Samples

Figure 1

Figure 1. Baikal teal captured at Donglim Reservoir, showing A) neurologic signs of torticollis, ataxia, and limb paresis; B) hemorrhage and necrosis in heart muscle; C) edema and congestion of lung; and D)...

During January–June 2014, a total of 771 wild bird carcassess were submitted to the Animal and Plant Quarantine Agency in Anyang, South Korea (Table 1). On January 17, many dead or sick wild birds were found around Donglim Reservoir in southwestern Korea. Three sick Baikal teals showing neurologic symptoms, including torticollis, ataxia, and limb paresis, were captured and euthanized (Figure 1, panel A). Over a 5-day period, the bodies of 119 Baikal teals, 9 bean geese, and 1 coot were collected from near the Donglim Reservior for necropsy. On January 22 and January 27, a total of 5 dead Baikal teals were found near the Geumgang River in midwestern South Korea (Table 2). Another 634 dead birds were found in other parts of the country. Necropsies were performed on all dead birds; trachea, kidney, cecal tonsil, pancreas, liver, intestine, heart, and lung were collected for virus isolation. Parenchymal tissues were collected for histopathologic analysis from 8 Baikal teals, 2 bean geese, and 1 whooper swan showing gross lesions and 3 bean geese and 2 mallard ducks not showing gross lesions. Collected tissues were fixed for 24 hours in 10% buffered neutral formaldehyde and processed for paraffin embedding. Bacterial culture was performed by using standard methods. The stomach contents were subjected to toxicology testing, as described previously (12).

Virus Isolation and Identification

Tissue samples from wild birds were inoculated into specific pathogen free embryonated chicken eggs (9–11 days of gestation), and influenza viruses were identified by using a hemagglutination assay and reverse transcription PCR. Virus identification was confirmed by sequence analysis, as described previously (10). In addition, molecular pathotyping was performed by nucleotide sequence analysis of the hemagglutinin cleavage site within the H5 subtype.

Histopathology and Immunohistochemistry

Paraffin-embedded sections were cut (5 μm), dewaxed, and stained with hematoxylin and eosin. Duplicate sections were immunohistochemically analyzed to determine the distribution of influenza virus antigens in individual tissues. Briefly, sections were stained with a mouse monoclonal antibody against influenza A virus nucleoprotein (MCA-400; AbD Serotec, Duesseldorf, Germany), followed by a biotinylated goat anti-mouse IgG secondary antibody. Bound antibodies were detected with an avidin-biotin detection system (Ventana Medical Systems, Tucson, AZ, USA). The RedMap kit (Ventana Medical Systems) served as the substrate chromogen.

Wild Bird Carcasses

Of a total of 771 wild birds, HPAI A(H5N8) viruses were isolated from 167. For the other 604 birds, test results for other avian influenza viruses were negative (Table 1). Bacterial (Escherichia coli, Staphylococcus aureus, and Salmonella Typhimurium), parasitic (nematodes, cestodes), and fungal infections were diagnosed for 29, 9, and 3 birds, respectively. We found that 73% of birds died of noninfectious causes. Agrochemicals, including monocrotophos, phosphamidon, carbofuran, diazinon, carbosulfan, endosulfan, parathion, dichlorvos, and methomyl, were found in the stomach contents of 222 birds; gunshot wounds, trauma (road kill or fracture), or miscellaneous (cachexia, dehydration, or suffocation) were the cause of death for 12, 103, and 32 birds, respectively. For 194 wild birds, the cause of death could not be determined because of postmortem autolysis, putrefaction, or both.

Observation of Gross Lesions and Isolation of HPAI Virus

During January–March 2014, a total of 167 wild birds of 8 species were infected with HPAI A(H5N8) virus. All 148 infected Baikal teals showed evidence of multifocal necrosis in the pancreas and liver, pulmonary congestion and edema, subepicardial hemorrhage, and myocarditis (Figure 1, panels B–D), and H5N8 virus was isolated from the trachea, cecal tonsil, kidney, liver, lung, pancreas, and heart. Monocrotophos poisoning was also diagnosed for 20 Baikal teals collected in Chungnam Province. Although no lesions were visible in the organs of 9 bean geese found near Donglim Reservoir, H5N8 virus was identified in the trachea and kidney of all 9. These birds also contained high concentrations of monocrotophos. Necropsy of 2 bean geese and 1 whooper swan found during February–March revealed distinct lesions in the pancreas and kidney; H5N8 virus was isolated from the trachea, cecal tonsil, and pancreas. However, no gross lesions associated with HPAI virus infection were found in the organs from a mallard and a white-fronted goose; for these birds, the cause of death seemed to have been peritonitis and gunshot wounds. HPAI A(H5N8) virus infection was found in 1 coot, 2 little grebes, and 1 great egret; however, because of postmortem changes, no gross lesions associated with HPAI virus infection were identified (Table 2). All H5N8 virus isolates showed an HPAI virus motif (LREK[R]RRKR/GLF) at cleavage sites of hemagglutinin.

Histopathologic and Immunohistochemical Findings

Baikal Teals

Figure 2

Figure 2. Histopathologic and immunohistochemical (IHC) testing results for Baikal teal. A) Focal necrosis in pancreas (hematoxylin and eosin [H&E] stain). B) Avian influenza virus antigen in necrotic pancreatic acini ([IHC stain). C)...

Histologic examination revealed lesions in the pancreas, kidney, brain, and lung of all 8 birds examined. The pancreas showed moderate to severe, multifocal to confluent acinar necrosis, and virus antigen was detected in necrotic cells (Figure 2, panels A, B). Glomerular capillaries showed evidence of diffuse thrombosis and mild necrosis of tubules along with crystalline urate; virus antigen was detected in the tubular epithelium and glomerular capillary endothelium (Figure 2, panels C, D). Mild lymphocytic perivascular cuffing and loss of Purkinje cells were observed in the cerebrum and cerebellum, and virus antigen was detected in ependymal cells and epithelium of the choroid plexus and in cerebellar Purkinje cells. The lungs showed evidence of marked congestion, edema, and hemorrhage, and thrombosis was found in the alveolar capillaries. Influenza virus antigen was observed in a few capillary endothelial cells and macrophages in the alveolar lumen. Mild multifocal necrosis of hepatocytes and a lymphocytic infiltrate were also observed, and massive amounts of virus antigen were distributed within the sinusoidal endothelium and in necrotic hepatocytes within the liver. No lesions were visible in the trachea, intestine, muscle, spleen, or heart, and no antigen-positive cells were found (Table 3).

Bean Geese

Figure 3

Figure 3. Histopathologic and immunohistochemical (IHC) testing results for bean goose. A) Diffuse necrotizing pancreatitis (hematoxylin and eosin [H&E] stain). B) Avian influenza virus antigen in necrotic pancreatic cells (IHC stain). C) Segmental...

In 2 of 3 bean geese examined, major histopathologic lesions were found in the same organs as in the Baikal teals. Moderate multifocal pancreatic necrosis was also observed. Myocardial myofibers showed evidence of segmental necrosis, and mildly swollen nuclei, focal necrosis, and virus antigen were detected in the heart (Figure 3, panels C, D). Also observed were randomly distributed foci of neuronal necrosis and mild to moderate lymphocytic perivascular cuffing in the cerebrum and a paucity of cerebellar Purkinje cells and focal necrosis in the cerebellum. Staining was positive for virus antigen in neurons, glial cells, dendritic cells, granule cells, and Purkinje cells (Figure 3, panels E–H). Moreover, renal tubular necrosis and crystalline urinary casts were observed in the kidney, and virus antigen was detected in the tubular epithelium. No lesions were evident in intestine, skeletal muscle, or spleen (Table 3).