M. W. G. van de Bildt

The 2000 Canine Distemper Epidemic in Caspian Seals (Phoca caspica): Pathology and Analysis of Contributory Factors

More than 10,000 Caspian seals (Phoca caspica) were reported dead in the Caspian Sea during spring and summer 2000. We performed necropsies and extensive laboratory analyses on 18 seals, as well as examination of the pattern of strandings and variation in weather in recent years, to identify the cause of mortality and potential contributory factors. The monthly stranding rate in 2000 was up to 2.8 times the historic mean. It was preceded by an unusually mild winter, as observed before in mass mortality events of pinnipeds. The primary diagnosis in 11 of 13 seals was canine distemper, characterized by broncho-interstitial pneumonia, lymphocytic necrosis and depletion in lymphoid organs, and the presence of typical intracytoplasmic inclusion bodies in multiple epithelia. Canine distemper virus infection was confirmed by phylogenetic analysis of reverse transcriptase-polymerase chain reaction products. Organochlorine and zinc concentrations in tissues of seals with canine distemper were comparable to those of Caspian seals in previous years. Concurrent bacterial infections that may have contributed to the mortality of the seals included Bordetella bronchiseptica (4/8 seals), Streptococcus phocae (3/8), Salmonella dublin (1/8), and S. choleraesuis (1/8). A newly identified bacterium, Corynebacterium caspium, was associated with balanoposthitis in one seal. Several infectious and parasitic organisms, including poxvirus, Atopobacter phocae, Eimeria- and Sarcocystis-like organisms, and Halarachne sp. were identified in Caspian seals for the first time.

Antibodies to selected pathogens in free-ranging terrestrial carnivores and marine mammals in Canada

Antibody titres to selected pathogens (canine adenovirus [CAV-2], feline herpesvirus [FHV], phocine herpesvirus [PHV-1], canine distemper virus, dolphin morbillivirus [DMV], phocine distemper virus [PDV], parainfluenza virus type 3 [P13], rabies virus, dolphin rhabdovirus [DRV], canine coronavirus, feline coronavirus, feline leukaemia virus, Borrelia burgdorferi and Toxoplasma gondii) were determined in whole blood or serum samples from selected free-ranging terrestrial carnivores and marine mammals, including cougars (Felis concolor), lynxes (Felis lynx), American badgers (Taxidea taxus), fishers (Martes pennanti), wolverines (Gulo gulo), wolves (Canis lupus), black bears (Ursus americanus), grizzly bears (Ursus arctos), polar bears (Ursus maritimus), walruses (Odobenus rosmarus) and belugas (Delphinapterus leucas), which had been collected at several locations in Canada between 1984 and 2001. Antibodies to a number of viruses were detected in species in which these infections have not been reported before, for example, antibodies to cav-2 in walruses, to pdv in black bears, grizzly bears, polar bears, lynxes and wolves, to DMV in grizzly bears, polar bears, walruses and wolves, to P13 in black bears and fishers, and to DRV in belugas and walruses.

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 northem 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 11 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 EUSA. The concentration of ΣDDTS 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.

Confirmation and Phylogenetic Analysis of Rabbit Hemorrhagic Disease Virus in Free-living Rabbits from the Netherlands

The number of free-living European rabbits (Oryctolagus cuniculus) in the Netherlands has declined dramatically in recent years. Although rabbit hemorrhagic disease virus (RHDV) infection has been implicated as a possible cause of this decline, the definitive diagnosis has not been reported. We examined three free-living rabbits found dead in the Netherlands in 2004 by use of gross pathology, histopathology, immunohistochemistry, and reverse transcriptase polymerase chain reaction. We subsequently compared the identified virus with RHDV from elsewhere in the world by phylogenetic analysis. There was widespread necrosis, hemorrhage, or both in liver, kidney, spleen, and lungs of all three rabbits, consistent with RHDV infection. The presence of RHDV in affected tissues was demonstrated by immunohistochemistry and reverse transcriptase polymerase chain reaction. The RHDV from the Netherlands showed the highest identity, 99%, with a strain from France in 2000, and fitted in genogroup G5. These results prove that RHDV infection causes mortality of free-living rabbits in the Netherlands and suggest that RHDV strains circulating in free-living rabbits in the Netherlands and France have a common source or that one has originated from the other.

Cetacean morbilliviruses are phylogenetically divergent

Summary.We performed a phylogenetic comparison of porpoise morbillivirus (PMV) and dolphin morbillivirus (DMV) isolates from porpoises and dolphins respectively according to criteria adopted by the World Health Organization for the phylogenetic comparison of measles viruses. PMV and DMV were more divergent than the most distantly related measles virus strains, thus challenging the classification of PMV and DMV as two strains of a single species, cetacean morbillivirus.

Identification of morbilliviruses of probable cetacean origin in carcases of Mediterranean monk seals (Monachus monachus)

Two morbilliviruses were isolated from carcases of Mediterranean monk seals (Monachus monachus) which had died in coastal areas of Greece and Mauritania. They were characterised as being closely related to the previously identified dolphin and porpoise morbilliviruses on the basis of their serological cross-reactivities in immunofluorescence assays, and sequence homologies in their N and P genes. The results suggest that morbilliviruses of aquatic mammals may cross barriers between species of different orders.

Replication of 2 Subtypes of Low-Pathogenicity Avian Influenza Virus of Duck and Gull Origins in Experimentally Infected Mallard Ducks

Many subtypes of low-pathogenicity avian influenza (LPAI) virus circulate in wild bird reservoirs, but their prevalence may vary among species. We aimed to compare by real-time reverse-transcriptase polymerase chain reaction, virus isolation, histology, and immunohistochemistry the distribution and pathogenicity of 2 such subtypes of markedly different origins in Mallard ducks (Anas platyrhynchos): H2N3 isolated from a Mallard duck and H13N6 isolated from a Ring-billed Gull (Larus delawarensis). Following intratracheal and intraesophageal inoculation, neither virus caused detectable clinical signs, although H2N3 virus infection was associated with a significantly decreased body weight gain during the period of virus shedding. Both viruses replicated in the lungs and air sacs until approximately day 3 after inoculation and were associated with a locally extensive interstitial, exudative, and proliferative pneumonia. Subtype H2N3, but not subtype H13N6, went on to infect the epithelia of the intestinal mucosa and cloacal bursa, where it replicated without causing lesions until approximately day 5 after inoculation. Larger quantities of subtype H2N3 virus were detected in cloacal swabs than in pharyngeal swabs. The possible clinical significance of LPAI virus-associated pulmonary lesions and intestinal tract infection in ducks deserves further evaluation.

Identification and characterization of a novel adenovirus in the cloacal bursa of gulls

Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Larus argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species.

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.

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.