Infected with H5N1 Avian Influenza Virus in Akita, Japan, in 2008

NOTE Pathology

Pathology of Whooper Swans (Cygnus cygnus) Infected with H5N1 Avian Influenza Virus in Akita, Japan, in 2008

Shuji OGAWA1), Yu YAMAMOTO2), Manabu YAMADA2), Masaji MASE2) and Kikuyasu NAKAMURA2)*

1)Akita Central Livestock Hygiene Service Center, 1–15–5, Terauchiuchihirune, Akita 011–0904 and 2)National Institute of Animal Health, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan

(Received 11 December 2008/Accepted 30 May 2009)

ABSTRACT. Two (1 adult and 1 young bird) of 4 H5N1-highly-pathogenic-avian-influenza (HPAI)-virus-infected whooper swans in Akita, Japan, in 2008 were investigated pathologically. Macroscopically, white spots with hemorrhages were scattered in the pancreas in the adult bird. Histologically, the adult bird had severe necrotizing pancreatitis and mild nonpurulent encephalitis. The young bird had severe nonpurulent encephalitis and nonpurulent enteric ganglionitis, and intestinal venous wall thickening. Virus antigens were detected in the lesions of pancreatitis in the adult bird and of encephalitis in adult and young birds. These findings suggest that the swans died or became moribund due to neurological disorders and necrotizing pancreatitis caused by H5N1 HPAI virus infection. KEY WORDS: avian influenza, pathology, whooper swan. J. Vet. Med. Sci. 71(10): 1377–1380, 2009

Waterfowl are well known reservoirs of avian influenza necropsied. Following a postmortem examination, the liv- virus [10] and are generally infected with the virus without ers, spleen, kidneys, heart, lungs, trachea, gastrointestinal clinical diseases. However, waterfowl (e.g. geese, ducks, tract, pancreas, and brain were removed and then fixed in and swans) infected with H5N1 highly pathogenic avian 20% buffered formalin (Table 1). Unfortunately, the pan- influenza (HPAI) virus were found dead in Hong Kong in creas of the young swan was not collected. All tissue sam- late 2002 [3]. The Asian lineage of H5N1 avian influenza ples were then embedded in paraffin, sectioned at 4 m, and virus, representative of the Qinghai Lake strain [2, 4, 9], was stained with hematoxylin and eosin (HE). prevalent among the waterfowl in China, Mongolia, and Mouse monoclonal antibody specific for type A influenza Siberia. That virus had invaded Europe during 2005 and virus matrix antigen (MCA401, AbD Serotic, MorphoSys 2006, where it infected swans in particular [1, 7, 12]. In UK, Ltd., Oxford, UK) was used as the primary antibody in April 2008, dead and moribund whooper swans (Cygnus the immunoperoxidase technique for detecting HPAI virus cygnus) were observed in Towada Lake of Akita, northern in formalin-fixed, paraffin-embedded sections. A Histofine Japan. H5N1 HPAI virus was isolated from these birds. simple stain MAX-PO (M) kit (Nichirei Inc., Tokyo, Japan) Pathological investigation of the wild birds infected with was used according to the manufacturer's instructions [5]. H5N1 virus is important for evaluating the infectious route After staining, the sections were counter-stained with hema- and mechanism of the virus causing mortality and clinical toxylin. signs. However, there are no reports on the pathology of Macroscopically, white spots with hemorrhages were swans naturally infected with H5N1 virus, except for two observed in the pancreas of the adult swan. No significant case reports of an outbreak among swans naturally infected gross lesions were observed in the young swan. Histologi- with Qinghai Lake lineage H5N1 HPAI virus in Germany cal lesions and distribution of influenza virus in various [12] and Hungary [7] in 2006. This paper describes the organs in the adult bird (Case No. 1) and the young bird pathology of H5N1-HPAI-virus-infected whooper swans (Case No. 2) are listed in Table 1. found dead or moribund in Akita, Japan, in 2008. Histologically, extensive degeneration and necrosis of Four whooper swans were found dead (three adults) or pancreatic exocrine cells (acinar cells) (Fig. 1) with hemor- moribund (one young) in Towada Lake in Akita Prefecture rhages, congestion, and heterophilic infiltration were of north Japan in 2008. Towada Lake is known as a resting observed in the adult bird. Most acinar cells decreased and place for whooper swans coming from Russia from winter lost secretory granules. Mild perivascular cuffing of lym- to spring. The strain of H5N1 HPAI virus (A/whooper phocytes and plasma cells was observed in the cerebrum, swan/Akita/1/2008) was isolated from these samples. The optic lobe, and medulla oblongata. Focal necrosis of paren- molecular biology of the virus strain is reported elsewhere chyma with ventriculitis and meningitis was observed in the [13]. cerebrum. Hyaline substances in the sinusoids of liver, Two of the four birds were examined pathologically. One myocardial degeneration, and mild thickening of blood ves- adult (Case No.1) and one young bird (Case No.2) were sels in the intestine were also observed. In the young bird, an extensive necrotic area was apparent *CORRESPONDENCE TO: NAKAMURA, K., National Institute of Animal Health, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan. in the cerebrum. Ischemic necroses of nerve cells were e-mail: [email protected] observed within the lesions. Perivascular cuffing of lym- 1378 S. OGAWA ET AL.

Table 1. Histological lesions and immunohistochemical detection of influenza virus antigens in the whooper swans Organs Lesions Case No.1 Case No.2 Liver Hyalin in sinusoids +a), A–b) +, A– ,Pe) Spleen –, A– NE Kidney NEc) –, A– Heart Myocardial degeneration +, A+ +, A+, Loss of myocardial fibers, P Lung Congestion NE –, A–, P Trachea NE –, A– Pancreas Necrotizing pancreatitis +++, A+++ NE Brain Nonpurulent encephalitis +, A++ +++, A+++ Cerebrum +, A++ +++, A+++ Cerebellum –, A– ++, A++ Optic lobe +, A– –, A– Medulla oblongata +, A+ ++, A++ Cecum, Enteric ganglionitis –, A–,Vd) +, A+, V, P Small intestine a) Severity of lesions (no, mild, moderate, severe = –, +, ++, +++). b) A=antigens of influenza virus. Severity of antigen distribution (no, mild, moderate, severe =A –, A +, A++, A+++). c) NE = not examined. d) V= venous thickening. e) P = Parasitic infection (schistosome). phocytes and plasma cells was also detected in the cerebrum creatitis, and hepatic necrosis [7]. The present cases had no (Fig. 2). There were necrosis of nerve cells and perivascular hepatic necrosis. Macroscopical and histological lesions of cuffing of lymphocytes and plasma cells in the medulla the present case are similar to those of the cases in Germany oblongata. The nerve cells were necrotic and disappeared in and Hungary except for hepatic necrosis. the granular cell and Purkinje cell layers of the cerebellum. Few reports describe the localization of the virus antigens Degenerative ganglion cells with infiltration of mononu- in the intestinal nerve plexus of birds infected with HPAI clear cells were detected in the submucosal nerve plexus of virus. Brown et al. [1] reported the pathogenicity and virus the cecum and small intestine (Fig. 3). Focal degeneration shedding in four species of swans (black swans, trumpeter of myocardial fibers and focal loss of myocardial fibers swans, whooper swans, and mute swans) and two species of were observed. Hyaline substances were seen in the sinuso- geese inoculated intranasally with HPAI virus (A/whooper ids of liver. Marked thickening of venous walls (Fig. 4) in sean/Mongalia/244/2005). The Qinghai Lake lineage H5N1 the small intestine and cecum was observed with intravascu- HPAI virus was isolated from a dead whooper swan in Mon- lar schistosome-like parasites (Fig. 5). In addition, the same golia in 2005. Brown et al. referred briefly to the virus anti- parasites were detected in the parenchyma of the liver and in gen in the intestinal parasympathetic ganglia of trumpeter the endocardium of the heart, and within the blood vessels swans, whooper swans and mute swans only in the table of of the lung. The parasite eggs were surrounded by pyogran- their report [1]. They also detected virus antigens in the ulation tissues in the liver, heart, and intestines. brain [1]. These findings are interesting. Generally HPAI Immunohistochemically, diffuse influenza virus matrix virus replicates within endothelial cells and spreads via the antigens were found in the degenerative and necrotic pan- vascular or lymphocytic systems to infect and replicate in creatic acinar cells (Fig. 6) of the adult bird, necrotic neu- variety of cell types in various organs [10]. However, there rons (Fig. 7) in the cerebrum and medulla oblongata of adult is possibility that the influenza virus of intestinal nerve and young birds, and degenerative cardiac myocytes of the plexus may have been introduced from brain lesions through heart of adult and young birds. In addition, antigens were the spinal cord or from the intestinal epithelium in the detected in the intestinal submucosal nerve plexus (Fig. 8) swans. of the young bird. Marked thickening of blood vessel walls associated with In the present study, the adult bird had severe necrotizing schistosome infection has been reported in swans [8, 15]. pancreatitis and mild nonpurulent encephalitis. The young Schistosomes migrate through the viscera and can be found bird had severe nonpurulent encephalitis, nonpurulent within mesenteric, renal, cloacal, and portal blood vessels myenteric ganlionitis, and intestinal venous wall thickening [15]. Van Bolhus et al. [15] detected obliterative endophle- with intravenous schistosome flukes. The dead swans (mute bitis with schistosomes in the lumens of veins of intestines swans and whooper swans) in Germany had nonpurulent in five dead mute swans. They speculated that vascular encephalomyelitis, pancreatic necrosis, and hepatic necrosis lesions associated with schistosomes infection contributed with virus antigens [12]. In Hungary, mute swans had non- to the swans’ emaciation and death. Randall and Reece [8] purulent encephalitis, myocardial necrosis, necrotizing pan- also referred to the same lesions, but they believed them to PATHOLOGY OF SWANS BY H5N1 AIV IN JAPAN IN 2008 1379

Fig. 1. Degeneration and necrosis of acinar cells with heterophils in pancreas. Case No.1. HE. Bar=200 m. Fig. 2. Perivascular cuffing of lymphocytes and plasma cells in cerebrum. Case No.2. HE. Bar=200 m. Fig. 3. Degeneration of ganglion cells with lymphocytic infiltration in the submucosal plexus of cecum. Case No.2. HE. Bar=400 m. Fig. 4. Severe thickening of vemous walls in serosa of small intestine. Case No.2. HE. Bar=200 m. Fig. 5. Schistosomatidae flukes in the vein of serosa of small intestine. Case No.2. HE. Bar=200 m. Fig. 6. Virus antigens in the acinar cells of pancreas. Case No.1. Immunoperoxidase staining. Bar=200 m. Fig. 7. Virus antigens in necrotic neurons within the lesions of cerebrum. Case No.2. Immunoperoxidase staining. Bar=400 m. Fig. 8. Virus antigens in the necrotic ganglion cells of cecum. Case No.2. Immunoperoxidase staiming. Bar=400 m. 1380 S. OGAWA ET AL. indicate thickening of arteries. We believe that the venous 5. Nakamura, K., Yamada, M., Yamaguchi, S., Mase, M., Narita, walls thickened, and as a result, hypertrophic veins looked M., Ohyama, T. and Yamada, M. 2001. Proliferation of lung like “arteries”. We often experienced the venous thickening macrophages in acute fatal viral infections in chickens. Avian and schistosomiasis in whooper swans found dead in Japan Dis. 45: 813–818. (unpublished data). Venous thickening with schistosomia- 6. Nakamura, K., Imada, T., Imai, K., Yamamoto, Y., Tanimura, N., Yamada, M., Mase, M., Tsukamoto, K. And Yamaguchi, S. sis of the present cases may induce the blood disturbance 2008. Pathology of specific-pathogen-free chickens inoculated and weakness for the swans. with H5N1 avian influenza viruses isolated in Japan in 2004. Generally, the chickens are more susceptible for HPAI Avian Dis. 52: 8–13. virus than other birds [10]. Severe encephalitis and pancre- 7. 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