Acute fatal sarcocystosis hepatitis in an Indo-Pacific bottlenose dolphin (Tursiops aduncus) in Hong Kong.

Unlike most species in the genus Sarcocystis, Sarcocystis canis has a broad intermediate host range. Its life cycle is incompletely known and most reports are from the USA. Here we report fatal hepatitis in a 4year old male Indo-Pacific bottlenose dolphin (Tursiops aduncus) from Hong Kong associated with a S. canis-like infection. Diagnosis was made based on clinical presentation, histopathology, transmission electron microscopy (TEM), and molecular characterization. Microscopically, S. canis-like like infection was confined to the liver. Immature and mature schizonts were found in hepatocytes and the parasite was associated with generalized hepatic necrosis. By TEM, schizonts divided by endopolygeny, and merozoites lacked rhoptries. Molecular characterization of parasites present in liver and brain tissues at the cox1 gene showed a high degree of identity (97-98%) and clustered together with Sarcocystis canis, S. lutrae, S. arctica, S. speeri, S. turdusi, and S. rileyi in a phylogenetic study. This is the first report of S. canis-like infection from Asia.

Is dolphin morbillivirus virulent for white-beaked dolphins (Lagenorhynchus albirostris)?

The virulence of morbilliviruses for toothed whales (odontocetes) appears to differ according to host species. In 4 species of odontocetes, morbilliviruses are highly virulent, causing large-scale epizootics with high mortality. In 8 other species of odontocetes, including white-beaked dolphins (Lagenorhynchus albirostris), morbilliviruses have been found as an incidental infection. In these species, the virulence of morbilliviruses is not clear. Therefore, the admission of 2 white-beaked dolphins with morbillivirus infection into a rehabilitation center provided a unique opportunity to investigate the virulence of morbillivirus in this species. By phylogenetic analysis, the morbilliviruses in both animals were identified as a dolphin morbillivirus (DMV) most closely related to that detected in a white-beaked dolphin in Germany in 2007. Both animals were examined clinically and pathologically. Case No. 1 had a chronic neural DMV infection, characterized by polioencephalitis in the cerebrum and morbillivirus antigen expression limited to neurons and glial cells. Surprisingly, no nervous signs were observed in this animal during the 6 months before death. Case No. 2 had a subacute systemic DMV infection, characterized by interstitial pneumonia, leucopenia, lymphoid depletion, and DMV antigen expression in mononuclear cells and syncytia in the lung and in mononuclear cells in multiple lymphoid organs. Cause of death was not attributed to DMV infection in either animal. DMV was not detected in 2 contemporaneously stranded white-beaked dolphins. Stranding rate did not increase in the region. These results suggest that DMV is not highly virulent for white-beaked dolphins.

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.

Neurological signs in juvenile harbour seals (Phoca vitulina) with fatal phocine distemper.

In 2002, the northern European harbour seal (Phoca vitulina) population experienced an epidemic of phocine distemper virus (PDV) in which 22,000 seals died. Clinical signs were recorded in 20 harbour seal pups admitted to the Seal Rehabilitation and Research Centre with clinical disease, and they were diagnosed PDV infection-positive by RT-PCR postmortem. All 20 had respiratory signs, 14 had conjunctivitis and 10 had neurological signs. Severe neurological signs were one of the criteria for euthanasia during the epidemic, and many pups that were euthanased were not included in this study owing to the lack of complete datasets. Neurological signs were therefore among the most prevalent signs of fatal PDV infection in harbour seal pups. The lymphoid depletion reported in dead seals during the epidemic was not reflected in the total mononuclear leucocyte count of the seal pups, but they had an absolute granulocytosis, thrombocytosis, anaemia, and high total white blood cell counts. When first examined, 11 of the pups had a positive serum IgG titre, and four had a positive serum IgM titre. High levels of PDV-specific serum IgG antibodies were not correlated with an absence of clinical signs or longer survival.

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.

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.

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.

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.

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 [PI3], 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 (Fellis concolor), lynxes (Fellis 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 PI3 in black bears and fishers, and to DRV in belugas and walruses.

Seal gammaherpesviruses: identification, characterisation and epidemiology.

Phocid herpesvirus type 2 (PhHV-2), tentatively classified as a gammaherpesvirus, has been isolated from European and American harbour seals (Phoca vitulina). Here we describe the isolation and the molecular as well as biological characterisation of different PhHV-2 isolates from harbour seals and grey seals (Halichoerus grypus). Of 522 harbour seals and 231 grey seals that had been admitted to the seal research and rehabilitation centre in Pieterburen, The Netherlands, between 1992 and 2000, 38 and 18%, respectively, proved to have PhHV-2 neutralising antibodies. PhHV-2 was isolated from peripheral blood mononuclear cells (PBMCs) of 12 and 28% of these seropositive animals, respectively, and 26 and 56% of these cell samples, respectively, were positive by PCR analysis. Analysis of amino acid sequences of PCR products and of the growth characteristics of different PhHV-2 isolates indicated that harbour and grey seals are infected with distinct gamma-herpesviruses, which however, may co-circulate between the two species.

Genetic characterization of wild-type measles viruses circulating in suburban Khartoum, 1997-2000.

Measles remains endemic in many East African countries, where it is often associated with high morbidity and mortality. We collected clinical specimens from Sudanese measles patients between July 1997 and July 2000. Sequencing of the 3' 456 nucleotides of the nucleoprotein gene from 33 measles virus (MV) isolates and 8 RNA samples extracted from clinical specimens demonstrated the presence of a single endemic MV strain with little sequence variation over time (overall nucleotide divergence of 0 to 1.3%). This was confirmed by sequencing of the complete H gene of two isolates from 1997 and two from 2000, in which the overall divergence ranged between 0 and 0.5%. Comparison with MV reference strains demonstrated that the viruses belonged to clade B, genotype B3, and were most closely related to a set of viruses recently isolated in Nigeria. Our study demonstrates a remarkable genetic stability of an endemically circulating MV strain.

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.

Mass die-Off of Caspian seals caused by canine distemper virus.

Thousands of Caspian seals (Phoca caspica) died in the Caspian Sea from April to August 2000. Lesions characteristic of morbillivirus infection were found in tissue specimens from dead seals. Canine distemper virus infection was identified by serologic examination, reverse transcriptase- polymerase chain reaction, and sequencing of selected P gene fragments. These results implicate canine distemper virus infection as the primary cause of death.

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.

Serological and virological characterization of clinically diagnosed cases of measles in suburban Khartoum.

Measles continues to be a major childhood disease in terms of global morbidity and mortality. In the main areas of its endemicity the only available means of diagnosis are based on clinical criteria: the presence of a maculopapular rash and fever accompanied by cough, coryza, and/or conjunctivitis. We have studied 38 clinically diagnosed cases of measles in Khartoum, Sudan, by means of serology, reverse transcriptase PCR (RT-PCR) on throat swabs and virus isolation from lymphocytes. On the basis of serology, 28 patients were diagnosed as having an acute measles virus (MV) infection, while in 10 cases the clinical symptoms proved to have other causes. It was shown that in cases with low serum immunoglobulin M (IgM) levels, an additional measurement of IgG or virus-neutralizing antibodies was necessary to discriminate between patients with an acute MV infection sampled during an early stage of the disease and patients who had experienced an MV infection in the more distant past. The serological laboratory diagnosis was validated by an MV-specific RT-PCR: for all confirmed measles cases tested a fragment of the correct size which hybridized with a third MV-specific primer could be amplified, while all serologically negative cases were also RT-PCR negative. MV could be isolated from 17 out of 23 of the serologically confirmed cases, demonstrating that virus isolation is less reliable as a diagnostic tool than serology or RT-PCR. This study stresses the urgent need for a rapid diagnostic field test for measles.

Morbilliviruses in Mediterranean monk seals.

Two morbilliviruses were isolated from Mediterranean monk seals (Monachus monachus), one from a stranded animal in Greece and the other one from carcasses washed ashore during a mass die-off in Mauritania. From both viruses N and P gene fragments were sequenced and compared to those of other known morbilliviruses. The monk seal morbilliviruses most closely resembled previously identified cetacean morbilliviruses, indicating that interspecies transmission from cetaceans to pinnipeds has occurred.

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.

Fusion protein gene nucleotide sequence similarities, shared antigenic sites and phylogenetic analysis suggest that phocid distemper virus type 2 and canine distemper virus belong to the same virus entity.

Nucleotide sequencing of the fusion protein (F) gene of phocid distemper virus-2 (PDV-2), recently isolated from Baikal seals (Phoca sibirica), revealed an open reading frame (nucleotides 84 to 2075) with two potential in-frame ATG translation initiation codons. We suggest that the second in-frame ATG triplet at positions 264 to 266 initiates the translation, resulting in a protein of 537 amino acid residues with a calculated M(r) of 63,035. The putative F1/F2 cleavage site, located approximately 100 amino acid residues from the N terminus, is identical to those of the F proteins of phocid distemper virus-1 (PDV-1) isolated from European harbour seals (Phoca vitulina) and of canine distemper virus (CDV). A full scale comparison of morbillivirus F genes reveals that the conserved F0 extracellular protein-encoding region contains a large number of non-expressed mutations, suggesting that this part of the protein is under strong functional constraints. Phylogenetic analysis of morbillivirus F gene nucleotide sequences revealed a closer evolutionary relationship between PDV-2 and CDV than between PDV-1 and PDV-2. These data were supported by cross-reactivity patterns of PDV-2 and CDV obtained with monoclonal antibodies to structural proteins of PDV-1 and CDV, and suggest that PDV-2 is a strain of CDV, resulting from a trans-species infection.