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.     

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).     

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.     

Quantitative analysis of the 2002 phocine distemper epidemic in the Netherlands

Phocine distemper virus (PDV) caused thousands of deaths among harbor seals (Phoca vitulina) from the North Sea in 1988 and 2002. To examine the effects of different factors on the pathology of phocine distemper, we performed necropsies and laboratory analyses on 369 harbor seals that stranded along the Dutch coast during the 2002 PDV epidemic. Diagnostic tests for morbillivirus infection indicated a differential temporal presence of morbillivirus in lung and brain. Seals of 3 years or older were significantly more often IgG positive than younger seals. The most frequent lesions in PDV cases were bronchopneumonia, broncho-interstitial pneumonia, and interstitial emphysema. Extra-thoracic emphysema was rare in <1-year-olds compared with older seals, even though severe pneumonia was more common. PDV cases generally had empty stomachs and less blubber than by-caught seals from before the epidemic. In PDV cases involving older animals, lung, kidney, and adrenal weights were significantly increased. Bordetella bronchiseptica was isolated from lungs in two thirds of the PDV cases examined. Our results indicate that brain should be included among the tissues tested for PDV by RT-PCR; that either phocine distemper has a longer duration in older seals or that there are age-related differences in immunity and organ development; that dehydration could play a role in the course and outcome of phocine distemper; and that bacterial coinfections in lungs are more frequent in PDV cases than gross lesions suggest. These results illustrate how quantitative analysis of pathology data from such epidemics can improve understanding of the causative disease.     

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.     

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.     

Pathological findings indicative of distemper in European seals

The first recorded cases of the recent epizootic were harbour seals observed at the Danish island of Anholt, 12 April 1988. The disease then spread throughout the sea waters of north-western Europe. The total mortality in Europe up to November 1988, was estimated to be at least 17,000 seals. The mortality rate in Danish-Swedish waters was about 60%. Autopsies including sampling for histology of most organs were performed on 37 harbour seals and 12 grey seals, collected mainly at the Swedish west coast and in the southern Baltic. In most of the harbour seals and in three of the grey seals we found histological changes in the upper and lower respiratory tracts, in the lower urinary tract and in the lymphatic system consistent with those diagnostic of distemper viral infection in the canine. These diagnostic criteria were: presence of intracytoplasmic eosinophilic inclusion bodies of epithelial cells of the trachea and the urinary bladder, interstitial pneumonia, and atrophy of lymphatic organs due to depletion of lymphocytes. Our findings in pathology of a canine distemper-like disease in the seals were presented in late August 1988, together with the Dutch findings in virology by Dr. Osterhaus and collaborators.     

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 among seals (Phoca vitulina) during the 1988 epidemic in the Bay of Heligoland. II. Serogological investigations reflecting a previous phocine distemper epidemic in a seal orphanage

Of 57 seals hospitalized in the Norddeich Orphanage, 37 (65%) had died until 22nd of August, 1988, when the first collection of blood samples from the survivors commenced. All the sera including those obtained at a later date from the remaining animals had invariably higher neutralizing antibody titres against a phocine distemper virus (PDV) isolate than against canine distemper virus (CDV). The difference of mean titres was calculated to be more than 1.5 x log10 serum dilution. Peak titres demonstrated by a direct neutralization peroxidase-linked antibody (NPLA) assay reached 1/90,000. Attempts to isolate PDV in seal kidney cell cultures from heparinized blood samples collected from convalescent animals were not successful. From the increase in antibody titre following the last fatal case it was concluded that the devastating epidemic sweeping through the Norddeich Orphanage was primarily due to phocine distemper.     

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.     

Phenotypic and genotypic properties of Streptococcus equi subsp. zooepidemicus isolated from harbor seals (Phoca vitulina) from the German North Sea during the phocine distemper outbreak in 2002

The present study was designed to identify and compare 32 beta-hemolytic streptococci isolated from 28 different harbor seals of the German North Sea during the phocine distemper outbreak in 2002. The bacteria were identified as Streptococcus equi subsp. zooepidemicus based on cultural, biochemical, serological and molecular studies. Epidemiological investigations by PCR restriction fragment length polymorphism analysis of the 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of the chromosomal DNA of the strains by pulsed field gel electrophoresis revealed that all 32 strains appeared to be identical. These results indicate that a single bacterial clone seemed to be distributed among the harbor seal population of the German North Sea during this outbreak.     

Canine distemper virus--an agent looking for new hosts

Canine distemper is caused by the canine distemper virus (CDV), a RNA virus belonging to the genus Morbillivirus of the family Paramyxoviridae. The genus Morbillivirus includes measles virus, Rinderpest virus and peste-des-petits-ruminants virus. The host spectrum of CDV, which includes numerous families of Carnivores, has been changed in the last years and distemper-like diseases have been observed in numerous other species. These include epidemics in large felids, marine mammals and javelinas. Different viruses have been isolated from pinnipeds including a seal-specific isolate, termed phocine distemper virus 1, PDV-1, and a CDV strain, named PDV-2. Retrospective analysis of previous epidemics among marine mammals in various regions of the world provide evidence for the occurrence of so far unrecognized morbillivirus epidemics. In some including the mass mortalities of Baikal and Caspian seals and of large felids in the Serengeti, terrestrial carnivores including dogs and wolves have been suspected as a vector for the infectious agent. However, in other epidemics among marine mammals the source of infection remains unknown including both seal epidemics in northwestern Europe in 1988 and 2002. It remains to be determined whether a morbillivirus from other marine mammals or terrestrial carnivores caused the infection in this unprotected seal populations.     

Seroepidemiological survey of morbillivirus infection in Kuril harbor seals (Phoca vitulina stejnegeri) of Hokkaido, Japan

Serological analysis was performed to detect morbillivirus infection in Kuril harbor seals in Hokkaido, Japan. Serum samples were collected from the seals at Nosappu (231 sera), Akkeshi (16), and Erimo (75) between 1998 and 2005. Antibodies to phocine distemper virus (PDV) were detected by ELISA in seals from Nosappu and Erimo. Antibodies to PDV were found in 56% (5/9) of the sampled seals from Nosappu in 1998, versus only 5% (3/66) for 2003, 1% (1/79) for 2004, and 1% (1/77) for 2005. These suggest epidemic caused by the virus in or before 1998. As antibody-positive seals included juvenile seals in 2003 and 2005, sporadic infections of the virus are thought to have occurred in recent years. In Erimo, antibodies to PDV were found in 50% (14/28) of sampled seals in 2004, versus only 13% (1/8) for 1999, 7% (1/15) for 2003, and 0% (0/24) for 2005. These suggest sporadic infection by the virus before 2003 and the epizootic between after autumn in 2003, when samples of 2003 were collected, and 2004. Since antibodies to canine distemper virus (CDV) were detected in one adult seal from Nosappu in each year from 2003 to 2005, sporadic infections of the virus were suggested. There were no difference in incidence of seals with antibodies to the viruses between males and females and between juveniles and adults.     

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.     

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.     

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.     

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.     

On the structure of the adrenal gland of the common seal (Phoca vitulina vitulina)

The adrenal gland is a vitally important endocrine gland that occupies a central role in the regulatory mechanisms of the body metabolism. Environmental stress factors lead to permanent strain and overload of the body resulting in structural alterations of the adrenals that in turn are followed by hormonal imbalances. This leads to an increased susceptibility to bacterial and viral diseases. The recurrence of numerous fatalities in the different seal populations of the North Sea (during the years 1988, 1989 and 2002), of the Baikal Lake and Caspian Sea (during the years 2000 and 2001) were the motive for a morphological investigation of the species-specific structure of the adrenal gland of the common seal in order to differentiate environmental stress-induced pathological alterations from the physiological structure of this organ. The study was based on adrenals of 112 common seals (Phoca vitulina vitulina) using light microscopic and transmission and scanning electron microscopic methods. The phocine adrenal gland displays several structural characteristics. Originating from the connective tissue organ capsule, narrow and broad septa intersperse the adrenal cortex. These septa contain blastemata as a reserve for the regeneration of hormone-producing cortical cells. Such blastemata are also occurring in the form of an intermediate zone in between the zona glomerulosa and zona fasciculata in the phocine adrenal cortex. Another species-specific characteristic is an inverse part of the adrenal cortex encircling the central vein of the organ. These structural features have to be considered in assessment and definition of pathological alterations of the adrenals as observed in the form of exhausted blastema cell pools in the adrenocortex of seals perished in the mentioned phocine mass mortalities.