Circulation of a novel strain of dolphin morbillivirus (DMV) in stranded cetaceans in the Mediterranean Sea

Abstract

Dolphin morbillivirus (DMV) has been responsible for several outbreaks of systemic infection and has resulted in cetacean strandings in the Mediterranean. In August-October 2016, seven striped dolphins (Stenella coeruleoalba) stranded on the Sicilian coastline (Italy) tested positive for DMV. Tissue samples from brain, lung, pulmonary lymph nodes, heart, spleen, liver, stomach, intestine, kidneys and urinary bladder, as well as blowhole swabs, were collected during necropsy for molecular diagnostics and pathology studies. Extracted tissue RNA was screened for DMV by real-time reverse transcription polymerase chain reaction (PCR). Some tissues exhibited microscopic lesions that were consistent with DMV infection on histopathological and immunohistochemical grounds. Conventional reverse transcription PCR to target partial nucleoprotein and phosphoprotein genes yielded sequences used to genetically characterize the associated DMV strain. DMV RNA was detected by both PCR assays in all tested tissues of the seven dolphins, which suggests systemic infections, but was absent from another dolphin stranded on the Sicilian coastline during the same period. The partial phosphoprotein and nucleoprotein gene sequences from the positive dolphins were 99.7% and 99.5% identical, respectively, to the DMV sequences recently observed in cetaceans stranded on the Spanish Mediterranean. Our study suggests that this DMV strain is circulating in the Mediterranean.

Figure 1

Stranding locations of the eight striped dolphins (Stenella coeruleoalba) that were stranded on the Sicilian coast in 2016. Black dots mark the location of strandings. The stranding date (month-day-year) is indicated next to each animal’s identification number. The map was created by the MapInfo Professional v8.5 software.

Figure 2

Cerebral microscopic lesions and results of immunohistochemistry against dolphin morbillivirus (DMV) in the striped dolphins (Stenella coeruleoalba) stranded in Sicily coast, Italy, in 2016. (A) Animal 1: Multifocal gliosis in the frontex cortex. H&E. Scale bar: 50 µm. (B) Animal 1: Serial slide of area shown in A. Massive presence of DMV antigen in neuronal bodies and punctate staining in the neutrophile. DMV immunostaining. Scale bar: 50 µm. (C) Animal 1: Perivascular infiltration of leukocytes, gemistocytes and syncytial cells in cerebral cortex. H&E. Scale bar: 50 µm. (D) Animal 1: serial slide of the area shown in C. Presence of DMV antigen in cerebral cortex perivascular area. DMV Immunostaining. Scale bar: 50 µm. (E) Animal 2: Glial proliferation (circle) and gemistocytic astrocytes (arrows) in the parietal cortex. H&E. Scale bar: 50 µm. (F) Animal 6: Malacia and glial proliferation (circle) in medulla oblongata. H&E. Scale bar: 50 µm. (G) Animal 7: Gliosis and hyperplasia (circle) of blood vessels in the medulla oblongata. H&E. Scale bar: 50 µm. (H) Animal 7: serial slide of the area shown in G. Presence of DMV antigen (circles) in the medulla oblongata. DMV immunostaining. Scale bar: 50 µm.

Figure 3

Phylogenetic analysis. Maximum likelihood trees based on a 250-nucleotides (nt 401–650) fragment of DMV phosphoprotein (P) and on a 192-nucleotides (nt 754–945) fragment of DMV nucleoprotein (N) gene sequences. The name of each sequence comprises the virus strain name (DMV, dolphin morbillivirus; PMV, porpoise morbillivirus; PWMV, pilot whale morbillivirus), GenBank accession number, cetacean species host (Sc, Stenella coeruleoalba; Gm, Globicephala melas; Gma, Globicephala macrorhynchus; Tt, Tursiops truncatus; Zc, Ziphius cavirostris; Bp, Balaenoptera physalus; Ip, Indopacetus pacificus; La, Lagenorhynchus albirostris; Pp, Phocoena phocoena, Dd, Delphinus delphis), year and geographic area of stranding (At, Atlantic Ocean; Me, Mediterranean Sea; No, North Sea; Pa, Pacific Ocean). Black dots (●) indicate the sequence obtained from one of the seven striped dolphins (Stenella coeruleoalba) stranded on the Sicilian coastline, Italy, in 2016.