Signalment:  

Whooper swan, Cygnus cygnusThis wild swan was found dead at the northern lake of Japan in May, 2008. The local veterinarian found the feces of this bird were influenza virus positive using a convenient test kit. The carcass was transported to our university and dissected within our P3 facility.

Gross Description:  

Diffusely the lungs showed severe congestive edema with edematous thickening of the pleura. Petechial hemorrhages were scattered on the pericardium and pancreas. Pericardial fluid was mildly increased and accompanied mild edematous thickening of pericardium and cardiac sac. The brain was congested.

Histopathologic Description:

Several glial nodules (Fig. 1-1) were scattered in the CNS. The nodules sometimes contained karyorrhexis or hyperchromatosis of nuclear wall of glial cells, rod cells, satellitosis to neuronophagia of nerve cells, and minute malacic foci. The karyorrhexis of glial cells and rod cells were also sparsely distributed throughout the CNS. The nuclei of perivascular cells (pericytes and astrocytes) were sometimes swollen, and perivascular inflammatory cell infiltration was indiscernible.

Imunohistochemistry using rabbit polyclonal antibody against highly pathogenic avian influenza virus of H5N2 subtype as primary antibody revealed viral antigens in the nuclei of astrocytes (arrow head in Fig. 1-2 and 1-3), microglial cells (arrows in Fig. 1-2), and nerve cells (arrows in Fig. 1-3) within and around the glial nodules. 

Besides the brain, lymphocytic necrosis in the spleen, mild fibrinous bronchopneumonia and focal necrosis of exocrine pancreas were found with viral antigens in alveolar epithelial cells, bronchial epithelial cells and exocrine pancreatic cells. Inflammatory cell infiltration was minimal in these lesions.

Morphologic Diagnosis:  

Nonpurulent encephalitis, diffuse, mild, influenza virus infection.

Lab Results:  

Highly pathogenic avian influenza virus of H5N1 subtype was isolated from the brain, lungs, trachea, colon and pancreas of the birds. HA titers of the virus in each organ were between 32-256, and the titer of the brain was 128.

Condition:  

Highly pathogenic avian influenza

Contributor Comment:  

The threat of highly pathogenic avian influenza virus of H5N1 subtype to humans as well as domestic and wild birds is a great concern of human public health and fowl industry in worldwide magnitude.(1,10) The virus first emerged in 2003 in east and southeast Asian countries. Many human cases have been reported in Indonesia, Vietnam and China. Japan suffered the outbreaks of H5N1 infection in domestic fowl seven times from 2004 to 2007. All of these outbreaks were rapidly controlled by thorough culling. In April to May of 2008, a total of three Whooper swans were found dead in the northern lakes of Japan and highly pathogenic avian influenza viruses of H5N1 subtype were isolated from them. These birds were migrating from southern Asian countries to Siberia. The present case was one of the three cases. Genomic analysis on the isolated viruses revealed the sequence of these isolates were almost identical and were remote from those of the previous outbreaks in 2004 and 2007 in Japan.

Pathological changes of birds due to highly pathogenic avian influenza of H5N1 subtype are necrotic and hemorrhagic changes are centered in the CNS, pancreas, lungs, liver, adrenals, heart and lymphoid organs.(2-4,9,10) The CNS lesions in the present case were very mild and were at an early stage of encephalitis in comparison with previous reports on experimental or non-migratory birds.(2,4)

Birds infected with highly pathogenic avian influenza rapidly develop viremia, and then the virus infects and damages vascular endothelial cells (3,8,9) resulting in hemorrhagic and edematous changes in various organs and tissues including the skin and skeletal muscles. Necrotic and apoptotic changes of parenchymal cells of organs follow. In the CNS, the virus antigen first appears in vascular endothelial cells, then extends to astroglia and nerve cells.(8, 9) In mice, the virus causes neither viremia nor endothelial damage. It invades the CNS via peripheral nerves.(5,7)

JPC Diagnosis:  

Cerebrum: Neuronal necrosis, subacute, multifocal, mild with multiple glial nodules .

Conference Comment:  

Influenza viruses are important pathogens in both humans and animals, and their ability to cross species barriers is of major concern to medical professionals. Influenza viruses are in the family Orthomyxoviridae, encompassing the genera Influenza A, B, and C, and Thogotovirus. Viruses from the genus influenza A infect humans, horses, pigs, seals, birds, whales, and mink; influenza B viruses infect only humans; influenza C viruses infect humans and swine; thogotoviruses are tick borne viruses found mainly in Africa, Asia, and Europe.(6)

The reservoir for influenza A is waterfowl, and the virus causes an asymptomatic infection in these species with replication in the intestinal epithelium and subsequent fecal shedding. These problematic waterfowl often spread influenza via migratory routes, and the virus has a chance to exchange genes with other novel influenza viruses during these sojourns creating a potential for a new, virulent influenza virus.(6) Swine are important intermediate hosts because they can get both influenza A and C, and thus create an environment for viral genetic rearrangement. Influenza viruses can either undergo genetic drift, (point mutation) or genetic shift (genetic segment reassortment) to create new strains of virus. Most combinations of influenza virus are non-pathogens, but when a drift or shift occurs creating a novel, virulent virus, pandemics such as the 1918 outbreak are the result.(6)

Highly pathogenic avian influenza in chickens and turkeys, also known colloquially as fowl plague, often causes death with little to no clinical warning. If birds survive the initial stage of disease they clinically present with severe respiratory distress along with cyanosis of the unfeathered skin to include the comb and wattles(6) In birds, unlike mammals, influenza replicates in both the respiratory and gastrointestinal tracts. Virulent strains cause viremia with resultant necrosis of lymphoid and gastrointestinal tissue, pancreatitis, myositis, and encephalitis. Petechial hemorrhages are commonly found in the digestive, respiratory and cardiac tissues because of viral damage to endothelial cells.(6)

References:

1. Kishida N, Sakoda Y, Isoda K, Matsuda K, Eto M, Sunaga Y, Umemura T, Kida H: Pathogenicity of H5 influenza viruses in ducks. Arch Virol 150:1383-1392, 2005
2. Kobayashi Y, Horimoto T, Kawaoka Y, Alexander DJ, Itakura C: Pathological studies of chickens experimentally infected with two highly pathogenic avian influenza viruses. Avian Pathol 25:285-304, 1996
3. Kwon YK, Joh SJ, Kim MC, Lee YJ, Choi JG, Wee SH, Sung HW, Kwon JH, Kang MI, Kim JH: Highly pathogenic avian influenza in Magpies (Pica pica sericea) in South Korea. J Wildlife Dis 41:618-623, 2005
4: Le Gall-Recule G, Briand FX, Schmitz A, Guionie O, Massin P, Jestin V: Double introduction of highly pathogenic H5N1 avian influenza virus into France in early 2006. Avian Pathol 37: 15-23, 2008. 
5. Park CH, Ishinaka M, Takada A, Kida H, Kimura T, Ochiai K, Umemura T: The invasion routes of neurovirulent A/Hong Kong/483/97(H5N1) influenza virus into the central nervous system after respiratory infection in mice. Arch Vorol 147:1425-1436, 2002
6. Matsuda K, Park CH, Sunden Y, Kimura T, Ochiai K, Kida H, Umemura T: The vagus nerve is one route of transneural invasion for intranasally inoculated influenza A virus in mice. Vet Pathol 41:101-107, 2004
7. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ: Orthomyxoviridae. In: Veterinary Virology, 3rd ed., pp. 459-46. Academic Press, San Diego, California, 1999
8. Park CH, Ozaki H, Takada A, Kida H, Ochiai K, Umemura T: Primary target cells of virulent strains of type A influenza virus in chicken embryos. Avian Pathol 30:269-272, 2001
9. Silvano FD, Yoshikawa M, Shimada A, Otsuki K, Umemura T: Enhanced neuropathogenicity of acian influenza A virus by passages through air sac and brain of chicks. J Vet Med Sci 59:143-148, 1996
10. Teifke JP, Klopfleisch R, Globig A, Starick E, Hoffmann B, Wolf PU, Beer M, Mettenleiter TC, Harder TC: Pathology of natural infections by H5N1 highly patyogenic avian influenza virus in Mute (Cygnus olor) and Whooper (Cygnus Cygnus) swans. Vet Pathol 44:137-143, 2007

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1-1, Cerebrum, Whooper Swan

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