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).     

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.     

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.     

Efficacy of ivermectin and moxidectin against Otostrongylus circumlitus and Parafilaroides gymnurus in harbour seals (Phoca vitulina)

Verminous bronchopneumonia caused by infection with Otostrongylus circumlitus and Parafilaroides gymnurus is an important cause of death during the rehabilitation of harbour seals (Phoca vitulina). During the winter of 2000/01, 35 juvenile harbour seals with severe clinical signs of verminous bronchopneumonia were treated with either 0.2 mg/kg ivermectin orally or 0.2 mg/kg moxidectin subcutaneously, and monitored for 30 days. The efficacy of the anthelmintics was determined by the pattern of larval excretion (Baermannisation) and the progress of the clinical signs. Both anthelmintics had reduced larval excretion by at least 99 per cent after 10 days, but the seals' rapid breathing rate and and dyspnoea returned to normal more quickly in the animals treated with moxidectin. The pharmacokinetics of the anthelmintics were determined by solid-phase extraction, and high-performance liquid chromatography with fluorescence detection. Moxidectin had a mean (sd) residence time of 9.04 (2.12) days compared with 4.83 (1.14) days for ivermectin.     

Polioencephalomalacia in captive harbour seals (Phoca vitulina)

In a colony of 11 harbour seals (Phoca vitulina Linné 1758) two episodes of central nervous disorders occurred within 2 years causing fatalities in seven adult animals. Clinical signs comprised dyspnoea, anorexia, apathy, incoordination and lateral recumbency. Vitamin B complex therapy was successful once. Pathomorphological examination of seven carcasses revealed acute and subacute malacia of the cerebellar grey matter. Additional acute malacic lesions located in the cerebral cortices and basal ganglia were observed. Mesencephalic nuclei were less severely affected and displayed acute changes. Despite intense search for environmental toxins and infectious agents, the cause of the fatalities remained undetermined. However, the type and pattern of the lesions are most suggestive of a thiamine deficiency.     

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.     

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.     

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.     

Use of a tiletamine-zolazepam mixture to immobilise wild grey seals and southern elephant seals

A mixture of tiletamine and zolazepam at a combined dose of 1 mg/kg was a reliable and safe agent for immobilising wild grey seals (Halichoerus grypus) and southern elephant seals (Mirouga leonina). The agent had a number of advantages over all the other agents used previously.     

Streptococcus phocae infections associated with starvation in Cape fur seals

Mortalities and abortions associated with starvation occurred at Cape Cross, Namibia, in Cape fur seals (Arctocephalus pusillus pusillus). Affected seals showed lethargy and emaciation, and the most common pathological signs were those of a respiratory infection, both in adults and offspring. Streptococcus phocae was isolated from adult seals, a cub and aborted foetuses.     

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.     

Histopathologic and immunocytochemical studies of distemper in seals

Thousands of harbor seals (Phoca vitulina) died in European seas during 1988. Respiratory distress and oculonasal discharge were common clinical signs. We necropsied 76 affected seals. The main necropsy finding was severe pneumonia. Microscopic lung changes were characterized by proliferation of type II pneumocytes, filling of alveolar lumina with serofibrinous exudate, leukocytes, and macrophages, and necrosis of bronchial and bronchiolar epithelium. Intracytoplasmic and intranuclear acidophilic inclusion bodies characteristic of morbillivirus infection were seen in bronchial and bronchiolar epithelial cells. Microscopic lesions of non-suppurative demyelinating encephalitis were seen in the brain. There was degeneration and necrosis of neurons, focal gliosis, perivascular cuffing, and patchy demyelination. Many neurons and astrocytes contained intracytoplasmic and intranuclear inclusions. Using an immunoperoxidase technique, we detected morbillivirus antigen in many tissues including lung, brain, spleen, and urinary bladder. The origin of the seal morbillivirus is unknown.     

Natural causes of death in non-suckling grey seals (Halichoerus grypus)

Thirty-four grey seals which died of natural causes were examined. They ranged in age from aborted fetuses to adults, but suckling pups were excluded from the study. The commonest primary cause of death was pneumonia and a variety of parasitoses occurred as secondary lesions.     

An outbreak of leptospirosis in seals (Phoca vitulina) in captivity

An outbreak of leptospirosis in seals (Phoca vitulina) in captivity is described. In a zoo in The Netherlands 5 adult seals died within 12 days. At necropsy all animals showed signs of acute septicaemia, consistent with acute leptospirosis. Serological examination of one animal was positive for antibodies against Leptospira interrogans serovar Icterohaemorrhagiae and the serologically closely related serovar Copenhageni. Polymerase chain reaction was positive in one other animal. 8 nutria (Myocastor coypus) were examined, serologically, through bacteriological culture and PCR. 81,8% (9/11) were serologically positive for Leptospira. The seals and nutria were housed in the same water system.     

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.     

A study on the effect of different rifle calibres in euthanisation of grey seals (Halichoerus grypus) in seal traps in the Baltic Sea

Background

In recent years, the euthanasia of seals has been discussed internationally and concern has been raised regarding the use of rifles, the effect of different calibres, and which calibres are sufficient for humane euthanasia. This study therefore investigated the effect of different firearm calibres on euthanasia of grey seals (Halichoerus grypus) in traps, and provides information for the development and refinement of regulations for hunting seals in the wild.

Findings

The effect of different calibres was studied in 19 seals shot in the head and neck at close range. All seals were necropsied and radiographed to characterize the injuries caused by the bullets. All tested calibres, 5.6 mm bullet diameter or larger, and .12 shotgun, were sufficiently effective to cause severe skull fractures, meningeal haemorrhages and instant death.

Conclusions

Rifles with 5.6 mm bullet diameter or larger, and a .12 shotgun loaded with a slug fired at close range to the head and neck of grey seals all caused instant death and can therefore be recommended for hunting seals in the wild.     

Prevalence of Brucella pinnipediae in healthy hooded seals (Cystophora cristata) from the North Atlantic Ocean and ringed seals (Phoca hispida) from Svalbard

Investigations for Brucella-infections were conducted in 29 hooded seals (Cystophora cristata) caught between Svalbard and Greenland (North Atlantic Ocean; Greenland Sea) autumn 2002, and from 20 ringed seals (Phoca hispida) caught in Billefjord, Svalbard, spring 2003. All animals were apparently healthy and were caught in their natural habitat. Bacteriology on tissue samples from ringed seals was negative, whereas Brucella sp. were recovered in tissues from 11 of the 29 hooded seals (38%), with the highest tissue prevalence in spleen (9/29) and lung lymph nodes (9/24). Anti-Brucella antibodies were detected in sera from 9 hooded seals (31%) (EDTA-modified Slow Agglutination test of Wright, Rose Bengal test, Complement Fixation Test, and Protein-A ELISA). The bacterial isolates all belonged to the genus Brucella according to classical biotyping and PCR analysis based on Insertion Sequence IS711, and were shown to be typical marine mammal strains, based on the occurrence of an IS711 element downstream of the bp26 gene. Their dependency on CO2 for growth, and the presence of one copy each of the omp2a and omp2b gene finally classified them as Brucella pinnipediae. Furthermore, all the hooded seal isolates showed an A+ M+ agglutination profile, which is different from the profile of reference seal strain 2/94 (harbour seal, Phoca vitulina). Thus, these results indicate that B. pinnipediae may contain different biovars. The present results suggest that infection with B. pinnipediae is enzootic in this population. Since the hooded seal is commercially hunted and consumed in Norway, the pathological impact of such infections and their zoonotic potential should be further addressed.     

Anaesthesia of three young grey seals (<Emphasis Type="Italic">Halichoerus grypus</Emphasis>) for fracture repair

Three young grey seals (Halichoerus grypus) were presented separately for fracture repair to the veterinary teaching hospital of University College Dublin. The seals were premedicated with a combination of pethidine, midazolam and atropine; anaesthesia was induced with propofol via the front flipper vein and maintained with sevoflurane or isoflurane in oxygen. One of the seals did not breathe spontaneously after anaesthesia; a cardiac arrest, resulting in death, occurred after several hours of mechanical ventilation. Post-mortem examination revealed a severe lungworm infestation and parasitic pneumonia in this animal. The two other seals recovered uneventfully from anaesthesia.