Juvenile, male harbor seal (Phoca vitulina).In 2014, an increased mortality occurred among harbor seals (Phoca vitulina) in northwestern European waters. Hundreds of carcasses were washed up on the shores of Sweden, Denmark, and Germany.(4,10,17) Along the Wadden sea coast of Lower Saxony, Germany, more than 320, and along the coast of Schleswig-Holstein, Germany, more than 2100 dead harbor seals were counted by the end of 2014.(11) The population size in the German part of the Wadden sea is approximately 12,000 animals. (19) This necropsied seal was found moribund on the beach of the north Freisian coast close to the town of B+â-+sum. The animal was euthanized humanely.
Necropsy revealed a poor nutritional status and severe generalized muscular atrophy. The poorly retracted lungs displayed severe congestion, diffuse consolidation, and multifocal firm nodular areas of gray-yellow discoloration with varying numbers of metazoan parasites. Additionally, there was severe, diffuse alveolar and interstitial emphysema. The pulmonary lymph nodes were markedly enlarged, and the tonsils were moderately swollen. There was no content in the stomach.
The tissue from the lung displays a mild diffuse hyperplasia of bronchial epithelial cells with loss of cilia. There are mildly scattered karyopyknotic cells (necrosis) within the epithelial lining of the deep airways associated with few intraepithelial lymph-ocytes and occasional mitotic figures. Bronchial lumina are partially filled with cellular debris, neutrophils, foamy macrophages and desquamated epithelial cells. Bronchial glands show severe hydropic swelling, karyorrhexis and pyknosis of epithelial cells (degeneration and necrosis) with accumulation of cellular debris within the lumina. In the peri-bronchial and perivascular interstitium, there is a moderate to severe infiltration of lymphocytes, plasma cells and macrophages extending multifocally into the interalveolar interstitium. Most alveolar lumina are filled either with macrophages and desquamated pneumocytes or neutrophils and macrophages. Occasionally, few to moderate numbers of erythrocytes are present in alveolar lumina. Furthermore, accumulation of eosinophilic homogenous or fine fibrillar material is present in alveoli (fibrin), sometimes in a membranous shape, along the alveolar wall. Interalveolar capillaries are engorged with red blood cells. Multi-focally, venous vessels show partial occlusion by eosinophilic material attached to the vascular wall with infiltrating fibroblasts and endothelial lining on the surface. Multifocally, there are cross and longitudinal sections of larval nematode structures with a diameter of up to 50 μm and a length of about 200 μm surrounded by a cuticle. In the body cavity, a digestive tract is present. Restricted to few lobules, there are distended alveoli with ruptured interalveolar septae as well as distended interlobular septae.
Lung: Pneumonia, broncho-interstitial, moderate, chronic, multifocal, lympho-histiocytic and plasmacytic with hyperplasia of bronchial epithelium; bronchitis, moderate, acute, multifocal, necrotizing with necrotizing adenitis of bronchial glands; pneumonia, suppurative, multifocal, moderate; multifocal subtotal vascular thrombosis; pulmonary endoparasitosis with larval stages of nematodes.
Lung tissue and tracheal swab were positive for influenza A virus using an influenza A-specific PCR for the matrix protein. Virus culture on embryonated chicken eggs and Madin-Darby canine kidney (MDCK) cells resulted in isolation of influenza A virus. Using specific primer sets for the hemagglutinin and neuraminidase genes with subsequent sequencing of the amplicons allowed classification into the subtype H10N7 of the influenza A virus.
Immunolabeling of influenza A virus nucleoprotein revealed specific antigen staining in the respiratory tract.
Immunohistochemistry for morbillivirus nucleoprotein and reverse transcription PCR for morbillivirus were negative.
Microbiologically, moderate colony numbers of Escherichia coli and low numbers of Acinetobacter pittii, Streptococcus phocae and Stenotrophomonas maltophila were isolated from the lung.
Parasitologically, the lung worms Otostrongylus circumlitus and Parafilaroides gymnurus, as well as the heart worm Acanthocheilonema (previously termed Dipetalonema) spirocauda, were identified in the lungs.
Influenza A, harbor seal
The seal suffered from severe pneumonia caused by a concurrent infection with influenza A virus H10N7, pulmonary endoparasites, and several bacterial species. Besides the lung, morphologic changes associated with the influenza A virus infection were present only in the upper respiratory tract characterized by acute necrotizing rhinitis and tracheitis. Immunohistologically, influenza A virus nucleoprotein was demonstrated intralesionally in the cytoplasm and nuclei of epithelial cells of bronchi and bronchial glands. Additionally, it was present in nasal and tracheal respiratory epithelium.
Avian influenza A viruses are known to cross species barriers and infect various mammalian species, including man and pinnipeds.(2,4,9,10,13) Different influenza virus A subtypes have previously been reported to cause fatal mass mortalities of harbor seals, including subtypes H5N1, H3N8 and H5N4.(2,4,9,13) However, subtype H10N7 has not been isolated from seals before. Infections with the subtype H10N7 have been reported in man in Egypt and Australia,(3,7) and various avian species including chicken,(3,14) ducks,(14) mallards,(17) and wild birds.(16) According to the predicted amino acid sequence, this subtype is regarded as a low pathogenic avian influenza (LPAI) strain with a low zoonotic potential.(11,17) However, virus detection has been reported in spleen samples of affected seals from Denmark and a more virulent potential of this H10N7 subtype cannot be excluded completely.(11)
The influenza epidemic in 2014 in northern European waters started with an increased mortality among harbor seals in March in Sweden, and swapped in July to Denmark,(11) and reached Germany in October and The Netherlands in November. More than 2400 dead harbor seals were counted in German coastal waters.(4,12) The origin and mode of transmission of this avian influenza virus remains undetermined. Phylogenetic analysis revealed a close relationship to various influenza A viruses detected in wild birds. Specifically, the hemagglutinin and neuraminidase genes were genetically most closely related to subtype H10N7 viruses recently found in migratory ducks in Georgia, Egypt, and the Netherlands.(4) The seals may be infected oronasally with the virus through direct or indirect contact with wild birds or their droppings, because they share the same shoreline habitats as waterfowl.(11,18) Hemagglutinin is the attachment protein of influenza viruses, binding to sialylated glycans on the host cell surface. Avian and swine influenza viruses bind to α2,3 linked sialic acid. In contrast, human and several mammalian influenza A viruses bind to sialylated glycans with an α2,6 linkage to galactose.(1,2)
Virus-associated destruction of epithelial cells in the respiratory tract probably caused a predisposition for secondary bacterial infection resulting in suppurative pneumonia. Together with the parasitic burden in the lung, respiratory insufficiency is regarded as a main cause of death. Bacteriologically, Escherichia coli, Acinetobacter pittii, Streptococcus phocae and Stenotrophomonas maltophila were isolated in the present case. Acinetobacter pittii and Streptococcus phocae are regarded as opportunistic pathogens. Similarly, Streptococcus phocae has been isolated from seals affected with phocine distemper, but has also been associated with starvation and abortion in Cape fur seals (Arctocephalus pusillus pusillus).(7) Interestingly, bacteriological culture of lung samples from other seal carcasses with influenza A H10N7 infection resulted in isolation of bacteria, e.g. Bordetella bronchiseptica and Streptococcus equi ssp. zooepidemicus that were not isolated from lung samples of seal carcasses investigated during the annual health monitoring program. However, these bacterial species were exclusively isolated during the seal die-offs in 2002 and 2014 as a secondary pathogen (Siebert, pers. communication).
Commonly reported nematodes in the lung of harbor seals are Parafilaroides gymnurus, Otostrongylus circumlitus, and the heartworm Acanthocheilonema (previously termed Dipetalonema) spirocauda.(15) Nematodic infections of the respiratory tract of harbor seals represent a common finding, particularly in juvenile individuals.(15) The epithelial hyperplasia of deep airways, as well as the vascular thrombosis, may be related to the parasitic infestation in this case.
As differential diagnosis for infectious seal mass die-offs, phocine distemper virus (PDV) infection has to be considered. Phocine distemper was excluded in this case by reverse transcription PCR (RT-PCR) and immunohistochemistry. The total Wadden Sea seal population has reached a so far unprecedented population size of more than 39,000 animals. A serological survey revealed a lack of antibodies against phocine distemper indicating a high susceptibility for phocine distemper.(6) The current prevalence of influenza virus antibodies in the German harbor seal population is unknown.
1. Lung: Pneumonia, bronchointerstitial, necrotizing and fibrinous, diffuse, severe, with submucosal gland necrosis, alveolar and interlobular emphysema, and organizing fibrin thrombi.
2. Lung: Larval nematodes, few (variable across sections).
Influenza A virus affects many species and waterfowl are considered the natural reservoir host for many subtypes. Subtypes which result in infection of mammals often occur through transmission from waterfowl. These viruses replicate in the intestinal tract of birds and fecal-oral transmission is the primary route of infection. In a rash of harbor seal deaths in Denmark reported in conjunction with the outbreak from which this case originated, (discussed above) animals were also affected with a necrotizing bronchopneumonia with the presence of bacteria in alveoli. Influenza virus A (H10N7) was also isolated in those cases; additionally, bacterial isolates included Pseudomonas aeruginosa with variable growth of Streptoccus equi subsp. zooepidemicus. The pulmonary lesions were attributed to primary infection with influenza A, complicated by secondary bacterial infection, as seen in this case.(11)
During the outbreak described above, which primarily resulted in seal deaths in Sweden, Denmark and Germany, only low numbers of seals were found dead in the Netherlands; the precise reason for variable mortality is unclear. A study measuring antibody levels of H10N7 (which in some references is termed seal influenza A) in captured or rehabilitated seals in the Netherlands found detectable antibodies in 41% of pups, 10% of weaners and 58% of adults or subadults, indicating infection with this virus may be widespread. Antibody titers were also found in adult grey seals. In subadult harbor and grey seals sampled prior to the 2014-2015 outbreak, only a small number had antibody titers against the virus, which may indicate absence of widespread herd immunity at the start of the outbreak.(5)
The conference histologic description was similar to the contributors histologic description above. There was extensive discussion regarding the vascular changes in the section. There are distinctive fibrin thrombi partially occluding vessel lumina, but there are also vessels which appear to have near complete occlusion by fibroblasts and/or macrophages that are surrounded by concentric layers of fibrous tissue. Participants postulated the vascular changes may represent variable chronicity of the lesions, with the older, mature thrombi demonstrating partial to complete recanalization. Multifocally, hyaline membranes line the alveolar surface and are more prominent in the less atelectatic areas of the lung. Alveolar and interlobular septal emphysema were also described. Multifocally there is hyperplasia of bronchial and bronchiolar epithelium, with loss of cilia in many areas. The contributor provides a detailed description and excellent history regarding the events surrounding this case.
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