Viral skin diseases in odontocete cetaceans: gross, histopathological, and molecular characterization of selected pathogens.

Fifty-five skin lesions from 31 stranded cetaceans along the Canary coasts (2011-2021) were submitted to macroscopic, histological, and molecular analyses to confirm infection by cetacean poxvirus, herpesvirus and cetacean morbillivirus. They were macroscopically categorized into eight categories with respective subcategories according to their color, shape, size, and consistency. Cetacean poxvirus was detected in 54.54% of the skin lesions through real-time and conventional PCRs based on the DNA polymerase gene. Additionally, herpesvirus and morbillivirus were currently detected from 43.63 and 1.82% of the cutaneous lesions, respectively. Coinfection of poxvirus and herpesvirus was detected in nine of them (16.36%), which makes the present study the first to report coinfection by both pathogens in skin lesions in cetaceans. A plausible approach to histopathological characterization of poxvirus-and herpesvirus-positive skin lesions was established. Hyperkeratosis, acanthosis, ballooning degeneration, and intracytoplasmic inclusion bodies in vacuolized keratinocytes through the stratum spinosum were common findings in poxvirus skin lesions. Alphaherpesvirus was associated with a prominent acanthotic epidermis, moderate necrosis, multifocal dyskeratosis, and irregular keratinocytes with both cellular and nuclei pleomorphism. The common histopathological findings of both pathogens were observed in coinfection lesions. However, those associated with herpesvirus were considerably more remarkable. Relationships between molecular and microscopic findings were observed for the lesions that showed tattoo-like and tortuous patterns. Further multidisciplinary diagnostic studies of infected skin lesions are needed to understand the epidemiology of these emerging infectious diseases.

Cerebral nasitremiasis in a Blainville's beaked whale (Mesoplodon densirostris) stranded in the Canary Islands.

Ten species within the genus Nasitrema (subfamily Nasitrematinae, family Brachycladiidae) have been reported infecting a wide variety of odontocetes worldwide, although there is still a lack of information about their presence in beaked whales (BWs). Nasitrema spp. are commonly described inhabiting the pterygoid sinus, the tympanic cavities, and the middle and inner ear; although aberrant migrations through the brain have been also reported. This trematode may cause different type of lesions, ranging from mild to severe saculitis, neuritis, otitis, and/or meningoencephalitis that may impede cetaceans to survive in the wild, resulting in incoordination, loss of equilibrium, and echolocation dysfunction ending in a stranding event. The presence of Nasitrema sp. was found in an adult female Blainville beaked whale stranded death in Fuerteventura, Canary Islands, on November 2016. The most relevant gross finding was a severe chronic-active multifocal pyogranulomatous and necrotizing encephalitis. Histologically, multiple areas of necrosis, pyogranulomatous and eosinophilic inflammation, haemorrhages and occasional cholesterol crystals were found associated with parasitic structures compatible with an adult trematode and its eggs. Molecular analysis, based on a quantitative polymerase chain reaction (qPCR) of the brain tissue sample detected 99% homology with a partial sequence of the NADH dehydrogenase subunit 3 (ND3) gene of Nasitrema delphini. In addition, liver, kidney, prescapular lymph node and brain samples were positive to herpesvirus (conventional nested PCR). Evidence of the presence of this parasite was not found in any of the 54 beaked whales (n = 54) stranded on the Canary Islands between 1999 and 2017, specifically 35 Cuvier's BWs and 19 specimens belonging to the Mesoplodon genus. To our knowledge, the current study represents the first description of a nasitremiasis in a member of the Ziphiidae family.

Molecular Characterization of Herpesviral Encephalitis in Cetaceans: Correlation with Histopathological and Immunohistochemical Findings.

Herpesviruses are causative agents of meningitis and encephalitis in cetaceans, which are among the main leading known natural causes of death in these species. Brain samples from 103 stranded cetaceans were retrospectively screened for the presence of herpesvirus DNA in the brain. Molecular detection of Cetacean Morbillivirus was performed in HV positive brain cases. Histopathologic evaluation of brain samples included the presence or absence of the following findings (n = 7): meningitis, perivascular cuffings, microgliosis, intranuclear inclusion bodies, malacia, neuronal necrosis and neurophagic nodules, and haemorrhages. Histological evidence of the involvement of other etiological agents led to complementary analysis. We detected the presence of alpha and gamma-HVs in 12 out of 103 (11.6%) brain samples from stranded cetaceans of five different species: one bottlenose dolphin, six striped dolphins, three Atlantic spotted dolphins, one Cuvier's beaked whale, and one common dolphin. Pathogenic factors such as viral strain, age, sex, and the presence of co-infections were analysed and correlated with the brain histopathological findings in each case. Herpesvirus was more prevalent in males, juveniles, and calves and a 41.6% incidence of co-infections in the brain was detected in our study: three with Dolphin Morbillivirus, one with Staphilococcus aureus septicaemia and one with Brucella spp.

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

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.

The Validation of a Non-Invasive Skin Sampling Device for Detecting Cetacean Poxvirus.

Poxvirus-like lesions are widely used as a potential health indicator in cetaceans, although for this application, corroboration of Poxvirus skin disease is imperative. Aiming to address skin biopsies intrusiveness, a preliminary investigation of a non-invasive skin sampling procedure to molecularly detect CePV-1 in 12 tattoo-like-lesions from two free-ranging stranded cetaceans in the Canary Islands was performed. Skin lesions were brushed with cytology cell samplers (CCSs) and placed into 1.5 mL microcentrifuge tubes with 1 mL of RNAlaterTM Stabilization Solution. For factual comparisons, DNA extractions from sloughed skin obtained with CCS and biopsies from the same lesions were accomplished with DNA Tissue Kit STM (QuickGene, Kurabo, Japan). Moreover, a second DNA extraction from sloughed skin with DNeasyTM Blood and Tissue Kit (Qiagen, Inc., Valencia, CA, USA) was performed to ascertain kit suitability for CCS. Molecular detection of CePV-1 was performed through a real-time PCR. As a result, a 91.7% and 83.3% rates of positivity were obtained with biopsies and CCS through Quickgene, respectively, compared to the rate of 100% using CCS with Qiagen. Accordingly, CCS is a reliable non-invasive sampling device to obtain sufficient genetic material to be analyzed for CePV-1 in tattoo-skin-lesions as well as for other purposes in cetaceans under human care.

Contribution to Herpesvirus Surveillance in Beaked Whales Stranded in the Canary Islands.

Herpesviruses (HVs) (Alpha- and Gammaherpesvirinae subfamilies) have been detected in several species of cetaceans with different pathological implications. However, available information on their presence in beaked whales (BWs) is still scarce. In this study, a total of 55 BWs (35 Ziphius cavirostris and 20 animals belonging to the Mesoplodon genus) were analyzed. Samples (n = 294) were obtained from BWs stranded along the coasts of the Canary Islands (1990-2017). Molecular detection of HV was performed by means of a conventional nested PCR based on the DNA polymerase gene. Herpesvirus was detected in 14.45% (8/55) of the analyzed BWs, including 2 positive animals from a previous survey. A percentage positivity of 8.57% was found within the Cuvier's BW group, while the percentage of positivity rose to 25% within the Mesoplodon genus group (three M. densirostris, one M. europaeus, and one M. bidens). All the obtained sequences from this study belonged to the Alphaherpesvirinae subfamily, from which three are considered novel sequences, all of them within the Mesoplodon genus group. In addition, to our knowledge, this is the first description of HV infection in Gervais' and Sowerby's BWs. Three out of eight HV-positive BWs displayed histopathological lesions indicative of active viral replication.

Histopathological Differential Diagnosis of Meningoencephalitis in Cetaceans: Morbillivirus, Herpesvirus, Toxoplasma gondii, Brucella sp., and Nasitrema sp.

Infectious and inflammatory processes are among the most common causes of central nervous system involvement in stranded cetaceans. Meningitis and encephalitis are among the leading known natural causes of death in stranded cetaceans and may be caused by a wide range of pathogens. This study describes histopathological findings in post-mortem brain tissue specimens from stranded cetaceans associated with five relevant infectious agents: viruses [Cetacean Morbillivirus (CeMV) and Herpesvirus (HV); n = 29], bacteria (Brucella sp.; n = 7), protozoa (Toxoplasma gondii; n = 6), and helminths (Nasitrema sp.; n = 1). Aetiological diagnosis was established by molecular methods. Histopathologic evaluations of brain samples were performed in all the cases, and additional histochemical and/or immunohistochemical stains were carried out accordingly. Compared with those produced by other types of pathogens in our study, the characteristic features of viral meningoencephalitis (CeMV and HV) included the most severe and frequent presence of malacia, intranuclear, and/or intracytoplasmic inclusion bodies, neuronal necrosis and associated neuronophagia, syncytia and hemorrhages, predominantly in the cerebrum. The characteristic features of Brucella sp. meningoencephalitis included the most severe and frequent presence of meningitis, perivascular cuffing, cerebellitis, myelitis, polyradiculoneuritis, choroiditis, ventriculitis, vasculitis, and fibrinoid necrosis of vessels. The characteristic features of T. gondii meningoencephalitis included lymphocytic and granulomatous encephalitis, tissue cysts, microgliosis, and oedema. In the case of Nasitrema sp. infection, lesions are all that we describe since just one animal was available. The results of this study are expected to contribute, to a large extent, to a better understanding of brain-pathogen-associated lesions in cetaceans.

Retrospective Study of Fishery Interactions in Stranded Cetaceans, Canary Islands.

Estimating cetacean interactions with fishery activities is challenging. Bycatch and chronic entanglements are responsible for thousands of cetacean deaths per year globally. This study represents the first systematic approach to the postmortem investigation of fishery interactions in stranded cetaceans in the Canary Islands. We retrospectively studied 586 cases necropsied between January 2000 and December 2018. Of the cases with a known cause of death, 7.4% (32/453) were due to fishery interactions, and the Atlantic spotted dolphin (Stenella frontalis) was the most affected species [46.9% (15/32)]. Three types of fishery interactions were recognized by gross findings: bycatch [65.6% (21/32)], chronic entanglements [18.8% (6/32)], and fishermen aggression [15.6% (5/32)]. Among the bycaught cases, we differentiated the dolphins that died because of ingestion of longline hooks [23.8% (5/21)] from those that died because of fishing net entrapments [76.2% (16/21)], including dolphins that presumably died at depth due to peracute underwater entrapment (PUE) [37.5% (6/16)], dolphins that were hauled out alive and suffered additional trauma during handling [43.8% (7/16)], and those that were released alive but became stranded and died because of fishery interactions [18.7% (3/16)]. Gross and histologic findings of animals in each group were presented and compared. The histological approach confirmed gross lesions and excluded other possible causes of death. Cetaceans in good-fair body condition and shallow diving species were significantly more affected by fishery interactions, in agreement with the literature. Low rates of fishery interactions have been described, compared with other regions. However, within the last few years, sightings of entangled live whales, especially the minke whale (Balaenoptera acutorostrata) and Bryde's whale (B. edeni), have increased. This study contributes to further improvement of the evaluation of different types of fishery interactions and may facilitate the enforcement of future conservation policies to preserve cetacean populations in the Canary Islands.

Lipids of lung and lung fat emboli of the toothed whales (Odontoceti).

Lipids are biomolecules present in all living organisms that, apart from their physiological functions, can be involved in different pathologies. One of these pathologies is fat embolism, which has been described histologically in the lung of cetaceans in association with ship strikes and with gas and fat embolic syndrome. To assess pathological lung lipid composition, previous knowledge of healthy lung tissue lipid composition is essential; however, these studies are extremely scarce in cetaceans. In the present study we aimed first, to characterize the lipids ordinarily present in the lung tissue of seven cetacean species; and second, to better understand the etiopathogenesis of fat embolism by comparing the lipid composition of lungs positive for fat emboli, and those negative for emboli in Physeter macrocephalus and Ziphius cavirostris (two species in which fat emboli have been described). Results showed that lipid content and lipid classes did not differ among species or diving profiles. In contrast, fatty acid composition was significantly different between species, with C16:0 and C18:1ω9 explaining most of the differences. This baseline knowledge of healthy lung tissue lipid composition will be extremely useful in future studies assessing lung pathologies involving lipids. Concerning fat embolism, non-significant differences could be established between lipid content, lipid classes, and fatty acid composition. However, an unidentified peak was only found in the chromatogram for the two struck whales and merits further investigation.

Neurobrucellosis in a common bottlenose dolphin (Tursiops truncatus) stranded in the Canary Islands.

BACKGROUND: Brucella spp. isolation is increasingly reported in cetaceans, although associated pathologies, including lesions of the musculoskeletal and nervous systems, are less frequently described. Concerning the nervous system, Brucella sp. infection causing meningitis, meningoencephalitis or meningoencephalomyelitis have been extensively reported in striped dolphins (Stenella coeruleoalba), and less frequently in other cetacean species. CASE PRESENTATION: A juvenile female common bottlenose dolphin (Tursiops truncatus) was found stranded alive in Lanzarote (Canary Islands, Spain) in 2005, but died shortly after. On physical examination, the dolphin showed a moderate body condition and was classified as code 2 (fresh dead) at the time of necropsy. The main gross findings were severe multiorgan parasitism, thickened and congested leptomeninges, and (sero)fibrino-suppurative and proliferative arthritis of the shoulder joint. Histopathological examination revealed the distinct features of a sub-acute systemic disease associated with Cetacean Morbillivirus (CeMV) infection. However, brain lesions diverged from those reported in systemic CeMV infection. This led to suspect that there was a coinfecting pathogen, based on the characteristics of the inflammatory response and the lesion distribution pattern in the central nervous system. Brucella sp. was detected in the brain tissue by PCR and Brucella antigen was demonstrated by immunohistochemistry in the brain and shoulder joint lesions. CONCLUSIONS: The zoonotic potential of marine mammal strains of Brucella has been demonstrated both in natural and laboratory conditions. In this study, PCR detected Brucella sp. in the brain of a common bottlenose dolphin stranded in the Canary Islands; the dolphin was also co-infected with CeMV. This is the first detection of Brucella sp. infection in a stranded cetacean in this archipelago. Therefore, we stress the importance of taking adequate measures during the handling of these species to prevent the transmissions of the infection to humans.

Morbillivirus infection in Risso's dolphin Grampus griseus: a phylogenetic and pathological study of cases from the Canary Islands.

The earliest evidence of cetacean morbillivirus (CeMV) infection dates from 1982, when the dolphin morbillivirus strain (DMV) was identified in bottlenose dolphins Tursiops truncatus stranded in the mid-Atlantic region. Since then, CeMV has been detected globally in at least 26 species of mysticetes and odontocetes, causing widespread mortality and a wide range of pathological effects. In the Canary Islands, DMV and pilot whale morbillivirus have been detected in cetacean species, including short-finned pilot whales Globicephala macrorhynchus and bottlenose dolphins. Risso's dolphins Grampus griseus have been reported year-round in waters of the Canary Islands and are considered a resident species. No information is currently available on CeMV prevalence in this species in this ocean region. We searched for evidence of CeMV infection in 12 Risso's dolphins stranded in the Canary Islands from 2003 to 2015 by means of histopathology, PCR and immunohistochemistry. PCR revealed 2 CeMV-positive animals (16.6%). Phylogenetic analysis showed that the strains from the 2 positive specimens were phylogenetically quite distant, proving that more than 1 strain infects the Risso's dolphin population in this region. We also determined that the strain detected in one of the specimens mainly circulated in the northeastern Atlantic Ocean from 2007 to 2013.