Kátia R. Groch

Cetacean Morbillivirus: Current Knowledge and Future Directions

We review the molecular and epidemiological characteristics of cetacean morbillivirus (CeMV) and the diagnosis and pathogenesis of associated disease, with six different strains detected in cetaceans worldwide. CeMV has caused epidemics with high mortality in odontocetes in Europe, the USA and Australia. It represents a distinct species within the Morbillivirus genus. Although most CeMV strains are phylogenetically closely related, recent data indicate that morbilliviruses recovered from Indo-Pacific bottlenose dolphins (Tursiops aduncus), from Western Australia, and a Guiana dolphin (Sotalia guianensis), from Brazil, are divergent. The signaling lymphocyte activation molecule (SLAM) cell receptor for CeMV has been characterized in cetaceans. It shares higher amino acid identity with the ruminant SLAM than with the receptors of carnivores or humans, reflecting the evolutionary history of these mammalian taxa. In Delphinidae, three amino acid substitutions may result in a higher affinity for the virus. Infection is diagnosed by histology, immunohistochemistry, virus isolation, RT-PCR, and serology. Classical CeMV-associated lesions include bronchointerstitial pneumonia, encephalitis, syncytia, and lymphoid depletion associated with immunosuppression. Cetaceans that survive the acute disease may develop fatal secondary infections and chronic encephalitis. Endemically infected, gregarious odontocetes probably serve as reservoirs and vectors. Transmission likely occurs through the inhalation of aerosolized virus but mother to fetus transmission was also reported.

Phocine Distemper Virus: Current Knowledge and Future Directions

Phocine distemper virus (PDV) was first recognized in 1988 following a massive epidemic in harbor and grey seals in north-western Europe. Since then, the epidemiology of infection in North Atlantic and Arctic pinnipeds has been investigated. In the western North Atlantic endemic infection in harp and grey seals predates the European epidemic, with relatively small, localized mortality events occurring primarily in harbor seals. By contrast, PDV seems not to have become established in European harbor seals following the 1988 epidemic and a second event of similar magnitude and extent occurred in 2002. PDV is a distinct species within the Morbillivirus genus with minor sequence variation between outbreaks over time. There is now mounting evidence of PDV-like viruses in the North Pacific/Western Arctic with serological and molecular evidence of infection in pinnipeds and sea otters. However, despite the absence of associated mortality in the region, there is concern that the virus may infect the large Pacific harbor seal and northern elephant seal populations or the endangered Hawaiian monk seals. Here, we review the current state of knowledge on PDV with particular focus on developments in diagnostics, pathogenesis, immune response, vaccine development, phylogenetics and modeling over the past 20 years.

Skeletal abnormalities in humpback whales Megaptera novaeangliae stranded in the Brazilian breeding ground

Skeletal tissues of 49 humpback whales Megaptera novaeangliae that stranded between 2002 and 2011 along the Abrolhos Bank seashore and its adjacent waters in Brazil were studied. Twelve (24.5%) animals presented pathological changes in one or more bones. Degenerative changes and developmental malformations were most frequent (10.2% each), followed by inflammatory/infectious and traumatic lesions (8.2% each). Infectious diseases led to severe lesions of the caudal vertebrae of 2 whales. In one of these individuals, the lesions involved 6 caudal vertebrae, leading to ankylosis of 3 vertebrae. Degenerative changes were observed in the vertebral columns of 3 animals, involving the joints of 13 ribs of 1 individual, and in the humerus of 1 whale. Traumatic lesions, such as osseous callus in the ribs, were observed in 4 animals. In 1 whale, the rib showed severe osteomyelitis, possibly resulting from the infection of multiple fractures. Developmental abnormalities such as spina bifida on 3 cervical vertebrae of 1 whale, fusion of spinal processes on thoracic vertebrae of 1 individual and fusion of the first 2 ribs unilaterally or bilaterally in 4 animals were found. Chronic infectious conditions found in the axial skeleton may have restrained spinal mobility and had detrimental effects on the general health of the animals, contributing to stranding and death. To our knowledge, this is the first systematic study on skeletal lesions in stranded humpback whales.

Lacaziosis-like disease in Tursiops truncatus from Brazil: a histopathological and immunohistochemical approach

Cetacean lacaziosis-like disease or lobomycosis-like disease (LLD) is a chronic skin condition caused by a non-cultivable yeast of the order Onygenales, which also includes Lacazia loboi, as well as Paracoccidioides brasiliensis and P. lutzii, respectively responsible for lacaziosis and paracoccidioidomycosis in humans. Complete identification and phylogenetic classification of the LLD etiological agent still needs to be elucidated, but preliminary phylogenetic analyses have shown a closer relationship of the LLD agent to Paracoccidioides spp. than to L. loboi. Cases of LLD in South American cetaceans based on photographic identification have been reported; however, to date, only 3 histologically confirmed cases of LLD have been described. We evaluated multiple tissue samples from 4 Tursiops truncatus stranded in the states of Santa Catarina (n = 3) and Rio Grande do Sul (n = 1), southern Brazil. Macroscopically, all animals presented lesions consistent with LLD. Hematoxylin-eosin, periodic acid-Schiff, Grocotts methenamine silver, and Mayers mucicarmin stains were used for histological evaluation. Microscopically, numerous refractile yeasts (4-9 µm in diameter) were observed in skin samples (4/4), and for the first time in dolphins, also in a skeletal muscle abscess (1/4). Immunohistochemistry using anti-P. brasiliensis glycoprotein gp43 as a primary antibody, which is known to cross-react with L. loboi and the LLD agent, was performed and results were positive in all 4 cases. We describe 3 new cases of LLD in cetaceans based on histopathology and immunohistochemistry. This is the first report of LLD in the muscle of cetaceans.

Reports of strandings and sightings of bottlenose dolphins (Tursiops truncatus) in northeastern Brazil and Brazilian oceanic islands

This study revises and updates information on bottlenose dolphin Tursiops truncatusstrandings and sightings in northeastern Brazil. A total of 72 strandings were recorded from 1992 to 2010, and 51 sightings were recorded from 1988 to 2010 along the states from Ceara to Bahia, including Fernando de Noronha and Sao Pedro and Sao Paulo archipelagos, Rocas Atoll and Abrolhos Bank. Most strandings were recorded in the states of Pernambuco, Bahia and Rio Grande do Norte. The number of reports was higher during summer, but no statistically significant difference was found between summer and other seasons. The majority of the stranded animals were adults (70.6%). Stranded calves were not observed. Total body length ranged from 138 to 321cm: 138 to 310cm for males; and 168 to 288cm for females. The average adult body length was 281cm (SD = 0.22). For stranding events where sex was determined (n = 29), a highly statistically significant difference was found between the number of males (n = 21) and females (n = 8). A total of six animals (8.3%) showed evidence of fisheries interactions such as net marks or removal of meat, blubber and eyes. Twenty-two sightings were recorded in the northeastern region, mostly in small islands and Rocas Atoll. This finding shows that the bottlenose dolphin is mainly an oceanic species in the region. More efforts are needed to improve our understanding of the populations and conservation status of bottlenose dolphins in this region.

Coinfection by Streptococcus phocae and cetacean morbillivirus in a short-beaked common dolphin Delphinus delphis.

We describe gross, histopathological, and immunohistochemical features of Streptococcus phocae and cetacean morbillivirus coinfection in a short-beaked common dolphin Delphinus delphis. Major gross findings were cutaneous purulent nodules in the tail fluke, vegetative mitral valve endocarditis, and presumed postpartum pyometra. Histologic examination revealed bacterial septicemia characterized by widespread intravascular coccoid bacterial emboli. These were associated with fibrinonecrotizing to pyogranulomatous dermatitis and panniculitis, embolic pneumonia, neutrophilic and lymphoplasmacytic meningochoroiditis, random neutrophilic hepatitis, lymphoplasmacytic myocarditis and epicarditis, necrotizing adrenalitis, suppurative endometritis, and multicentric reactive lymphadenopathy. Bacteriology and molecular analysis with sequencing of the 16S rRNA gene identified S. phocae from lung, brain, and adrenal gland tissue. Immunohistochemical analysis for morbillivirus detection revealed positive immunolabeling in the epithelium of the choroid plexus of the fourth ventricle. Published reports on S. phocae infection in cetaceans are rare, and pathological details are limited. The present case indicates that S. phocae has potential pathogenic capacity in common dolphins. The pathogenesis is proposed to have involved cutaneous penetration after a skin trauma, leading to initial cutaneous disease and eventual systemic infection.

A simultaneous diagnosis and genotyping method for global surveillance of cetacean morbillivirus

Cetacean morbillivirus (CeMV) is considered one of the most important viral pathogens in cetaceans. CeMV outbreaks of lethal disease have repeatedly been observed in Europe, the Americas, and Australia, while large herds of gregarious species were found to be the likely reservoirs and sources of CeMV infection to susceptible species in the Atlantic and Pacific Oceans. Furthermore, three new strains were detected recently in Hawaii, Brazil and Australia. To clarify the real global distribution of CeMV and possible carriers, we showed a novel technique successfully diagnosing and distinguishing different virus strains (DMV, PWMV and novel CeMVs) using FFPE samples from 1996 to 2011. This efficient method that combines qRT-PCR and high resolution melting (HRM) could be applied to the future retrospective global studies for better understanding of different prevalence and outbreak conditions among ocean basins and the mechanism of variable host response to pathogens.

Pathology and causes of death in stranded humpback whales (Megaptera novaeangliae) from Brazil

This study describes the pathologic findings of 24 humpback whales (Megaptera novaeangliae) found stranded along the Brazilian coast from 2004 to 2016. Eighteen (75%) animals evaluated were found stranded alive. From these, 13 died naturally on shore and five were euthanized. Six died at sea and were washed ashore. Of the 24, 19 (79.2%) were calves, four (16.7%) were juveniles, and one (4.2%) was an adult. The most probable cause of stranding and/or death (CSD) was determined in 23/24 (95.8%) individuals. In calves, CSD included neonatal respiratory distress (13/19; 68.4%), infectious disease (septicemia, omphaloarteritis and urachocystitis; 3/19; 15.8%), trauma of unknown origin (2/19; 10.5%), and vehicular trauma (vessel strike; 1/19; 5.3%). In juveniles and adult individuals, CSD was: emaciation (2/5; 40%), sunlight-thermal burn shock (1/5; 20%); and discospondylitis (1/5; 20%). In one juvenile, the CSD was undetermined (1/5; 20%). This study integrates novel findings and published case reports to delineate the pathology of a South-western Atlantic population of humpback whales. This foundation will aid in the assessment of the population health and establish a baseline for development of conservation policies.

Two decades of monitoring in marine debris ingestion in loggerhead sea turtle, Caretta caretta, from the western Mediterranean

Anthropogenic marine debris is one of the major worldwide threats to marine ecosystems. The EU Marine Strategy Framework Directive (MSFD) has established a protocol for data collection on marine debris from the gut contents of the loggerhead sea turtle (Caretta caretta), and for determining assessment values of plastics for Good Environmental Status (GES). GES values are calculated as percent turtles having more than average plastic weight per turtle. In the present study, we quantify marine debris ingestion in 155 loggerhead sea turtles collected in the period 1995-2016 in waters of western Mediterranean (North-east Spain). The study aims (1) to update and standardize debris ingestion data available from this area, (2) to analyse this issue over two decades using Zero-altered (hurdle) models and (3) to provide new data to compare the only GES value available (off Italian waters). The composition of marine debris (occurrence and amounts of different categories) was similar to that found in other studies for the western Mediterranean and their amounts seem not to be an important threat to turtle survival in the region. Model results suggest that, in the study area, (a) period of stranding or capture, (b) turtle size and (c) latitude are significant predictors of anthropogenic debris ingestion (occurrence and amount) in turtles. The GES value for late juvenile turtles (CCL>40 cm) has decreased in the last ten years in the study area, and this is very similar to that obtained in Italian waters. We also provide a GES value for early juvenile turtles (CCL≤40 cm) for the first time. Recommendations arising from this study include ensuring use of (1) the standardized protocol proposed by the MSFD for assessing marine debris ingestion by loggerhead sea turtles and (2) the ecology of the turtles (neritic vs oceanic), rather than their size, to obtain GES values.

Immunohistochemical investigation of the cross-reactivity of selected cell markers in formalin-fixed, paraffin-embedded lymphoid tissues of Franciscana ( Pontoporia blainvillei )

A considerable amount of knowledge on natural and anthropogenic pathologic conditions affecting different cetacean species has been gained over the last decades. Nonetheless, the immunopathological bases for most of these processes have been poorly documented or remain unknown. Comparative immunopathological investigations in these species are precluded by the limited number of specific antibodies, most of which are not commercially available, and the reduced spectrum of validated and/or cross-reactive ones. To partially fill in this gap of knowledge, a set of commercially available primary antibodies were tested for cross-reactivity against leukocytes and cytokines in formalin-fixed, paraffin-embedded (FFPE) lymphoid tissues (lymph nodes, spleen and thymus) of three bycaught, apparently healthy and fresh Franciscanas (Pontoporia blainvillei) using immunohistochemistry. On the basis of similar region specificity within the lymphoid organs, cellular morphology and staining pattern with human control tissues, 13/19 primary antibodies (caspase 3, CD3, CD57, CD68, FoxP3, HLA-DRα, IFNγ, IgG, IL4, IL10, Lysozyme, TGFβ and PAX-5) exhibited satisfactory cross-reactivity. Our results expand the spectrum of suitable cross-reactive primary antibodies in FFPE cetacean tissues. Further comparative immunopathological studies focused on infectious diseases and ecotoxicology may benefit from establishment of baseline expression of immunologically relevant molecules in various cetaceans species.