary in the brain in Figure 1B (HE, 100x, bar = 10 µm), with an intracytoplasmic viral inclusion body inside a glial cell (arrowhead) in Figure 1C (HE, 100x, bar = 10 µm). |
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| Figure 2. Phylogenetic tree | 
Morbillivirus phylogeny inferred by maximum likelihood (ML) analysis of the nucleoprotein (N), fusion protein (F), and hemagglutinin (H) genes combined in a single dataset of 503 nucleotides. DMV sequences identified in this study are labeled (closed circle = harbor seal; closed triangle = stranded dolphins). DMV, Dolphin Morbillivirus; CeMV, Cetacean Morbillivirus; CDV, Canine Distemper Virus; PDV, Phocine Distemper Virus; PPRV, Peste-des-Petits-Ruminants Virus; RPV, Rinderpest Virus; MeV, Measles Virus. The infected species and the year of stranding or sequence submission for cetacean morbilliviruses are indicated, if available. |
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Acknowledgements
The authors wish to thank the personnel of the Mediterranean Marine Mammals Tissue Bank for their assistance with the necropsies and laboratory analyses.
* Presenting author
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