Molecular Diagnosis of Cetacean Morbillivirus in Beaked Whales Stranded in the Canary Islands (1999-2017)

Abstract

A retrospective survey for detecting the cetacean morbillivirus (CeMV) was carried out in beaked whales (BWs) stranded in the Canary Islands (1999-2017). CeMV is responsible for causing worldwide epizootic events with the highest mass die-offs in cetaceans, although the epidemic status of the Canarian Archipelago seems to be that of an endemic situation. A total of 319 tissue samples from 55 BWs (35 Cuvier's BWs and 20 specimens belonging to the Mesoplodon genus) were subjected to the amplification of a fragment of the fusion protein (F) and/or phosphoprotein (P) genes of CeMV by means of one or more of three polymerase chain reactions (PCR). RNA integrity could not be demonstrated in samples from 11 animals. Positivity (dolphin morbillivirus strain (DMV)) was detected in the skin sample of only a subadult male Cuvier's BW stranded in 2002, being the earliest confirmed occurrence of DMV in the Cuvier's BW species. The obtained P gene sequence showed the closest relationship with other DMVs detected in a striped dolphin stranded in the Canary Islands in the same year. A phylogenetic analysis supports a previous hypothesis of a cross-species infection and the existence of the circulation of endemic DMV strains in the Atlantic Ocean similar to those later detected in the North-East Atlantic, the Mediterranean Sea and the South-West Pacific.

Keywords: Canary Islands; PCR; Ziphiidae; Ziphius cavirostris; beaked whales; cetaceans; morbillivirus.

Figure 1

Map of the geographical distribution of beaked whales stranded in the Canary Islands between 1997 to 2017. Stranding sites for each beaked whale is identified by its case number. The species are represented with different colours.

Figure 2

Maximum likelihood phylogenetic tree for the nucleotide sequences of the phosphoprotein (P) gene. The phylogenetic tree consists of 102 sequences from reported cases of cetacean morbillivirus. To construct the tree, were designed the Neighbour-Join and BioNJ algorithms along with the Kimura 2-parameter model and Gamma distribution to model the evolutionary rate differences among sites [5 categories (+G, parameter = 0.7797)]. The Bootstrap method was performed to resample 500 replicates and evaluate the reliability of the tree. The accession number from GenBank, the host, the sample of detection, the date of collection, and the geographic area of stranding were used to identify the nucleotide sequences. Abbreviations: ENoAt (Northeast Atlantic Ocean); WAt (West Atlantic Ocean); CeAt (Central Atlantic Ocean); SoAt (South Atlantic Ocean); Me (Mediterranean Sea); Pa (Pacific Ocean); NPa (North Pacific Ocean); No (North Sea); CaspS (Caspian Sea).

Figure 3

Maximum likelihood phylogenetic tree for the nucleotide sequences of the fusion protein (F) gene. The phylogenetic tree consisting of 52 sequences from reported cases of cetacean morbillivirus. To construct the tree, we designed the Neighbour-Join and BioNJ algorithms along with the Tamura 3-parameter model and Gamma distribution to model the evolutionary rate differences among sites (five categories (+G, parameter = 0.5319)). The Bootstrap method was performed to resample 500 replicates and evaluate the reliability of the tree. The accession number from GenBank, the host, the sample of detection, the date of collection and the geographic area of each stranding were used to identify the nucleotide sequences. Abbreviations: ENoAt (North-East Atlantic Ocean); WAt (West Atlantic Ocean); CeAt (Central Atlantic Ocean); Me (Mediterranean Sea); No (North Sea).

Figure 4

Histological and Immunohistochemistry evaluation of PCR-positive skin sample from a Cuvier’s Beaked whale (CET 182). (A) Histopathology. Most of the keratinocytes of the stratum spinosum exhibit ballooning (freeze artefact). HE stain, ×10. Inset: Detail of the ballooning (*). HE stain, ×40. (B) Immunohistochemistry. CDV immunostaining was not observed. Immunohistochemistry stain, X 10. Inset: detail of lack of specific immunostaining against CDV in keratinocytes (*). Immunohistochemistry stain, ×40. (C) Immunohistochemistry. Positive control for CDV antibody in laryngeal tonsil sample. Immunostaining was observed in the cytoplasm of epithelial cells. Immunohistochemistry for morbillivirus using an antibody to CDV nucleoprotein., ×20. Inset: detail of specific immunostaining against CDV in epithelial cells (*). ×60.