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.     

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.     

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.     

Distemper virus as a cause of central nervous disease and death in badgers (Meles meles) in Denmark

During the summer of 2002 a distemper-like disease was observed in the free-ranging badger population in Denmark. It was characterised by grand seizures, abnormal behaviour and death; the badgers all had severe chronic pneumonia and some had non-suppurative encephalomyelitis. In this study, eight of the affected badgers were examined by gross pathological, histological, immunohistological, bacteriological, parasitological and virological methods, and were diagnosed with distemper; canine distemper virus was identified.     

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.     

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.     

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.     

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.     

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.     

Canine distemper virus-induced depletion of uninfected lymphocytes is associated with apoptosis

Canine distemper virus (CDV), a negative stranded RNA morbillivirus, causes a multisystemic disease in dogs, which is associated with a severe immune suppression. The aim of the study was to examine the influence of early CDV infection on leukocyte depletion, lymphopenia and virus-induced cell death in dogs infected with a virulent CDV strain. From 10 infected dogs, peripheral blood leukocytes were harvested periodically, phenotyped and analyzed for CDV antigen content and apoptosis using Annexin V-FITC and propidium iodide labeling. CDV infection induced a severe CD3+ T cell and CD21+ B cell depletion in all animals at 3 days post-infection (d.p.i.). For dogs with severe distemper, developing virus persistence in the lymphoid tissue and central nervous system, this lymphopenia lasted until the end of the experiment. Increased levels of lymphocyte apoptosis were found at 3 d.p.i., and monocyte apoptosis at 6 d.p.i. This was more prominent in the group of animals with severe distemper. At 3 d.p.i. no leukocyte infection was detectable indicating that the early lymphocyte depletion and apoptosis was not a direct consequence of virus infection. Taken together, our results demonstrate that CDV-induced lymphopenia is an early event and that the degree of lymphocyte depletion correlates with the severity of disease and virus persistence in the lymphoid tissue and central nervous system.     

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.     

Granulomatous meningoence-phalomyelitis in 21 dogs

Granulomatous meningoencephalomyelitis (GME) is a well recognised disease entity affecting dogs. It manifests in a wide variety of clinical syndromes. The lesion is characterised by focal or disseminated non-caseating granulomas in the brain and spinal cord, non-suppurative meningitis and marked perivascular lymphoid cuffing. The clinical signs can be acute and rapidly progressive or can manifest as a chronic relapsing disease. In this survey, 12 clinical cases were referred to Murdoch University Veterinary Hospital and nine cases were submitted for necropsy. While cerebrospinal fluid examination in seven of these cases suggested inflammatory disease, necropsy confirmed the presence of GME. An immunohistochemical technique for detection of distemper virus antigen failed to identify the presence of distemper virus antigen in any of the cases. It was concluded that distemper virus was not involved in the aetiology of these 21 cases all of which were confirmed by post mortem examination.     

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.     

Comparative analyses of canine distemper viral isolates from clinical cases of canine distemper in vaccinated dogs

Sequence and phylogenetic analyses of three isolates of canine distemper virus (CDV) isolated from three dogs with a vaccination history were compared with the same analyses of vaccine virus isolated from a vaccine used for dogs. The three dogs showed clinical signs of a recent major type of CD in Japan, including oculonasal discharge and diarrhea, and pathological findings including non-suppurative encephalitis, pneumonia, mild gastroenteritis and lymphoid depletion. Inclusion bodies were in the stomach without inflammation and encephalitis was without clinical signs. One of the highest titers of CDV in different organs of the three dogs was commonly systemic lymphatic organs, including the spleen, lymph nodes and tonsils. New isolates of CDV joined to the clades of the Asia 1 group that is far from the vaccine group. These results surely indicate that wild strains of CDV from dogs with a vaccination history were not reversed vaccine virus, and that the dogs showed characteristics of recent CD in Japan.     

Evolutionary characteristics of morbilliviruses during serial passages in vitro: Gradual attenuation of virus virulence

The genus Morbillivirus is classified into the family Paramyxoviridae, and is composed of 6 members, namely measles virus (MV), rinderpest virus (RPV), peste-des-petits-ruminants virus (PPRV), canine distemper virus (CDV), phocine distemper virus (PDV) and cetacean morbillivirus (CeMV). The MV, RPV, PPRV and CDV have been successfully attenuated through their serial passages in vitro for the production of live vaccines. It has been demonstrated that the morbilliviral virulence in animals was progressively attenuated with their consecutive passages in vitro. However, only a few reports were involved in explanation of an attenuation-related mechanism on them until many years after the establishment of a quasispecies theory. RNA virus quasispecies arise from rapid evolution of viruses with high mutation rate during genomic replication, and play an important role in gradual loss of viral virulence by serial passages. Here, we overviewed the development of live-attenuated vaccine strains against morbilliviruses by consecutive passages in vitro, and further discussed a related mechanism concerning the relationship between virulence attenuation and viral evolution.     

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.     

Pathogenesis and immunopathology of systemic and nervous canine distemper

Canine distemper is a worldwide occurring infectious disease of dogs, caused by a morbillivirus, closely related to measles and rinderpest virus. The natural host range comprises predominantly carnivores. Canine distemper virus (CDV), an enveloped, negative-sense RNA virus, infects different cell types, including epithelial, mesenchymal, neuroendocrine and hematopoietic cells of various organs and tissues. CDV infection of dogs is characterized by a systemic and/or nervous clinical course and viral persistence in selected organs including the central nervous system (CNS) and lymphoid tissue. Main manifestations include respiratory and gastrointestinal signs, immunosuppression and demyelinating leukoencephalomyelitis (DL). Impaired immune function, associated with depletion of lymphoid organs, consists of a viremia-associated loss of lymphocytes, especially of CD4+ T cells, due to lymphoid cell apoptosis in the early phase. After clearance of the virus from the peripheral blood an assumed diminished antigen presentation and altered lymphocyte maturation cause an ongoing immunosuppression despite repopulation of lymphoid organs. The early phase of DL is a sequel of a direct virus-mediated damage and infiltrating CD8+ cytotoxic T cells associated with an up-regulation of pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α and IL-12 and a lacking response of immunomodulatory cytokines such as IL-10 and transforming growth factor (TGF)-β. A CD4+-mediated delayed type hypersensitivity and cytotoxic CD8+ T cells contribute to myelin loss in the chronic phase. Additionally, up-regulation of interferon-γ and IL-1 may occur in advanced lesions. Moreover, an altered balance between matrix metalloproteinases and their inhibitors seems to play a pivotal role for the pathogenesis of DL. Summarized, DL represents a biphasic disease process consisting of an initial direct virus-mediated process and immune-mediated plaque progression. Immunosuppression is due to early virus-mediated lymphocytolysis followed by still poorly understood mechanisms affecting antigen presentation and lymphocyte maturation.