Histopathologic and immunocytochemical studies of distemper in harbor porpoises

During 1988 thousands of harbor seals (Phoca vitulina) died in European seas as a result of morbillivirus infection. Six harbor porpoises (Phocoena phocoena) found stranded on the coast of Northern Ireland in late 1988 were submitted to our laboratory for necropsy. Pneumonia was the main necropsy finding in three of these animals. Microscopic lung lesions characterized by necrosis of bronchial and bronchiolar epithelium and infiltration of alveoli with leukocytes, lymphoid cells, macrophages, and multinucleate syncytia were seen in all six porpoises. Cytoplasmic and nuclear acidophilic inclusions characteristic of morbillivirus infection were common in bronchial and bronchiolar epithelial cells and in alveolar macrophages and syncytia. Brain alterations included degeneration and necrosis of neurons, microglial infiltration, and perivascular cuffing. There were cytoplasmic and nuclear acidophilic inclusions in many neurons. Immunoperoxidase staining of morbillivirus antigen was seen in many tissues including lung, brain, spleen, and urinary bladder. Alterations in our porpoises were similar to those seen in distemper in seals and many species of terrestrial mammals. Systemic viral disease has not previously been documented in Cetacea.     

Pathology of morbillivirus infection in striped dolphins (Stenella coeruleoalba) from Valencia and Murcia, Spain.

During the summer and fall of 1990 hundreds of striped dolphins (Stenella coeruleoalba) died in the Spanish Mediterranean as a result of morbillivirus infection. A pathological investigation was carried out on dolphins from Valencia and Murcia which were among the first to die in the epizootic. The dolphins were in poor body condition and pneumonia was the main necropsy finding. Microscopic lung lesions characterized by necrosis of bronchial and bronchiolar epithelium and infiltration of alveoli with macrophages, lymphocytes, neutrophils and multinucleated syncytia were seen in most dolphins. Cytoplasmic and nuclear eosinophilic viral inclusions were present in bronchial and bronchiolar epithelium and in syncytia. Focal granulomatous inflammation associated with nematodes was also present. Brain lesions included diffuse degeneration and necrosis of neurons, microgliosis, perivascular cuffing, formation of syncytia and focal demyelination. Cytoplasmic and nuclear eosinophilic inclusions were present in neurons and glial cells. There was severe lymphoid necrosis and depletion of spleen and lymph nodes and syncytia also occurred in lymph nodes. Biliary and transitional epithelium contained nuclear and cytoplasmic eosinophilic inclusions. Immunoperoxidase staining using monoclonal antibodies to phocine distemper virus confirmed the presence of morbillivirus antigens in lung and brain. The distribution and severity of lesions in striped dolphins are similar to those of distemper in seals, harbor porpoises and terrestrial mammals. The formation of syncytia in the lung and brain may be a useful pathological indicator of morbillivirus infection and may be used in the investigation of pinniped and cetacean strandings in North America.     

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.     

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.     

Morbillivirus infection in two common porpoises (Phocoena phocoena) from the coasts of England and Scotland.

Two common porpoises (Phocoena phocoena), one found stranded on the Isle of Sheppey, Kent, and the other in the Moray Firth, Scotland, in late 1990, were examined post mortem. Lesions of diffuse bronchointerstitial pneumonia were present in both animals; they were characterised by the infiltration of alveoli with leucocytes, macrophages and multinucleate syncytia, the necrosis of bronchial and bronchiolar epithelium, focal proliferation of type II pneumocytes and occasional acidophilic cytoplasmic inclusions in alveolar macrophages and syncytia. Lymphoid depletion was apparent in the spleen, thymus and lymph nodes of both porpoises. Other changes in the Isle of Sheppey porpoise included severe dacryoadenitis. Marked pharyngitis, oesophagitis and balanoposthitis were present in the Moray Firth porpoise. Immunoperoxidase staining revealed the presence of morbillivirus antigen in a range of epithelia from both porpoises. This is the first report of morbillivirus infection in cetaceans from the coast of Great Britain.     

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.     

Pathologic and immunocytochemical studies of morbillivirus infection in striped dolphins (Stenella coeruleoalba).

Hundreds of striped dolphins (Stenella coeruleoalba) died along the Spanish Mediterranean coast during the second half of 1990. We necropsied 58 dolphins. Partial collapse of the lungs with patchy atelectasis, subcutaneous edema, icterus, and stomatitis were the most prominent gross morphologic changes. Histologically, a bronchiolo-interstitial pneumonia was the most frequent lesion (72% of the animals). It was characterized by hyperplasia of alveolar epithelial type II cells and formation of multinucleate syncytia in alveolar and bronchiolar lumina. Other prominent lesions were encephalitis (69%), lymphoid depletion, and formation of multinucleate syncytia in the cortex of lymph nodes. The distribution of morbillivirus antigen was investigated in 23 well-preserved dolphins using a monoclonal antibody against the hemagglutinin glycoprotein of phocine distemper virus. Positive immunostaining was found in brain (77%), in lung (70%), and in mesenteric (61%), mediastinal (47%), and prescapular (45%) lymph nodes. Phocine distemper virus antigen was demonstrated less frequently in trachea, stomach, biliary epithelium, intestine, kidney, and mammary gland. Necrotizing-hemorrhagic pneumonia and encephalitis due to Aspergillus fumigatus were seen in three dolphins, whereas two animals had lesions of toxoplasmosis. Changes in our dolphins were similar to those caused by distemper in seals and porpoises. The origin of the dolphin virus and the relationships among dolphin, seal, and porpoise morbilli viruses are unknown.     

Morbillivirus infections in aquatic mammals: a brief overview

Since 1987, at least eight morbillivirus infection (MI) epidemics have caused mass mortality of several free-living pinniped and cetacean populations around the world. The responsible agents, all belonging to the genus Morbillivirus (family Paramyxoviridae), have been characterized as either "canine distemper virus" strains, infecting pinnipeds, or as three new morbilliviruses, namely "phocid (phocine) distemper virus" , "porpoise morbillivirus" and "dolphin morbillivirus" . The last two agents are currently gathered under the common denomination of "cetacean morbillivirus". At post-mortem examination, a commonly occurring macroscopic lesion is represented by more or less severe bilateral pneumonia, with consolidation, congestion and oedema of both lungs, which fail to collapse. Histologically, a non-suppurative broncho-interstitial pneumonia, characterized by type II pneumocyte hyperplasia and by formation of endobronchial, endobronchiolar and endoalveolar "Warthin-Finkeldey type" syncytia, as well as a multifocal, non-suppurative encephalitis, associated with a severe and generalized lymphoid tissue depletion, are common pathological findings. Furthermore, eosinophilic viral inclusions are often detected, at both the intracytoplasmic and intranuclear level, within bronchial and bronchiolar epithelial, pulmonary syncytial, neuronal and other cell types. These inclusions, along with lymphoid and other cellular elements, are often found to be immunohistochemically positive for morbillivirus antigen. Among the still debated, or even controversial issues regarding MI in sea mammals, the one related to the origin of their causative agents is of particular concern. Another intriguing issue regards the synergistic effects, if any, associated with chronic exposure to a number of environmental pollutants, such as organochlorines and heavy metals. In fact, it is also unknown whether and how these chemicals contribute towards modulating the pathogenic and pathogenetic activity primarily displayed by sea mammal morbilliviruses.     

An outbreak of post-vaccinal suspected distemper-like encephalitis in farmed ferrets (Mustela putorius furo)

Two outbreaks of an encephalitis apparently induced by an attenuated live distemper vaccine occurred in a large ferret breeding establishment in New Zealand. Approximately 350 of 6,000 young ferrets 16-22 weeks old died. Many were found dead with no premonitory signs, others showed severe neurological signs. Some with central nervous system (C.N.S.) signs recovered. Pathological examination showed no gross abnormalities except for a few with mild conjunctivitis, rhinitis and lung emphysema. Microscopically there was a moderate to massive non-inflammatory necrosis of hippocampal nerve cell bodies. In those animals which survived for several days with CNS signs there was also a mild to moderately severe non-supportive encephalitis, and in some of these distinct neuronal intranuclear and intracytoplasmic eosinophilic inclusion bodies were seen. Some ferrets also had a bronchiolitis with intracytoplasmic eosinophilic inclusion bodies in bronchiolar epithelium. All these lesions suggest that a distemper like condition was involved. About half of the ferrets also had a mild to severe inflammatory myocardial necrosis.     

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.     

An outbreak of post-vaccinal suspected distemper-like encephalitis in farmed ferrets (Mustela putorius furo)

Two outbreaks of an encephalitis apparently induced by an attenuated live distemper vaccine occurred in a large ferret breeding establishment in New Zealand. Approximately 350 of 6,000 young ferrets 16–22 weeks old died. Many were found dead with no premonitory signs, others showed severe neurological signs. Some with central nervous system (C.N.S.) signs recovered.

Pathological examination showed no gross abnormalities except for a few with mild conjunctivitis, rhinitis and lung emphysema. Microscopically there was a moderate to massive non-inflammatory necrosis of hippocampal nerve cell bodies. In those animals which survived for several days with CNS signs there was also a mild to moderately severe non-supportive encephalitis, and in some of these distinct neuronal intranuclear and intracytoplasmic eosinophilic inclusion bodies were seen. Some ferrets also had a bronchiolitis with intracytoplasmic eosinophilic mclusion bodies in bronchiolar epithelium. All these lesions suggest that a distemper like condition was involved. About half of the ferrets also had a mild to severe inflammatory myocardial necrosis.     

Epizootic of morbilliviral disease in common dolphins (Delphinus delphis ponticus) from the Black sea

Forty-seven common dolphins (Delphinus delphis ponticus) were stranded on the northern shores of the Black Sea between mid-July and early September 1994, more than in previous or subsequent years. Two of the 47 dolphins were examined in detail to try to determine the cause of the increased stranding rate. Their lesions included broncho-interstitial pneumonia with type II epithelial cell hyperplasia and multinucleate syncytial cells, neuronal necrosis, gliosis, and non-suppurative meningitis of the brain, necrotic stomatitis, gastroenteritis and cholangitis, and lymphoid depletion of the spleen and lymph nodes. The diseased tissues stained positive in an immunoperoxidase test, using a polyclonal antiserum to measles virus as the primary antibody, and electron microscopy showed that they contained regularly-shaped intranuclear particles about 22 nm in diameter. They were positive by the polymerase chain reaction (PCR) for the nucleoprotein gene of morbillivirus. However, there was no evidence of morbillivirus in frozen tissues either by virus isolation or by antigen capture ELISA. The concentration of sigma DDTS in the blubber of both dolphins was about 50 to 100 times higher than the levels in toothed cetaceans from the North Sea, North Atlantic Ocean, and Baltic Sea. The lesions were consistent with those found in other species with morbilliviral disease, and the positive immunoperoxidase test, PCR and electron microscopical examination confirmed a morbillivirus as the primary cause of these lesions.     

Morbillivirus in seals: isolation and some growth characteristics in cell cultures

The procedure used for the cultural isolation of morbillivirus from seals as well as some growth characteristics of isolates is described briefly. Emphasis was laid upon cytopathic changes typical for morbillivirus and observed in seal kidney cell culture subsequent to inoculation of organ tissue suspensions or buffy coat leucocytes. The modalities which resulted in the identification of 21 morbillivirus isolates from 16 seals by using direct fluorescent antibody (FA) or peroxidase linked antibody (PLA) techniques are outlined and illustrated.     

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.     

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.     

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.     

Pathogenesis of two strains of lion (Panthera leo) morbillivirus in ferrets (Mustela putorius furo)

Canine distemper virus (CDV) was previously considered to have a host range restricted to the canid family. In 1994, the virus was associated with sporadic outbreaks of distemper in captive felids. However, after severe mortality occurred in the Serengeti lions (Panthera leo), attention became focused on the pathogenesis of the virus and a concerted effort was made to identify the virus as CDV or a closely related feline morbillivirus. The present study was designed to explore the susceptibility of ferrets to challenge with two morbilliviruses isolated from lions and the protective effects of a modified-live mink distemper vaccine. Because mortality in ferrets infected with pathogenic CDV approaches 100%, the ferret was selected as a test animal. Two strains of lion morbillivirus were used as a challenge, A92-27/20 (California lion isolate) and A94-11/13 (Serengeti lion isolate). The two strains of lion morbillivirus were antigenically related to CDV (Rockborn strain), and ferrets were susceptible to both of the viruses when inoculated intraperitoneally. The inoculated ferrets were anorectic at 5-6 days postinoculation (PI), exhibited oculonasal discharge at 9-12 days PI, and became moribund at 12-22 days PI. Severe bilateral conjunctivitis was the typical clinical sign. Inclusion bodies characteristic of morbillivirus (eosinophilic, intranuclear, and intracytoplasmic) were distributed in many epithelial cells, including those of the skin, conjunctiva, gallbladder, liver, pancreas, stomach, trachea, lung, urinary bladder, and kidney. Virus was reisolated from selected lung tissues collected at necropsy and identified by CDV-specific immunofluorescence. Ferrets vaccinated with the mink distemper vaccine (Onderstepoort strain) were protected from challenge with the two lion strains, adding further support to the premise that the viruses are closely related to CDV.     

Nonsuppurative encephalitis in a dog

A 4-year-old male German Hunting Terrier presented with tremor, dyspnea, trismus, spasms of the musculature of the larynx and pharynx, and hypothermia and subsequently died despite intensive clinical care. Prior clinical signs included vomitus and diarrhea. Microscopic examination of the brain revealed a multifocal nonsuppurative brain stem encephalitis; a few intralesional neurons contained intranuclear inclusions. By immunohistochemistry, Aujeszky disease virus (Suid herpesvirus 1) antigen was detected in neurons in the brain and in ganglion cells of the trigeminal ganglia. Viral culture of brain tissue confirmed the presence of Aujeszky disease virus. Histopathologic findings in the brain with the identification of Aujeszky disease virus by immunohistochemistry and polymerase chain reaction are consistent with Aujeszky disease virus-induced encephalitis. Sequencing revealed a 100% homology of the isolated Aujeszky disease virus with Aujeszky disease virus isolates of wild boar from Eastern Germany.