The seal death in Danish waters 1988. 2. Virological studies.

Mass abortions and high mortality were observed in harbour seals in Danish waters during 1988. Severe pneumonia and emphysema were typical clinical and post-mortem findings. Virological studies were carried out to identify the cause of the epidemic. Although seal herpesvirus (SeHV) was isolated in 23 of 114 animals this virus was subsequently found not to be the primary cause of the disease. Following the observation of seroconversion against canine distemper virus (CDV) in diseased seals (Osterhaus & Vedder 1988) a CDV-like morbillivirus (phocine distemper virus, PDV) was identified in organs of diseased animals. It is concluded that the epidemic was caused by introduction of PDV into a highly susceptible population presumably free from morbillivirus infection. The origin of PDV remains unknown but evidence of prior morbillivirus infection has been found in arctic and antarctic seal populations.     

Morbillivirus infections of seals during the 1988 epidemic in the Bay of Heligoland: III. Transmission studies of cell culture-propagated phocine distemper virus in harbour seals (Phoca vitulina) and a grey seal (Halichoerus grypus): clinical, virological and serological results

Eight harbour seals (Phoca vitulina), two of them seronegative, six seropositive against PDV and a seronegative grey seal (Halichoerus grypus) were exposed to a low doses of a cell culture-propagated phocine distemper virus isolate (PDV 2558/Han 88). An intranasal route of inoculation was chosen. Clinical signs, resembling those of 1988's seal disease and seroconversion were observed in both seronegative harbour seals. One of them succumbed to the infection. The virus was not transmitted to another susceptible harbour seal which served as in-contact animal. Virus could be recovered from leucocytes of the diseased seals. Viremia was also present in a seropositive harbour seal that developed mild clinical signs; other seropositive seals were protected from clinical disease. The grey seal showed seroconversion upon inoculation, but did not develop any signs of disease. The humoral immune response of the seals plainly discriminated between homologous (PDV) and heterologous (canine distemper virus, CDV) virus as shown by virus neutralization tests and an antibody-binding assay (PLA).     

Round table on morbilliviruses in marine mammals.

Since 1988 morbilliviruses have been increasingly recognized and held responsible for mass mortality amongst harbour seals (Phoca vitulina) and other seal species. Virus isolations and characterization proved that morbilliviruses from seals in Northwest Europe were genetically distinct from other known members of this group including canine distemper virus (CDV), rinderpest virus, peste des petits ruminants virus and measles virus. An epidemic in Baikal seals in 1987 was apparently caused by a morbillivirus closely related to CDV so that two morbilliviruses have now been identified in two geographically distant seal populations, with only the group of isolates from Northwest Europe forming a new member of the genus morbillivirus: phocid distemper virus (PDV). Because of distemper-like disease, the Baikal seal morbillivirus was tentatively named PDV-2 in spite of its possible identity with CDV. The appearance of morbilliviruses in the Mediterranean Sea causing high mortality amongst dolphins should further increase the research activities on protection strategies for endangered species of marine mammals.     

Variations in the severity of phocid herpesvirus type 1 infections with age in grey seals and harbour seals

The data recorded during an outbreak of phocid herpesvirus type 1 infection among 19 harbour seals and 29 grey seals being nursed in a seal rehabilitation centre in The Netherlands in 1998 were used, together with data from similar outbreaks in previous years, to compare the clinical signs observed in the two species at different ages. The severity of the disease was inversely correlated with age in the harbour seals, and the infected harbour seals generally developed more severe clinical signs than the infected grey seals.     

Mass mortality in seals caused by a newly discovered morbillivirus

During a recent disease outbreak among harbour seals (Phoca vitulina) in the North and Baltic seas, more than 17,000 animals have died. The clinical symptoms and pathological findings were similar to those of distemper in dogs. Based on a seroepizootiological study, using a canine distemper virus (CDV) neutralization assay, it was shown that CDV or a closely related morbillivirus (phocid distemper virus-PDV) was the primary cause of the disease. The virus was isolated in cell culture from the organs of dead seals and characterized as a morbillivirus by serology (immunofluorescence neutralization and enzyme-linked immunosorbent assays) and by negative contrast electron microscopy. Experimental infection of SPF dogs resulted in the development of mild clinical signs of distemper and CDV-neutralizing antibodies. The disease was reproduced in seals by experimental inoculation of organ material from animals that had died during the outbreak. However, seals that had been vaccinated with experimental inactivated CDV vaccines were protected against this challenge. This fulfilled the last of Koch's postulates, confirming that the morbillivirus isolated from the seal organs, was the primary cause of the disease outbreak. The recent demonstration of the presence of a similar virus in Lake Baikal seals (Phoca sibirica), which infected these Siberian seals 1 year before the northwestern European seals were infected, raises new questions about the origin of this infectious disease in pinnipeds.     

Retrospective analysis of two phocine distemper epidemics in the North and Baltic Seas in 1988 and 2002

The relapse of the outbreak of the phocine distemper virus in the Danish island of Anholt this June, emphasizes the importance of the topic among experts. During the phocine distemper virus (PDV) epidemic in 1988, a total of 23,000 harbour seals (Phoca vitulina) died. In 2002 a second outbreak of PDV resulted in the death of more than 30,000 harbor seals. Both epidemics originated near the Danish island of Anholt and spread to adjacent colonies. Additional centres of infection were observed in the Dutch Wadden Sea far from the infected Danish seal populations. Arctic seals and grey seals were considered as vectors. Grey seal populations may serve as a reservoir for PDV or act as subclinically infected carriers of the virus between Arctic and North Sea seal populations. Mixed colonies of grey and harbour seals are widely distributed in the North and Baltic Seas. The role of environmental contaminants and their potential impact on immune function are discussed. The duration and geographical patterns of the two PDV epidemics are compared.     

Relatedness of Streptococcus equi subsp. zooepidemicus strains isolated from harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) of various origins of the North Sea during 1988-2005

The present study was designed to identify 15 beta-hemolytic streptococci isolated during a period between 1988 and 2005 from nine harbour seals and six grey seals from various origins of the North Sea. All isolates were identified as Streptococcus equi subsp. zooepidemicus. The bacteria were additionally investigated for relatedness by restriction fragment length polymorphism analysis of PCR amplified 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of chromosomal DNA of the strains by pulsed field gel electrophoresis. The molecular analysis yielded identical or closely related patterns within the strains of the present study and with the S. equi subsp. zooepidemicus strains isolated from harbour seals of German North Sea which were investigated previously [Akineden, O., Hassan, A.A., Alber, J., El-Sayed, A., Estoepangestie, A.T.S., L?mmler, C., Weiss, R., Siebert, U., 2005. Phenotypic and genotypic properties of S. equi subsp. zooepidemicus isolated from harbor seals (Phoca vitulina) from the German North Sea during the phocine distemper outbreak in 2002. Vet. Microbiol. 110, 147-152]. This indicates that this single or closely related bacterial clone existed during both phocine distemper virus epidemics in 1988 and 2002 and that a direct transmission of the strains has occurred between two seal species and between seal populations of far distant regions possibly with grey seals as a vector.     

Investigations on course and outcome of phocine distemper virus infection in harbour seals (Phoca vitulina) exposed to polychlorinated biphenyls. Virological and serological investigations.

The influence of polychlorinated biphenyls (PCBs) on phocine distemper virus (PDV) infections in harbour seals (Phoca vitulina) was studied. Six out of ten seals had been conditioned with a defined mixture of PCB-congeners for several weeks. Following exposure to the cell culture-propagated PDV isolate 2558/Han 88 the complete clinical picture of "1988 seal plague" was provoked in all ten seals inoculated. Four out of six PCB-conditioned seals and two out of four seals not loaded with PCBs succumbed to the infection within three weeks post inoculation. With regard to the clinical course, duration of cell-associated viremia, PDV-antigen distribution in tissues of fatally infected seals and the humoral immune response to PDV no differences between PCB-loaded and unloaded seals were recognized. Evidence was obtained that the pathogenesis of experimental PDV-infection in harbour seals shares some features with those of canine distemper in terrestrial carnivores. In contrast, however, to experimental distemper infection of gnotobiotic dogs prompt development of high titres of PDV-specific IgG did not correlate with recovery from infection.     

Occurrence of a canine distemper-like disease in aquarium seals

Outbreaks of a canine distemper-like acute disease brought high mortalities to seal populations in north-west Europe and Lake Baikal from late 1987 to 1988. During these outbreaks three seals which were introduced from Lake Baikal to an aquarium in Japan developed a distemper-like disease and other seals raised in the same room were similarly affected. Clinical signs of dead seals were anorexia, diphasic fever, dyspnea, and neuromuscular tics. Characteristic microscopic lesions of acute interstitial pneumonia seen in the lung were accompanied with hyperplasia and syncytial giant cell formation of type II pneumocytes. Eosinophilic intranuclear and cytoplasmic inclusion bodies were present in bronchial epithelial cells, type II pneumocytes, epithelial cells of bile ducts and interlobular ducts of pancreas, transitional epithelium of renal pelvis, and reticular cells of lymph nodes. Ultrastructure of inclusion bodies was similar to that seen in cells infected with morbilliviruses. Serum samples from recovered seals had virus-neutralization antibodies against canine distemper virus. The present cases were the first report of morbillivirus infection of aquarium seals in Japan.     

The seal death in Danish waters 1988. 1. Pathological and bacteriological studies.

During the seal epizootic in Danish waters in 1988 a total of 81 adult seals were necropsied. The cause of death was suppurative bronchopneumonia complicated by pleurisy. Histologically, an interstitial pneumonia with cytoplasmatic inclusion bodies typical of canine distemper was identified in many of the seals. The condition was in many cases complicated with a secondary infection with Bordetella bronchiseptica.     

Herpesvirus in harbour seals (Phoca vitulina): isolation, partial characterization and distribution

From post-mortem material (liver and lung) and leucocytes of four (3.6%) out of 112 examined harbour seals during the seal epizootic in 1988 six cytopathogenic viral isolates were obtained which were provisionally classified as herpes-like viruses. Results of physico-chemical and electron microscopic investigations suggested their relationship to the herpesvirus family. Serological examinations were carried out with sera from wildlife as well as captive animals using herpesvirus isolates from four different seals. The neutralization tests revealed as only moderate distribution of seropositive reagents up to 53% of the wildlife seal population. Amongst the seals in the orphanage of Norddeich a very small number of seropositive animals was found. The results obtained indicated a minor role of herpesviruses as primary cause of seal mortality in the North Sea during the 1988 season.     

Humoral immune responses in seals infected by phocine distemper virus

Recently the isolation and characterisation of a morbillivirus which caused high mortality in common seals (Phoca vitulina) in 1988 have been reported. Because of the clinical and pathological similarity of the disease in seals to that of distemper in dogs, the name phocine distemper virus (PDV) has been proposed. There are marked differences in the virus-induced proteins of PDV compared to other morbilliviruses and the humoral immune response of moribund and dead seals to PDV was restricted to some of the internal antigens of PDV, similar to the response described earlier for canine distemper virus infection in dogs.     

Morbillivirus infection of seals (Phoca vitulina) during the 1988 epidemic in the Bay of Heligoland. I. Mode, frequency and significance of cultural virus isolation and neutralizing antibody detection

From 16 (14%) out of 112 dead or euthanized seals originating from wildlife and seal orphanages phocine morbillivirus was isolated. The majority of viral isolates in cell cultures was obtained from lung homogenates of 15 out of 71 free-ranging seals (21%). The virus was isolated by longterm cultivation in roller cultures of seal kidney cells. The phocine morbillivirus was detected by typical cytopathogenic alteration and by peroxidase-linked antibody (PLA) assay, respectively. A neutralization test based on PLA was used for antibody detection in seals using a canine distemper virus (CDV) strain and in parallel one of the phocine morbillivirus isolates. All sera tested were proven to contain neutralizing antibodies of higher titres against the latter virus than against the CDV strain. Several seals furnished morbillivirus isolates and at the same time exhibited neutralizing antibodies of low to medium titres. No viral isolates were obtained from the majority of sick animals with moderate to high neutralizing titres (greater than 1/1,000). The significance of these findings is discussed in relation to the cause of the mass mortality amongst seals observed in 1988 in the Bay of Heligoland.     

A retrospective study of diagnosis in 109 cases of canine lower respiratory disease

Between 1983 and 1988, 109 cases of canine lower respiratory tract disease were examined and categorised according to clinical, radiographic and bronchoscopic findings and cytology of bronchial mucus. Non-specific chronic tracheobronchitis was diagnosed in 19 cases, bronchopneumonia (subacute or chronic) in 12 cases, bronchiectasis in 10 cases, parasitic bronchitis due to Oslerus osleri in 20 cases, eosinophilic bronchitis with or without bronchiectasis or alveolar infiltration in 25 cases, bronchial foreign bodies, mainly cereal ears, in 14 cases, primary neoplasia (adenocarcinomas) in six cases and miscellaneous disease in three cases. Features of these conditions are discussed. Although the aetiology of chronic bronchial disease may not always be ascertained, it is important to assign a dog to a diagnostic category so that the owner can be given a more accurate prognosis.     

Indications for both host-specific and introduced genotypes of Staphylococcus aureus in marine mammals

Staphylococcus aureus is present in the marine environment and causes disease in marine mammals. To determine whether marine mammals are colonized by host-specific strains or by strains originating from other species, we performed multi-locus sequence typing on ten S. aureus strains isolated from marine mammals in the U.K., the Netherlands, and the Antarctic. Four new sequence types of S. aureus were discovered. S. aureus strains from a southern elephant seal (n=1) and harbour porpoises (n=2) did not cluster with known S. aureus strains, suggesting that they may be host species-specific. In contrast, S. aureus strains from harbour seals (n=3), other harbour porpoises (n=3), and a grey seal (n=1) clustered with S. aureus strains previously isolated from domestic ruminants, humans, or birds, suggesting that these S. aureus strains in marine mammals were introduced from terrestrial species.     

A review of the 1988 European seal morbillivirus epizootic

An epizootic of morbillivirus infection killed thousands of common seals (Phoca vitulina) in European seas in 1988. Most of the affected seals had respiratory signs and the main post mortem finding was acute pneumonia. The histopathological changes were similar to those of canine distemper. Six common porpoises (Phocoena phocoena) found stranded on the coast of Northern Ireland in late 1988 had similar lesions. Morbillivirus infection also killed several thousand Siberian seals (Phoca siberica) in Lake Baikal in 1987 and 1988. A morbillivirus (phocine distemper virus) has been isolated from affected seals in several European countries and studies of the antigenicity of the virus indicate that it has several unique epitopes that distinguish it from the other known morbilliviruses. Biochemical studies of the viral proteins, RNA and nucleotide sequence confirm that it is a new morbillivirus. There is seroepizootiological evidence of morbillivirus infection in Greenland harp seals (Pagophilus groenlandicus), ringed seals (Phoca hispida) and Dutch common seals several years before the 1988 epizootic. Antibodies to a morbillivirus have also been found in bottlenose dolphins (Tursiops truncatus) from the eastern coast of the USA. Further studies are required to determine whether these sea mammal populations have been infected with phocine distemper virus.     

Fatal enterocolitis in harbour seals (Phoca vitulina) caused by infection with Eimeria phocae

Coccidiosis due to Eimeria phocae infection has been described in harbour seals (Phoca vitulina) from the western Atlantic population, but not in any detail in seals from the eastern Atlantic population. This paper describes fatal enterocolitis due to E phocae infection in three juvenile harbour seals at a rehabilitation centre in the Netherlands in July 2003. The clinical signs were lethargy, bloody faeces, and intermittent convulsions and muscle tremors just before they died; the nervous signs resembled those of nervous coccidiosis in calves. The main pathological finding was severe, diffuse, haemorrhagic enterocolitis; there were diffuse inflammatory changes in the lamina propria of the jejunal, ileal, caecal and colonic mucosa that were associated with the presence of the sexual stages and oocysts of a coccidian species identified as E phocae. A retrospective microscopical examination of intestinal tissues from 113 harbour seals that had died between 1999 and 2004 revealed one seal that was positive for E phocae.     

Uroendoscopy. Evaluation of the lower urinary tract.

Recent advances in uroendoscopy have allowed diagnostic evaluation of the lower urinary tract in most of our canine and feline patients. By providing a magnified view of the luminal surfaces of the lower urinary tract, uroendoscopy provides useful diagnostic information that is not readily available even by more invasive techniques.     

Characterisation of the European seal morbillivirus

The ELISA test originally developed for the detection of serum antibodies to rinderpest virus has been shown to detect cross-reacting antibodies in sera of diseased common and grey seals. Analysis of sera collected from various seal populations is in progress to establish the correlation between different morbillivirus neutralisation tests, ELISA tests and the disease status of the animals. RNA purified from post-mortem tissues removed from diseased seals has been analysed by hybridisation with cloned cDNAs made to various genes of canine distemper, peste des petits ruminants, rinderpest and measles viruses. This study has confirmed the presence of RNA sequences characteristic of a morbillivirus, but shown that the virus is not identical with any known morbillivirus. Work is in progress to determine the nucleotide sequences of clones carrying inserts homologous to morbillivirus genes isolated from cDNA made on a template of infected seal tissue RNA.