Unusually High Mortality in Waterfowl Caused by Highly Pathogenic Avian Influenza A(H5N1) in Bangladesh

Abstract

Mortality in ducks and geese caused by highly pathogenic avian influenza A(H5N1) infection had not been previously identified in Bangladesh. In June-July 2011, we investigated mortality in ducks, geese and chickens with suspected H5N1 infection in a north-eastern district of the country to identify the aetiologic agent and extent of the outbreak and identify possible associated human infections. We surveyed households and farms with affected poultry flocks in six villages in Netrokona district and collected cloacal and oropharyngeal swabs from sick birds and tissue samples from dead poultry. We conducted a survey in three of these villages to identify suspected human influenza-like illness cases and collected nasopharyngeal and throat swabs. We tested all swabs by real-time RT-PCR, sequenced cultured viruses, and examined tissue samples by histopathology and immunohistochemistry to detect and characterize influenza virus infection. In the six villages, among the 240 surveyed households and 11 small-scale farms, 61% (1789/2930) of chickens, 47% (4816/10 184) of ducks and 73% (358/493) of geese died within 14 days preceding the investigation. Of 70 sick poultry swabbed, 80% (56/70) had detectable RNA for influenza A/H5, including 89% (49/55) of ducks, 40% (2/5) of geese and 50% (5/10) of chickens. We isolated virus from six of 25 samples; sequence analysis of the hemagglutinin and neuraminidase gene of these six isolates indicated clade 2.3.2.1a of H5N1 virus. Histopathological changes and immunohistochemistry staining of avian influenza viral antigens were recognized in the brain, pancreas and intestines of ducks and chickens. We identified ten human cases showing signs compatible with influenza-like illness; four were positive for influenza A/H3; however, none were positive for influenza A/H5. The recently introduced H5N1 clade 2.3.2.1a virus caused unusually high mortality in ducks and geese. Heightened surveillance in poultry is warranted to guide appropriate diagnostic testing and detect novel influenza strains.

Keywords: Bangladesh; H5N1 subtype; clade 2.3.2.1a; duck; goose; histopathology; immunohistochemistry; influenza A virus; outbreaks.

Fig. 1

Location of villages affected with highly pathogenic avian influenza A(H5N1) virus and dates of onset of illness in ducks and geese in subdistricts of Netrokona District, Bangladesh, June–July 2011. Different colours within the district map indicate boundaries of subdistricts.

Fig. 2

Photographs of a duck with severe twisting of the head and neck (torticollis), Netrokona, Bangladesh, June–July 2011.

Fig. 3

Phylogeny of hemagglutinin (HA) gene sequences of HPAI H5N1 viruses collected from waterfowl experiencing unusually high mortality in Netrokona, Bangladesh during June–July 2011. Viruses detected in Bangladesh during this outbreak investigation are denoted by a triangle in clade 2.3.2.1a. Collapsed branches indicate other large clades of H5N1 virus with strain names not shown. HA clades are indicated by brackets. The tree was calculated using the neighbour-joining method with the Kimura 2-parameter distance model.

Fig. 4

Haematoxylin- and eosin-stained sections of the brain and pancreas of ducks and of the intestine of chickens infected with avian influenza A (H5N1) virus in Netrokona, 2011. (a) Brain – mild perivascular cuffing and increased glial cells. (b) Pancreas – acinar cell necrosis (c) Intestine – increased cellularity of the lamina propria in the villi.

Fig. 5

Immunohistochemistry staining for viral nucleoprotein (brown) of brain, pancreas and intestine of ducks infected with avian influenza A (H5N1) virus in Netrokona, Bangladesh, 2011. (a) Brain: moderate amount of nucleoprotein of avian influenza (AI) in the nucleus and cytoplasm of neurons (large arrow) and glial cells (small arrow). (b) Pancreas: large amount of AI antigen in the nucleus and cytoplasm of acinar cells (small arrow) and a few mononuclear inflammatory cells (large arrow) (c) Intestine: large amount of nucleoprotein of AI antigen in the nucleus and cytoplasm of subserosal and submucosal ganglia (arrows, myenteric plexus).