Necropsies of 95 harbor seals (P. vitulina) collected from July to December 2002 showed a moderate-to-severe pulmonary alveolar and interstitial emphysema and alveolar edema as the predominant findings. Additional lesions included mediastinal emphysema, gradually variable suppurative bronchopneumonia, and catarrhalic enteritis. Histologic lesions consisted of interstitial pneumonia with multinucleated syncytial cells and a moderate-to-severe lymphocytic depletion in the lymphoid tissues. Single animals had an acute, focal, nonsuppurative encephalitis (). In addition, neuronal necrosis and mild gliosis were observed. Cytoplasmic and nuclear acidophilic inclusion bodies were detected in respiratory epithelial cells, gastric surface mucous and chief cells, intestinal crypt epithelial cells, and hepatic and pancreatic duct epithelial cells. In the urogenital tract, inclusion bodies were observed in endometrial, vaginal, and epididymal epithelial cells as well as epithelial cells of the renal pelvis and urinary bladder. Occasionally, inclusion bodies were present in neuronal and glial cells of the central nervous system.
Figure 1 Tissue lesions from a harbor seal (Phoca vitulina) with phocine distemper virus infection. (A) Cerebral cortex with nonsuppurative encephalitis. Hematoxylin and eosin staining. (B) Immunohistochemical labeling of morbilliviral antigen in glandular epithelial (more ...)
Immunohistochemical analyses were performed by using a cross-reacting murine monoclonal antibody specific for the morbillivirus nucleoprotein. Morbillivirus antigen was demonstrated in 39 (45%) of the 86 cases. Morbillivirus antigen was detected in lung, trachea, stomach, intestine, liver, pancreas, kidneys, urinary bladder, female genital mucosa, and epididymal tubules (). In the lymphoid tissues, variable numbers of lymphocytes and macrophages of the follicular and parafollicular areas were positive. In affected areas of the brain, neurons and glial cells contained morbillivirus antigen in the nuclei and cytoplasm.
Screening for morbillivirus-specific nucleic acid in tissue samples from lung, spleen, and lymph nodes as well as in blood samples from 85 seals was performed by reverse transcription–polymerase chain reaction (RT-PCR). For this procedure, universal morbillivirus primers based on the conserved sequence of a 457-bp fragment of the phosphoprotein gene (8
) were used. PDV-specific RNA was detected in 46 (54%) of the 85 seals from German waters affected from July onward. Both PDV-specific RNA and morbillivirus antigen were detected in 33 (43%) of 77 animals. Seals with no detectable morbillivirus antigen or nucleic acid had pneumonia and endoparasitosis of varying degrees of severity or died of undetermined causes.
Sequence analysis of the RT-PCR product showed an identity of 97% compared to the Dutch isolate of 1988. The German isolate was 100% identical with the PDV isolate from the Netherlands and differed in 1 nt from the Danish isolate (6
) (not shown). Phylogenetic analysis showed that the phocine isolates from the two epidemics in European waters formed a discrete cluster, separated from the CDV isolates, including those from lion and Siberan seal ().
Figure 2 Unrooted neighbor-joining phylogenetic tree constructed by using 369 nt from the gene coding for the morbillivirus P protein. Alignments were calculated with CLUSTAL X (Version 1.8). Bootstrapping (values indicated in %) was performed with 1,000 replicates. (more ...)
Neutralization assays using the CDV strain Onderstepoort were performed to determine the titers of serum samples from 187 harbor seals from German waters, collected from 1996 until the outbreak of the epidemic in 2002 (9
). Because of the cytotoxicity of some serum samples, only titers of >
10 were considered positive. No neutralizing antibodies were found in 164 (88%) of 187 serum samples. Titers from 22 (12%) of the 187 animals ranged from 14 to 240 (mean 50.5, ± 52.6 standard deviation). One animal had a titer of 480.