ORIGINAL ARTICLE

Investigating a crow die-off in January–February 2011during the introduction of a new clade of highly pathogenic avianinfluenza virus H5N1 into Bangladesh

Salah Uddin Khan • LaShondra Berman • Najmul Haider • Nancy Gerloff • Md Z. Rahman •

Bo Shu • Mustafizur Rahman • Tapan Kumar Dey • Todd C. Davis • Bidhan Chandra Das •

Amanda Balish • Ausraful Islam • Jens P. Teifke • Nord Zeidner • Steven Lindstrom •

Alexander Klimov • Ruben O. Donis • Stephen P. Luby • H. L. Shivaprasad • Andrea B. Mikolon

Received: 15 April 2013 / Accepted: 28 July 2013 / Published online: 1 October 2013

� Springer-Verlag Wien 2013

Abstract We investigated unusual crow mortality in

Bangladesh during January-February 2011 at two sites.

Crows of two species, Corvus splendens and C. macro-

rhynchos, were found sick and dead during the outbreaks.

In selected crow roosts, morbidity was *1 % and mor-

tality was *4 % during the investigation. Highly patho-

genic avian influenza virus H5N1 clade 2.3.2.1 was

isolated from dead crows. All isolates were closely related

to A/duck/India/02CA10/2011 (H5N1) with 99.8 % and

A/crow/Bangladesh/11rs1984-15/2011 (H5N1) virus with

99 % nucleotide sequence identity in their HA genes. The

phylogenetic cluster of Bangladesh viruses suggested a

common ancestor with viruses found in poultry from India,

Myanmar and Nepal. Histopathological changes and

immunohistochemistry staining in brain, pancreas, liver,

heart, kidney, bursa of Fabricius, rectum, and cloaca were

consistent with influenza virus infection. Through our

limited investigation in domesticated birds near the crow

roosts, we did not identify any samples that tested positive

for influenza virus A/H5N1. However, environmental

samples collected from live-bird markets near an outbreak

site during the month of the outbreaks tested very weakly

positive for influenza virus A/H5N1 in clade 2.3.2.1-spe-

cific rRT-PCR. Continuation of surveillance in wild and

domestic birds may identify evolution of new avian influ-

enza virus and associated public-health risks.

Introduction

Bangladesh has reported highly pathogenic avian influenza

virus A (HPAI) H5N1 in domestic poultry since February

2007 [1]. From 2007 through 2011, 514 outbreaks were

reported in 51 out of 64 districts [2]. The hemagglutinin (HA)

of all H5N1 influenza A viruses isolated from the 2007-2009

outbreaks belonged to clade 2.2.2, with very limited (\5 %)

divergence in their genetic makeup [3]. HPAI H5N1 was not

reported in wild birds during this period.

S. U. Khan (&) � N. Haider � M. Z. Rahman � M. Rahman �A. Islam � N. Zeidner � S. P. Luby � A. B. Mikolon

CCD, International Centre for Diarrhoeal Disease Research,

Bangladesh (ICDDR, B), Dhaka, Bangladesh

e-mail: sukhanbd@gmail.com; khansu@icddrb.org

URL: www.icddrb.org

S. U. Khan

Department of Environmental and Global Health, College of

Public Health and Health Professions, University of Florida,

Gainesville, FL, USA

L. Berman � N. Gerloff � B. Shu � T. C. Davis � A. Balish �N. Zeidner � S. Lindstrom � A. Klimov �R. O. Donis � S. P. Luby � A. B. Mikolon

Centers for Disease Control and Prevention, (CDC), Atlanta,

GA, USA

T. K. Dey

Forest Department, Ministry of Environment and Forest, Dhaka,

Bangladesh

B. C. Das

Department of Livestock Services, Ministry of Fisheries and

Livestock, Dhaka, Bangladesh

J. P. Teifke

Friedrich-Loeffler-Institut (FLI), Federal Research Institute for

Animal Health, Greifswald-Insel Riems, Germany

H. L. Shivaprasad

California Animal Health and Food Safety Laboratory System,

Tulare branch, University of California, Davis,

Tulare, CA, USA

123

Arch Virol (2014) 159:509–518

DOI 10.1007/s00705-013-1842-0

In January-February 2011, Bangladesh reported wide-

spread outbreaks of HPAI A/H5N1 in domestic poultry [4].

During the same period, the Forest Department and the

Department of Livestock Services of Bangladesh received

multiple reports of crow die-offs from at least two

administrative divisions of the country (personal commu-

nication, Department of Livestock Services, Bangladesh)

[5]. As a part of outbreak response, members from the

Department of Livestock Services, Forest Department of

Bangladesh, International Centre for Diarrhoeal Disease

Research, Bangladesh (icddr,b), and Centers for Disease

Control and Prevention (CDC), Atlanta, conducted field

and laboratory investigations. We report here the results of

outbreak investigations conducted to determine the cause

of crow die-offs and subsequent studies to determine the

source of H5N1 infection.

Materials and methods

Outbreak settings

We investigated die-offs in two crow roosts from 17 to 23

January 2011 in Patuakhali (N22.35944 E90.34912), a

small town in southern Bangladesh, and from 13 to 15

February 2011 in the capital city of Dhaka (N23.73989

E90.39829). The sites are 153 kilometers apart. In Patu-

akhali, the crows roosted in orchards (approximately 200

trees) surrounded by dense human settlement, whereas in

Dhaka, a city with a population density of 35,000/square

kilometer, the crows roosted in a park (approximately

3000-5000 trees) [6].

Data and sample collection procedures

The field team consisted of veterinarians and veterinary

technicians from the Department of Livestock Services,

Forest Department, and icddr,b. We used the total count

method to estimate the number of healthy, sick, and dead

crows in both roosts at two time points [7]. We recorded

detailed clinical presentations of a subset of sick crows

found at the Patuakhali outbreak site three times a day for

two consecutive days. All of the sick crows that we

included in the clinical observation had fallen from their

roosts and were unable to fly. These crows were a subset of

the ‘‘sick crows’’ that were identified during the investi-

gation. The field investigation team kept the sick crows in

an enclosed place under the crow roost, providing adequate

food and water. However, the sick crows were free to roam

within the orchard and none of them were sacrificed. The

orchard where the crows roosted was in the backyard of a

household fenced by a brick wall that made the place

inaccessible to the neighbors. The household members

rarely entered their backyard. We also interviewed the

people whose dwellings were under the crow roost in Pa-

tuakhali to estimate frequencies of crow deaths and history

of poultry die-offs in their backyards during the outbreak.

We collected oropharyngeal and cloacal swab samples

from dead and sick crows found under the roosts and

pooled them in viral transport medium, one pool per crow.

We kept two carcasses of crows that had recently died for

necropsy and histopathology. We also collected cloacal

swab samples from three domestic ducks raised by

household members living under the crow roost in Patu-

akhali. At the same time, we collected environmental swab

samples from live-bird markets located nearest to the out-

break sites to investigate the spread of the virus to domestic

poultry: one market in Patuakhali in January 2011 and two

in Dhaka during February 2011. These markets were within

three kilometers of the outbreak sites and sold about 200 to

2000 birds per day, including native chickens, commer-

cially raised chickens, ducks, geese, and pigeons. Sampling

at each of the markets included swabs (n=10) from poultry

cages, feed and water trays, and fecal droppings from

poultry stalls. Swabs were pooled into one environmental

sample per market.

We conducted a follow-up investigation in wild birds

around the Patuakhali crow roost on 7-8 February 2011,

two weeks after the primary investigation. The team col-

lected oropharyngeal and cloacal swabs from five crows

lying on the ground under the roost and unable to fly. We

caught wild birds around the crow roost using mist nets in

their flyways [8], banded them, and released them at the

same location where they were captured, after collecting

oropharyngeal and cloacal swabs.

During clinical observation and sampling, the team used

appropriate personal protective equipment, including

gloves, goggles, and face masks, and they made regular use

of disinfection.

Virus isolation, sequencing, and phylogeny

We performed real-time reverse transcription polymerase

chain reaction (rRT-PCR) to identify the influenza A virus

targeting the matrix (M) gene and the HA for subtyping and

clade-specific rRT-PCR as described previously [9]. Virus

culture and isolation in eggs, RNA extraction and sequencing

procedures were performed as described previously [10]. We

performed phylogenetic analysis as described elsewhere [11].

Briefly, complete HA sequences were aligned to a subset of

reference sequences representing currently circulating South

East Asian H5 clades using BioEdit [12], and a neighbor-

joining phylogenetic tree with 1000 bootstrap replicates was

calculated with MEGA version 4 [13]. HA sequences were

submitted to GISAID (http://platform.gisaid.org) with

accession numbers: EPI448167, EPI448159, EPI448151,

510 S. U. Khan et al.

123

EPI448063, EPI448055, EPI448047, EPI465423,

EPI448031, EPI353381, and EPI353364.

Histopathology

We performed histopathology and immunohistochemistry

on two bird samples collected during the outbreak at the

Patuakhali site. Tissue samples were collected and fixed in

10 % buffered neutral formalin. The tissues were trimmed,

processed, embedded in paraffin, sectioned at 4 microme-

ters, stained with haematoxylin and eosin (H&E), and

examined by bright field microscopy.

Immunohistochemistry (IHC)

To study the distribution of nucleoprotein (NP) of AI virus

in various organs and cells, IHC was performed on selected

tissues according to a method described elsewhere [14].

Briefly, paraffin sections were mounted on positively

charged Super Frost Plus microscopic slides, dewaxed, and

rehydrated. The sections were pretreated with 10 mM citric

acid, pH 6.0, for 10 min at 110 �C in a decloaking chamber

(Biocare Medical, Concord, CA) and subsequently incu-

bated with the rabbit anti-NP serum, diluted 1:750 in Tris-

buffered saline. Tissue sections were stained using the

avidin-biotin-peroxidase complex method, with 3-amino-9-

ethylcarbazole (red), and with hematoxylin (blue) accord-

ing to the method by Klopfleisch et al. [14].

Ethical considerations

This investigation was part of an outbreak response to a

possible public-health threat and was not considered

human subject research. However, before initiating field

investigation and sampling, the team obtained informed

consent from the property owners where the crows roosted,

owners of the individual birds that were sampled, and the

live-bird market authorities where we performed environ-

mental sampling. We collected all of the dead crows found

under the roost in biohazard bags during the investigation

and incinerated them at icddr,b or at the local livestock

hospital facility. During the investigation, we requested the

inhabitants close to the crow roost not to come into contact

with dead or sick crows. The local livestock and forest

department officials regularly disinfected the surface

beneath the crow roost at both sites. The crow samples

were handled at biosafety level (BSL) II for initial

screening by rRT-PCT, and they were later transferred to

BSL III laboratory facilities for virus culture. The protocols

for sampling domestic and wild birds and collecting envi-

ronmental samples were approved by the Institutional

Review Boards of the International Centre for Diarrhoeal

Disease Research, Bangladesh.

Results

Morbidity and mortality in crows

The Patuakhali outbreak occurred at a crow roost with an

estimated population of 1,500 birds. Seventeen (1 %) of

the birds showed clinical signs of illness, and 57 (4 %)

were found dead during the one-week investigation. The

Patuakhali residents dwelling under tree canopies with

roosting crows first noticed unusually high crow mortality

in the first week of January 2011. The crow deaths con-

tinued for approximately one month. The Dhaka city out-

break involved about 900 crows that roosted in the trees of

a city park. Of these, five (0.6 %) appeared sick, and 39

(4 %) were found dead during the investigation. In the

Dhaka city outbreak site, crow deaths were first reported

during the first week of February and continued until mid-

March 2011. We identified two species of crows, house

crows (Corvus splendens) and large-billed crows (Corvus

macrorhynchos); once the crows became ill, they were

found dead within 24 hours.

Clinical features in crows

In Patuakhali, the seven sick crows we observed in cap-

tivity for two days were unable to fly, appeared drowsy,

had dilated pupils, and had whitish fecal material attached

to the plumage surrounding the cloaca. The birds showed

neurologic signs, including uncoordinated gait, tremors,

pronounced opisthotonos, and torticollis at the terminal

stage (Fig. 1). We observed two (29 %) crows with dis-

charges from the nares during the later stage of clinical

illness. The field team observed cannibalism and carcass

scavenging among the crows (Fig. 1). The average cloacal

temperature of the sick or moribund crows was 100.3 �F

(range: 94 �F to 107 �F) (37.94 �C; range: 34.4–41.7 �C).

Phylogeny of H5N1viruses from crows

We collected 31 pooled oropharyngeal-cloacal swabs: 11

from C. splendens and 20 from C. macrorhynchos from the

two outbreak sites. All tested positive for avian influenza

virus A/H5 by rRT-PCR. Virus was successfully isolated

from 8 out of 31 samples (C. splendens. n=4, and C.

macrorhynchos, n=4), including samples from Patuakhali

(n=4) and from Dhaka (n=4). Sequence analysis of the HA

and NA genes indicated that the eight isolates were influ-

enza virus A/H5N1, and the HA contained the multibasic

cleavage site motif (PQRERRRKR*G) characteristic of

HPAI. Preliminary phylogenetic analysis showed that these

viruses were almost identical to each other in their HA

gene sequences and were most closely related to viruses

with clade 2.3.2.1 HA genes (Fig. 2). All isolates were

Highly pathogenic avian H5N1 influenza virus in Bangladesh 511

123

closely related to A/duck/India/02CA10/2011 (H5N1) and

A/crow/Bangladesh/11rs1984-15/2011(H5N1) virus, with

99.8 % and 99 % nucleotide sequence identity, respec-

tively, in their HA genes [15, 16]. Although virus culture

for the remaining samples was not successful, HA and NA

genes were sequenced using RNA extracted from the

specimen, and phylogenetic analysis revealed that these

viruses also belonged to clade 2.3.2.1.

Dispersion of H5N1 to the domestic poultry and poultry

marketing chain

We identified 12 households with 80 backyard poultry

living under the crow roost in Patuakhali. The household

members reported that 43 (54 %) of their poultry became

sick and 28 (35 %) died during 12 to 22 January 2011.

However, during our investigation, the backyard poultry in

those households did not show clinical signs of illness, and

none were found dead. Three ducks were available during

the investigation, and cloacal samples from all of them

tested negative for influenza virus A by rRT-PCR. The

epidemiologic investigation did not identify additional

domestic or wild birds with clinical signs of disease in

areas surrounding crow roosts in Patuakhali or Dhaka

during the outbreak investigations.

Environmental swab samples collected from one live-

bird market from Patuakhali city during the month of the

outbreaks tested very weakly positive for influenza virus

A/H5N1 by clade-2.3.2.1-specific rRT-PCR. Partial HA

gene sequence data from the two Dhaka market samples

also indicated closest relationship to clade 2.2.2; however,

we failed to isolate virus from any of the environmental

samples from the Patuakhali and Dhaka live-bird market

samples.

Follow-up investigation in wild birds in Patuakhali

During the follow-up investigation on 7-8 February 2011 in

Patuakhali, the team visited the outbreak site. They cap-

tured two large-billed crows (C. macrorhynchos) and three

house crows (C. splendens) under the crow roost. These

birds appeared sick and were unable to fly; we detected

influenza virus A/H5N1 clade 2.3.2.1 from oropharyngeal

and cloacal swabs by rRT-PCR. Additionally, the team

captured 30 wild birds of five different species near the

crow roost: house crows (C. splendens, n=2), pintail snipes

(Gallinago stenura, n=7), greater painted snipes (Rostra-

tula benghalensis, n=12), Asian pied starling (Sturnus

contra, n=8), and a barn owl (Tyto alba, n=1). Of birds,

only the barn owl was apparently sick and unable to fly.

Oropharyngeal and cloacal swabs samples from all of these

apparently healthy birds and the barn owl tested negative

for influenza virus A by rRT-PCR.

Histopathology

Lesions in various organs of both birds were similar, with

minor variation between the two. In the brain, significant

lesions were confined to the cerebellum, brain stem and

medulla oblongata. Multifocal mild to severe enlargement,

degeneration and necrosis (characterized by red neurons)

and central chromatolysis of neurons including Purkinje

cells was observed. Some of the nuclei within the neurons

appeared enlarged and red, with homogenous composition

(Fig. 3a). Both cerebrum and cerebellum had mild multi-

focal increased glial cells, satellitosis, and neuronophagia

in the grey matter. Occasional perivascular cuffing and

mild hemorrhages scattered within the neuropil were

observed. The meninges were occasionally thickened due

to infiltration of lymphocytes.

Fig. 1 Sick and dead crows found under the roost in Patuakhali and

Dhaka city during January-February 2011. a A sick crow with ataxic

limbs found in the Dhaka city park, February 2011. b An apparently

healthy crow pecking on a sick crow under the roost in the Dhaka city

park, February 2011

512 S. U. Khan et al.

123

In the pancreas, severe multifocal foci of acute coagu-

lative necrosis of acinar cells were noted, with minimal

inflammation (Fig. 3b). The liver had acute mild multifocal

coagulative necrosis of hepatocytes accompanied by mild

infiltration of lymphocytes and macrophages (Fig. 3c). The

spleen had an increased number of mononuclear cells,

including plasma cells, as well as mild hemosiderosis.

Kidneys had mild multifocal acute necrosis of tubular

epithelial cells with no associated inflammation. The heart

had mild multifocal degeneration of myofibers with no

inflammation. An occasional segment of intestine demon-

strated mild necrosis of lymphocytes within Peyer’s

patches.

In one bird, the rectum/cloaca had increased cellularity

of the lamina propria, and the bursa of Fabricius had

changes ranging from cystic follicles to severe lymphoid

depletion.

Other organs, including the eyes, lungs, trachea, phar-

ynx, proventriculus, gizzard, intestine, gall bladder, testes

and skeletal muscles, did not have any significant lesions.

Immunohistochemistry (IHC)

IHC directed against the nucleoprotein (NP) of avian

influenza virus stained both the nucleus and cytoplasm in

various tests, and the results are summarized in Table 1.

Fig. 2 Phylogeny of HA gene

sequences of H5N1 (HPAI)

viruses, with the Bangladesh

viruses indicated in boldface.

Viruses that circulate in other

regions of South East Asia are

included and shown as

collapsed branches (triangles)

labeled with clade names (e.g.,

2.2.2). Clade 2.3.2.1 and the

lineages are indicated behind

the bracket. The tree was

calculated using the neighbor-

joining method with the Kimura

2-parameter distance model.

Numbers above nodes indicate

bootstrap values ([70 %) and

the tree was rooted at the

midpoint

Highly pathogenic avian H5N1 influenza virus in Bangladesh 513

123

Within the brain, neurons, including axons/dendrites,

stained well (Fig. 4a). Other cells such as glia, ependymal

and meningeal cells had scattered staining. Acinar cells in

the pancreas (Fig. 4b), hepatocytes in the liver (Fig. 4c),

myocardium of the heart (Fig. 5a), epithelial cells of the

bursa of Fabricius (Fig. 5b), and enterocytes of the rectum

and cloaca exhibited positive staining. Other cells such as

renal tubular epithelial cells of the kidney and epithelia

cells of the trachea also showed positive staining (data not

shown). Other organs on which IHC was performed,

including pharynx, lungs, proventriculus gizzard and

intestine, did not show staining for AI antigen.

Discussion

The H5N1 viruses identified in poultry from 2007 to early

2009 in Bangladesh belonged to clade 2.2.2 and circulated

in commercial and backyard poultry [3, 17]. Recent reports

indicated that clade 2.3.4.2 viruses are also circulating in

Bangladesh, but no die-offs in wild birds due to any of

these viruses had been reported [15, 18]. The high mor-

tality in crows caused by the newly introduced H5N1 clade

2.3.2.1 may indicate that members of this clade differ in

their virulence and host range, and crows may have

increased susceptibility to these viruses. H5N1 viruses with

Fig. 3 Histopathologic changes in the brain, pancreas, and liver of

crows infected with influenza virus A/H5N1 viruses in Bangladesh in

2011 (haematoxylin and eosin staining). a Brain: Neurons with red

and swollen nuclei and a mild spongy change in the neuropil and

increased numbers of glial cells due to avian influenza virus infection.

b Pancreas: Acute focal severe necrosis of acinar cells with minimal

mononuclear cell inflammation. c Liver: Acute focal necrosis of

hepatocytes with minimal inflammation

Table 1 Intensity of immunohistochemistry staining in various organs in the two dead crows from Bangladesh, 2011

Bird ID Brain Pancreas Liver Heart Kidney Trachea Rectum/Cloaca Bursa of Fabricius

3146 ??? ?? ?? ?? ? ? NE NE

3148 ? ??? ? ? ? ? ??? ???

? mild, ?? moderate, ??? severe. NE, not examined

Fig. 4 Immunohistochemistry staining of brain, pancreas, and liver

of crows infected with influenza A/H5N1 viruses in Bangladesh in

2011. a Brain: Large amounts of nucleoprotein of AI antigen in

neurons and glial cells and in a few axons and dendrites. b Pancreas:

Nucleoprotein of AI antigen in both the nuclei and cytoplasm of most

of the acinar cells. c Liver: Moderate amount of nucleoprotein of AI

antigen in the nucleus and cytoplasm of primarily hepatocytes

514 S. U. Khan et al.

123

clade 2.3.2.1 HA were first detected in a common magpie

in Hong Kong SAR in 2007 [19, 20]. Since then, and

especially in the past 3 years, viruses from this clade have

spread to Asia and Europe, including China, Japan, Mon-

golia, Vietnam, the Republic of Korea, Myanmar, India,

Nepal, the Russian Federation, Bulgaria, and Romania [16,

19–26]. Although the majority of clade 2.3.2.1 viruses that

were reported were in domestic poultry, seven out of ele-

ven countries reported this clade in wild birds [3]. In 2010,

Nepal was the first country in the South Asian region to

identify this clade in domestic poultry [18]. Although the

H5N1 clade 2.3.2.1 viruses were found to cause outbreaks

in India’s poultry population about a month after the out-

break in Bangladesh, the viruses were almost identical

[16]. This indicates the continuing emergence of this par-

ticular clade of H5N1 virus, causing rapid spread from one

location to another, as seen in other parts of the world [23].

Particularly in Southeast Asia, national and international

poultry trade and migratory wild birds are commonly

implicated in cross-border transmission of the virus, which

reemphasizes the need for continuation of wild and

domestic bird surveillance for influenza viruses [27].

Although there are several reports of HPAI H5N1

pathology in poultry, there has been only one report on

crows (Corvus macrorhynchos) [28]. Our findings suggest

that the lesions in the brain, pancreas, liver and other

organs of the crows were caused by HPAI virus, as con-

firmed by immunohistochemistry. Moreover, there was

good correlation between histopathology and IHC. Lesions

in the brain and pancreas were quite severe, and mild to

moderate in other organs. These lesions would explain the

deaths of the two crows. Both the histopathologic lesions

Fig. 5 Immunohistochemistry

staining of heart and bursa of

Fabricius of crows infected with

influenza A/H5N1 viruses in

Bangladesh in 2011. a Heart:

Large amounts of nucleoprotein

of AI antigen, primarily in the

sarcoplasm and in some nuclei

of cardiac myocytes. b Bursa of

Fabricius: Abundant

nucleoprotein of AI antigen in

the nucleus and cytoplasm of

epithelial cells

Highly pathogenic avian H5N1 influenza virus in Bangladesh 515

123

and IHC seen in various organs of these crows in our study

are in good agreement with the report of Tanimura et al.

[28].

Viruses from clade 2.3.2.1 diverged into three distinct

groups, referred to as Hubei-like (A/Hubei/1/2010),

including strains circulating in South East Asian countries

such as Vietnam, Hong Kong/6841-like (A/Hong Kong/

6841/2010) strains detected in Japan, Korea, Russia and

primarily other Far Eastern countries, and barn swallow-

like (A/barn swallow/Hong Kong/1161/2010) strains,

including recent viruses from Hong Kong and Vietnam

(Fig. 2) [18, 29]. The clade 2.3.2.1 viruses identified in

crows during investigations in Bangladesh were most clo-

sely related to the Hubei-like clade 2.3.2.1 viruses found in

India in 2011 [16], Nepal in 2011, and Myanmar in 2010

and 2011 [18, 20, 30]. Bangladesh viruses from the Hubei-

like cluster were highly similar in their HA sequences and

were derived from both poultry and crow outbreaks [18]

(Fig. 2). The phylogenetic cluster of Bangladesh viruses

with viruses from neighboring countries suggested a

common ancestor with Hubei-like viruses found in poultry

from India, Myanmar, and Nepal and in wild birds (crows)

in Nepal [14, 18, 20, 30].

The rapid spread of HPAI H5N1 clade 2.3.2.1 has been

associated with illness in both domestic and wild birds

[24]. Our findings also suggest that once a crow popula-

tion in a roost becomes infected, the virus continues to

circulate among crows for a period of months. The most

likely way for virus to be transmitted between the crows

could be cannibalism and close contact with the ill crows.

The mortality rate in crows was very high, in agreement

with previous reports [31]. During January-April 2011,

Bangladesh reported 159 outbreaks of H5N1 in domestic

poultry, mostly in commercial farms [4]. Live-bird mar-

kets are considered high-risk sites for harboring influenza

viruses, and the poultry marketing chain may lead to

spread to a wider geographic area in the country [32].

Although we could not prove that birds from live-bird

markets near the crow roosts were infected with the same

clade of influenza virus, the possibility of horizontal

transmission of the virus between infected crows and

poultry in the live-bird market cannot be ruled out. Since

we collected only 10 environmental swab samples from a

live-bird market, we may have missed circulating influ-

enza viruses in the poultry. However, using an identical

sampling technique in the live bird markets of Bangladesh

has proven sufficient to identify circulating influenza

viruses, including A/H5N1 [32]. Additionally, it is also

possible that the infected crows may spread the virus

locally while scavenging near backyard poultry produc-

tion facilities and the commercial farms. Crows may

scavenge within eight kilometers from their roosting place

[33]. Our results indicate that crows infected with H5N1

exhibited clinical signs and were unable to fly, and the

clinical outcome was death. The combination of these

factors may limit the ability of infected crows to spread

the A/H5N1 clade 2.3.2.1 virus to a short geographical

range when compared to the marketing chain of live-bird

markets.

We systematically investigated crow outbreaks in only

two locations in Bangladesh during January-February

2011. However, there were reports of several crow out-

breaks at other locations during the same time period that

we did not have the resources to investigate. Additionally,

we counted the number of sick and dead crows only in the

area under the crow roost; this approach may have under-

estimated the morbidity and mortality in the crow

population.

Since the 2.2.2, 2.3.2.1 and 2.3.4 clades share common

hosts and reservoirs, surveillance is important to identify

whether they will reassort, co-circulate and/or replace one

another [34]. Bangladesh continues to be at risk for intro-

duction and evolution of additional new influenza viruses.

Informing and educating the public may help avoid direct

contact with sick and dead wild birds and promote sanitary

disposal to limit infection of humans and poultry.

Acknowledgments This research study was funded by the United

States Centers for Disease Control and Prevention (Cooperative

Agreement 5U01CI000628). icddr,b acknowledges with gratitude the

commitment of CDC to its research efforts. The contents of this report

are solely the responsibility of the authors and do not necessarily

represent the official views of the CDC or the icddr,b. We also

acknowledge and appreciate the diligent technical assistance of

Department of Livestock and Forest Department field officers of

Bangladesh, the icddr,b field team, and the immunohistochemistry

team of Gabriele Czerwinski at FLI.

References

1. World Organization for Animal health (2007) Highly pathogenic

avian influenza, Bangladesh (Immediate notification)

2. OIE (2011) Highly pathogenic avian influenza, Bangladesh.

Follow-up report No. 1–33. World Organization for Animal

health

3. Loth L, Gilbert M, Osmani MG, Kalam AM, Xiao X (2010) Risk

factors and clusters of Highly Pathogenic Avian Influenza H5N1

outbreaks in Bangladesh. Prev Vet Med 96(1–2):104–113. doi:10.

1016/j.prevetmed.2010.05.013

4. OIE (2011) Highly pathogenic avian influenza, Bangladesh.

Follow-up report No. 26–32. World Organization for Animal

health

5. Star TD (2011) Crows dying, poultry culled: Fear of fresh round

of bird flu grips Barisal, Kishoreganj. http://www.thedailystar.

net/newDesign/print_news.php?nid=172776. Accessed 24 Mar

2011, 6 Feb 2008

6. BBS (2012) Bangladesh Bureau of Statistics. Population density of

Dhaka city, 2008. http://www.bbs.gov.bd/WebTestApplication/user

files/Image/SubjectMatterDataIndex/pk_book_09.pdf. Accessed 23

Feb 2012

516 S. U. Khan et al.

123

7. Behrouzi-Rad B (2010) Population Estimation and Breeding

Biology of the House Crow Corvus splendens on Kharg Island,

Persian Gulf. Podoces 5(2):87–94

8. Ralph CJ, Dunn EH (eds) (2004) Monitoring bird populations

using mist nets, Studies in Avian Biology, vol 29. Cooper

Ornithological Society, Lawrence

9. Kis Z, Jones J, Creanga A, Ferdinand K, Inui K, Gerloff N, Davis

CT, Nguyen T, Donis RO (2013) Real-time RT-PCR assay to

differentiate clades of H5N1 avian influenza viruses circulating in

Vietnam. J Virol Methods 193:452–458. doi:10.1016/j.jviromet.

2013.06.023

10. Szretter KJ, Balish AL, Katz JM (2006) Influenza: propagation,

quantification, and storage. Curr Protoc Microbiol Chapter 15:

Unit 15G 11. doi:10.1002/0471729256.mc15g01s3

11. Rivailler P, Perry IA, Jang Y, Davis CT, Chen LM, Dubovi EJ,

Donis RO (2010) Evolution of canine and equine influenza

(H3N8) viruses co-circulating between 2005 and 2008. Virology

408(1):71–79. doi:10.1016/j.virol.2010.08.022

12. Hall TA (1999) BioEdit: a user-friendly biological sequence

alignment editor and analysis program for Windows 95/98/NT.

Nucl Acids Symp Ser 41:95–98

13. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molec-

ular Evolutionary Genetics Analysis (MEGA) software version

4.0. Mol Biol Evol 24(8):1596–1599. doi:10.1093/molbev/

msm092

14. Klopfleisch R, Werner O, Mundt E, Harder T, Teifke JP (2006)

Neurotropism of highly pathogenic avian influenza virus

A/chicken/Indonesia/2003 (H5N1) in experimentally infected

pigeons (Columbia livia f. domestica). Vet Pathol 43(4):463–470

15. Islam MR, Haque ME, Giasuddin M, Chowdhury EH, Samad

MA, Parvin R, Nooruzzaman M, Rahman MM, Monoura P

(2011) New Introduction of Clade 2.3.2.1 Avian Influenza Virus

(H5N1) into Bangladesh. Transbound Emerg Dis 59(5):460–463.

doi:10.1111/j.1865-1682.2011.01297.x

16. Nagarajan S, Tosh C, Smith DK, Peiris JS, Murugkar HV, Sridevi

R, Kumar M, Katare M, Jain R, Syed Z, Behera P, Cheung CL,

Khandia R, Tripathi S, Guan Y, Dubey SC (2012) Avian Influ-

enza (H5N1) Virus of Clade 2.3.2 in Domestic Poultry in India.

PLoS One 7(2):e31844. doi:10.1371/journal.pone.0031844

17. Khan SU, Gurley ES, Rahman M, Jahangir Hossain M, Uz

Zaman R, Nasreen S, Azim T, Mikolon A, Azziz-Baumgartner E,

Luby SP (2010) Live bird market surveillance for avian influenza

in Bangladesh, 2007–2009. In: Abstract in the International

Conference on Emerging Infectious Diseases. Atlanta

18. WHO (2012) Antigenic and genetic characteristics of zoonotic

influenza viruses and development of candidate vaccine viruses

for pandemic preparedness. September 2012. http://www.who.int/

influenza/vaccines/virus/201209_h5h7h9_vaccinevirusupdate.pdf.

Accessed 21 July 2013

19. Smith GJ, Vijaykrishna D, Ellis TM, Dyrting KC, Leung YH,

Bahl J, Wong CW, Kai H, Chow MK, Duan L, Chan AS, Zhang

LJ, Chen H, Luk GS, Peiris JS, Guan Y (2009) Characterization

of avian influenza viruses A (H5N1) from wild birds, Hong Kong,

2004–2008. Emerg Infect Dis 15(3):402–407

20. FAO-EMPRES (2012) H5N1 HPAI, GLobal Overview, April–

June 2012. http://www.fao.org/docrep/016/ap387e/ap387e.pdf.

Accessed 21 July 2013

21. Li Y, Liu L, Zhang Y, Duan Z, Tian G, Zeng X, Shi J, Zhang L,

Chen H (2011) New avian influenza virus (H5N1) in wild birds,

Qinghai, China. Emerg Infect Dis 17(2):265–267. doi:10.3201/

eid1702.100732

22. L’Vov DK, Shchelkanov M, Prilipov AG, Deriabin PG, Fediak-

ina IT, Galkina IV, Kireev DE, Frolov AV, Akanina DS, Usa-

cheva OV, Shliapnikova OV, Poglazov AB, Morozova TN,

Proshina ES, Grebennikova TV, Zaberezhnyi AD, Iakovlev SS,

Shcherbakova LO, Shapovalov AB, Zhalin MV, Rudenko VP,

Pichuev AE, Litvin KN, Varkentin AV, Steshenko VV, Khari-

tonov SP, Samokhvalov EI, Al’khovskii SV, Aliper TI, Mar-

tynovchenko VV, Lysenko SN, Vlasov NA, Nepoklonov EA

(2008) Interpretation of the epizootic outbreak among wild and

domestic birds in the south of the European part of Russia in

December 2007. Vopr Virusol 53(4):18–23

23. Reid SM, Shell WM, Barboi G, Onita I, Turcitu M, Cioranu R,

Marinova-Petkova A, Goujgoulova G, Webby RJ, Webster RG,

Russell C, Slomka MJ, Hanna A, Banks J, Alton B, Barrass L,

Irvine RM, Brown IH (2011) First reported incursion of highly

pathogenic notifiable avian influenza A H5N1 viruses from clade

2.3.2 into European poultry. Transbound Emerg Dis 58(1):76–78.

doi:10.1111/j.1865-1682.2010.01175.x

24. Sakoda Y, Sugar S, Batchluun D, Erdene-Ochir TO, Okamatsu

M, Isoda N, Soda K, Takakuwa H, Tsuda Y, Yamamoto N,

Kishida N, Matsuno K, Nakayama E, Kajihara M, Yokoyama A,

Takada A, Sodnomdarjaa R, Kida H (2010) Characterization of

H5N1 highly pathogenic avian influenza virus strains isolated

from migratory waterfowl in Mongolia on the way back from the

southern Asia to their northern territory. Virology 406(1):88–94.

doi:10.1016/j.virol.2010.07.007

25. Uchida Y, Mase M, Yoneda K, Kimura A, Obara T, Kumagai S,

Saito T, Yamamoto Y, Nakamura K, Tsukamoto K, Yamaguchi S

(2008) Highly pathogenic avian influenza virus (H5N1) isolated

from whooper swans, Japan. Emerg Infect Dis 14(9):1427–1429

26. Wan XF, Nguyen T, Davis CT, Smith CB, Zhao ZM, Carrel M,

Inui K, Do HT, Mai DT, Jadhao S, Balish A, Shu B, Luo F, Emch

M, Matsuoka Y, Lindstrom SE, Cox NJ, Nguyen CV, Klimov A,

Donis RO (2008) Evolution of highly pathogenic H5N1 avian

influenza viruses in Vietnam between 2001 and 2007. PLoS One

3(10):e3462. doi:10.1371/journal.pone.0003462

27. Newman SH, Hill NJ, Spragens KA, Janies D, Voronkin IO,

Prosser DJ, Yan B, Lei F, Batbayar N, Natsagdorj T, Bishop CM,

Butler PJ, Wikelski M, Balachandran S, Mundkur T, Douglas

DC, Takekawa JY (2011) Eco-virological approach for assessing

the role of wild birds in the spread of avian influenza H5N1 along

the Central Asian Flyway. PLoS One 7(2):e30636

28. Tanimura N, Tsukamoto K, Okamatsu M, Mase M, Imada T,

Nakamura K, Kubo M, Yamaguchi S, Irishio W, Hayashi M,

Nakai T, Yamauchi A, Nishimura M, Imai K (2006) Pathology of

fatal highly pathogenic H5N1 avian influenza virus infection in

large-billed crows (Corvus macrorhynchos) during the 2004

outbreak in Japan. Vet Pathol 43(4):500–509. doi:10.1354/vp.43-

4-500

29. Creanga A, Thi Nguyen D, Gerloff N, Thi Do H, Balish A, Dang

Nguyen H, Jang Y, Thi Dam V, Thor S, Jones J, Simpson N, Shu

B, Emery S, Berman L, Nguyen HT, Bryant JE, Lindstrom S,

Klimov A, Donis RO, Davis CT, Nguyen T (2013) Emergence of

multiple clade 2.3.2.1 influenza A (H5N1) virus subgroups in

Vietnam and detection of novel reassortants. Virology

444:12–20. doi:10.1016/j.virol.2013.06.005

30. Saito T, Uchida Y, Myint WW, Thein WZ, Watanabe C, Take-

mae N, Mase M, Okamatsu M, Mar A, Mon CC, Gawng LT,

Sann K, Kyi TA, Yamaguchi S (2008) Characterisation of highly

pathogenic avian influenza viruses in Myanmar. Vet Rec 163

(24):722–723

31. Ellis TM, Dyrting KC, Wong CW, Chadwick B, Chan C, Chiang

M, Li C, Li P, Smith GJ, Guan Y, Malik Peiris JS (2009) Analysis

of H5N1 avian influenza infections from wild bird surveillance in

Hong Kong from January 2006 to October 2007. Avian Pathol

38(2):107–119. doi:10.1080/03079450902751855

32. Khan SU, Rahman M, Gurley ES, Mikolon AB, Rahman MZ, Uz

Zaman R, Jahangir Hossain M, Nasreen S, Widdowson M-A,

Azim T, Azziz-Baumgartner E, Luby SP (2011) Live bird market

environmental sampling: a tool for poultry influenza surveillance.

In: American Society for Tropical Medicine and Hygiene, 60th

Highly pathogenic avian H5N1 influenza virus in Bangladesh 517

123

annual meeting, December 4–8, 2011. Philadelphia Marriott

Downtown, Philadelphia

33. Sonerud Geir A, Hansen Henning, Smedshaug CA (2002) Indi-

viduarlo ostings trategies in a flock-living bird: movementa and

social cohesiono of hooded crows (Corvus corone comix) from

pre-roost gatheings to roost sites. Behav Ecol Sociobiol

51(4):309–318

34. WHO/OIE/FAO H5N1 Evolution Working Group (2012) Con-

tinued evolution of highly pathogenic avian influenza A (H5N1):

updated nomenclature. Influenza Other Respi Viruses 6(1):1–5.

doi:10.1111/j.1750-2659.2011.00298.x

518 S. U. Khan et al.

123