Kikuyasu Nakamura

Association of increased pathogenicity of Asian H5N1 highly pathogenic avian influenza viruses in chickens with highly efficient viral replication accompanied by early destruction of innate immune responses.

ABSTRACT The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.

Association between Pathogenicity of Infectious Bursal Disease Virus and Viral Antigen Distribution Detected by Immunohistochemistry

Highly pathogenic infectious bursal disease virus (IBDV) strains (Ehime/91, DV86) and a moderately pathogenic strain (J1) were compared in order to clarify the association between the pathogenicity of IBDV and viral antigen distribution. Virus target cells in the bursa, thymus, spleen, and bone marrow were examined by transmission electron microscopy. Although all strains caused similar bursal atrophy, the highly pathogenic strains brought about a greater decrease in the thymic weight index and more severe lesions in the cecal tonsil, thymus, spleen, and bone marrow. Immunohistochemical detection of IBDV antigen in tissues from chickens infected with Ehime/91 and DV86 strains showed a higher frequency of antigen-positive cells in the spleen and bone marrow. Transmission electron microscopy indicated the presence of viral particles in the cytoplasm of epithelial reticular cells in the thymus and monocytes in the bone marrow. The results show that pathogenicity of field strains of IBDV correlates with lesion production in non-bursal lymphopoietic organs. The results also suggest that pathogenicity of IBDV may be associated with virus antigen distribution in non-bursal lymphopoietic organs.

Comparative study of respiratory lesions in two chicken lines of different susceptibility infected with infectious bronchitis virus: histology, ultrastructure and immunohistochemistry.

Infectious bronchitis virus (IBV) was inoculated intranasally into line C and line 151 chickens, which were resistant and highly susceptible, respectively, to IBV infection, and the resulting respiratory lesions compared. Histologically, damage to the mucociliary system of the respiratory tract in line 151 persisted longer than in line C. Using the avidin-biotin-peroxidase complex method, IBV antigens were detected in histological sections of the tracheas of line 151 for a longer time than in line C. Ultrastructural studies confirmed the histological findings. Immuno-globulin G-, IgM- and IgA-containing cells were seen in the lamina propria and between epithelial cells in the trachea of both lines from at least 7 to 12 days after IBV inoculation but in greater numbers in line 151.

Highly pathogenic avian influenza virus (H5N1) isolated from whooper swans, Japan.

On April 21, 2008, four whooper swans were found dead at Lake Towada, Akita prefecture, Japan. Highly pathogenic avian influenza virus of the H5N1 subtype was isolated from specimens of the affected birds. The hemagglutinin (HA) gene of the isolate belongs to clade 2.3.2 in the HA phylogenetic tree.

Pathology of Fatal Highly Pathogenic H5N1 Avian Influenza Virus Infection in Large-billed Crows (Corvus macrorhynchos) during the 2004 Outbreak in Japan

Highly pathogenic H5N1 avian influenza viruses were isolated in 9 large-billed crows that died in Kyoto and Osaka prefectures in Japan from March to April in 2004. We studied 3 of the 9 crows using standard histologic methods, immunohistochemistry, and virus isolation. The most prominent lesions were gross patchy areas of reddish discoloration in the pancreas. The consistent histologic lesions included severe multifocal necrotizing pancreatitis, focal degeneration and necrosis of neuron and glial cells in the central nervous system, and focal degeneration of cardiac myocytes. All of these tissues contained immunohistochemically positive influenza viral antigens. The virus was isolated from the brain, lung, heart, liver, spleen, and kidney of the crows examined. Thus we concluded that highly pathogenic avian influenza virus was associated with clinical disease, severe pathologic changes, and death in the 3 crows.

Pathologic study of specific-pathogen-free chicks and hens inoculated with adenovirus isolated from hydropericardium syndrome.

The mortality and pathology caused by serotype 4 adenovirus, isolated from chickens with hydropericardium syndrome (HPS) in Japan, was investigated in specific-pathogen-free (SPF) chickens. One-day-old to 15-mo-old SPF chickens were inoculated intramuscularly, orally, and intranasally with liver homogenates from HPS chickens or isolated serotype 4 adenovirus. There were no clinical signs before death. The mortality rate in all groups of 1-day-old chicks was 100%, irrespective of the inoculum or inoculation route. Four-week-old chickens inoculated with liver homogenate also had a 100% mortality rate. Five-week-old chickens inoculated with cell culture of HPS adenovirus had a 40% mortality rate. The mortality rates of 7-mo-old hens inoculated with liver homogenates intramuscularly and orally were 75% and 25%, respectively. In 15-mo-old hens inoculated with liver homogenates intramuscularly, the mortality rate was 70%. Gross lesions were hydropericardium and swelling and congestion of the liver with occasional petechial hemorrhages. Histologically, the liver had diffuse or multifocal hepatic necrosis and hemorrhage with intranuclear inclusion bodies noted within hepatocytes. In the spleen, macrophages containing erythrocytes and yellow pigment were prominent in the red pulp. In the lung, a moderate diffuse macrophage infiltration was noted throughout the lung parenchyma, and these macrophages contained yellow pigment. In the pancreas of the chicks inoculated at 1 day old, there was multifocal necrosis of glands with intranuclear inclusion bodies. Intranuclear inclusion bodies were seen also in the gizzard, proventriculus, duodenum, cecum, kidney, and lung of the chicks inoculated at 1 day old. Immunohistochemically, the intranuclear inclusion bodies of various organs showed positive reactions against group I avian adenovirus. Adenovirus was recovered from the liver of chickens with HPS. This study indicates that HPS adenovirus is able to reproduce HPS lesions and mortality in SPF chicks and even adult chickens and that it is a highly pathogenic strain.

Histology, immunohistochemistry, and ultrastructure of hydropericardium syndrome in adult broiler breeders and broiler chicks.

Ten 250-day-old broiler breeders, seven 16-day-old broiler chicks, and three 25-day-old broiler chicks suffering from hydropericardium syndrome (HPS) in Japan were examined histologically, immunohistochemically, and ultrastructurally. Clinically, the chickens died suddenly without apparent signs. The mortality rates were 6.4%, 20.2%, and 26.1%, respectively. The common characteristic histologic lesion was necrosis of hepatocytes, accompanied by intranuclear inclusions of hepatocytes and hemorrhages. In the spleen, there were activation of macrophages in splenic sinus and ellipsoids and erythrophagocytosis in the splenic sinus. The interlobular interstitium of the lung showed marked edema. The air and blood capillary areas of parabronchi included many macrophages with yellow pigments. With immunoperoxidase staining, intranuclear inclusion bodies within degenerating hepatocytes stained positively for group I adenovirus antigen. Ultrastructurally, numerous viral particles (65-70 nm in diameter) were demonstrated in the intranuclear inclusions of hepatocytes. Group I adenovirus (serotype 4) was isolated from liver samples of adult broiler breeders and broiler chicks with HPS. This study suggests that HPS may be caused by group I adenovirus.

Avian influenza virus (H5N1) replication in feathers of domestic waterfowl.

We examined feathers of domestic ducks and geese inoculated with 2 different avian influenza virus (H5N1) genotypes. Together with virus isolation from the skin, the detection of viral antigens and ultrastructural observation of the virions in the feather epidermis raise the possibility of feathers as sources of infection.

Escherichia coli multiplication and lesions in the respiratory tract of chickens inoculated with infectious bronchitis virus and/or E. coli.

Escherichia coli numbers and histopathological changes were studied in the respiratory tract of line 151 chickens intranasally inoculated with infectious bronchitis virus (IBV) and/or virulent E. coli; this line is highly susceptible to IBV. Chickens inoculated with IBV alone showed increased numbers of E. coli in the trachea and had tracheitis, airsacculitis, and bronchiolitis. One of 17 chickens inoculated with IBV alone died with fibrinopurulent serositis. Chickens inoculated with IBV and E. coli had more severe and persistent respiratory lesions than those inoculated with IBV alone. E. coli was isolated from tracheas of chickens inoculated with IBV and E. coli more frequently than from chickens inoculated with IBV alone. In this group, 14 of 27 chickens died with tracheal plugs or with fibrinopurulent serositis. There was neither increased numbers of E. coli nor significant lesions in the respiratory tract of the group inoculated with E. coli alone. These results suggest that IBV may facilitate E. coli invasion into the lower respiratory tract of the chicken.

Pathology of Spontaneous Colibacillosis in a Broiler Flock

Forty-eight of 134 chickens collected from a flock on a broiler farm were diagnosed pathologically and microbiologically to have colibacillosis. Both acute septicemia (seven birds, 1 to 36 days old) and subacute serositis (41 birds, 5 to 57 days old) were found. The former consisted of necrosis with fibrinous exudates in the ellipsoids and lymphoid follicles of the spleen, and fibrinous thrombi in sinusoids of the liver with occasional necrosis of hepatic cells. The latter had fibrinopurulent inflammation with granulomatous changes in the serosal tissues—including the epicardium, pericardium, and hepatic peritoneal sac—accompanied by septicemic lesions in the spleen and liver. Respiratory lesions (airsacculitis, pneumonia, and tracheitis) were noted in most chickens affected with acute septicemia and subacute serositis. Degenerative changes also were observed in the bursa of Fabricius.