Avian Influenza (Bird Flu): Agricultural and
Last updated August 8, 2006
of Avian Influenza
Outbreaks of HPAI in Domestic Avian Populations
Status of H5N1 in Asia and Europe
As a Biological Weapon
Features in Domestic Birds
Diagnosis in Birds
Diagnosis in Birds
Control in Poultry
Definition of Avian Influenza
Avian influenza, which is caused by influenza A
viruses, can affect a variety of domestic and wild bird species.
Infection can range from asymptomatic to severe, depending on the
virulence of the virus and the susceptibility of the avian host.
Avian influenza in domestic chickens and turkeys is classified
according to disease severity, with two recognized forms: highly
pathogenic avian influenza (HPAI), also known as fowl plague, and
low-pathogenic avian influenza (LPAI). Avian influenza viruses that
cause HPAI are highly virulent and mortality rates in infected
flocks often approach 100%. While LPAI viruses are generally of
lower virulence, LPAI in flocks should be controlled because LPAI
viruses can serve as progenitors to HPAI viruses.
Notifiable avian influenza is defined by the World
Organization for Animal Health (OIE) as "an infection of poultry
caused by any influenza A virus of the H5 or H7 subtypes or by any
avian influenza virus with an intravenous pathogenicity index (IVPI)
greater than 1.2 (or as an alternative at least 75% mortality)" (see
OIE 2004). The OIE further classifies avian influenza as HPAI or
LPAI according to the following criteria:
- HPAI viruses have an IVPI in 6-week-old chickens
greater than 1.2 or, as an alternative, cause at least 75%
mortality in 4-to 8-week-old chickens infected intravenously. H5
and H7 viruses which do not have an IVPI of greater than 1.2 or
cause less than 75% mortality in an intravenous lethality test
should be sequenced to determine whether multiple basic amino
acids are present at the cleavage site of the hemagglutinin
molecule (HA0); if the amino acid motif is similar to that
observed for other HPAI isolates, the isolate being tested should
be considered as HPAI.
- LPAI are all influenza A viruses of H5 and H7
subtype that are not HPAI viruses.
According to the OIE International Animal Health Code,
countries that identify HPAI should report the occurrence to OIE
within 24 hours.
Several different avian influenza strains have been
shown to infect humans. These include viruses of the H5 subtype
(H5N1), the H7 subtype (H7N2, H7N3, H7N7), the H9 subtype (H9N2),
and the H10 subtype (H10N7). See the document, "Avian Influenza
(Bird Flu): Implications for Human Disease" on this Web site for
Viral Classification and Genetic Composition of Influenza
- Family: Orthomyxoviridae
- Enveloped virions are 80 to 120 nm in diameter,
are 200 to 300 nm long, and may be filamentous.
- They consist of spike-shaped surface proteins, a
partially host-derived lipid-rich envelope, and matrix (M)
proteins surrounding a helical segmented nucleocapsid (6 to 8
- The family contains five genera, classified by
variations in nucleoprotein (NP and M) antigens: influenza A,
influenza B, influenza C, thogotovirus, and isavirus.
- Genus: Influenzavirus A
- Consists of a single species: influenza A
- The multipartite genome is encapsidated, with
each segment in a separate nucleocapsid. Eight different
segments of negative-sense single-stranded RNA are present; this
allows for genetic reassortment in single cells infected with
more than one virus and may result in multiple strains that are
different from the initial ones (see References:
- The genome consists of 10 genes encoding for
different proteins (eight structural proteins and two
nonstructural proteins). These include the following: three
transcriptases (PB2, PB1, and PA), two surface glycoproteins
(hemagglutinin [HA] and neuraminidase [NA]), two matrix proteins
(M1 and M2), one nucleocapsid protein (NP), and two
nonstructural proteins (NS1 and NS2).
- The virus envelope glycoproteins (HA and NA) are
distributed evenly over the virion surface, forming
characteristic spike-shaped structures. Antigenic variation in
these proteins is used as part of the influenza A virus subtype
definition (but not used for influenza B or C viruses).
- Influenza A virus subtypes
- There are 16 different HA antigens (H1 to H16)
and nine different NA antigens (N1 to N9) for influenza A. Until
recently, 15 HA types had been recognized, but a new type (H16)
was isolated from black-headed gulls caught in Sweden and the
Netherlands in 1999 and reported in the literature in 2005 (see
- All known subtypes of influenza A can be found in
birds, and feral aquatic birds are the major reservoir for
influenza A viruses (see References:
Fouchier 2004). Severe disease from influenza generally does not
develop in feral birds; however, domestic chickens and turkeys
are susceptible to severe and potentially fatal influenza.
- H5 subtypes
- H5 subtypes include both HPAI and LPAI strains.
- H5N1 strains circulate among birds worldwide and
are responsible for the current outbreak in Asia and Europe
among poultry and other birds.
- H5N1 appears to be expanding its host range, has
caused a number of human deaths (see below), and the possibility
that H5N1 could mutate into a human pandemic strain is causing
worldwide concern (see References:
WHO: Epidemic and pandemic alert and response).
- H5N1 has been differentiated into genetic clades
on the basis of sequence data. Clade 1 includes human and bird
isolates from Vietnam, Thailand, and Cambodia and bird isolates
from Laos and Malaysia. Clade 2 viruses have been identified in
bird isolates from China, Indonesia, Japan, and South Korea (see
WHO Global Influenza Program Surveillance Network).
- H7 and H9 subtypes
- H7 includes HPAI and LPAI strains.
- H9 is only known to include LPAI strains.
- These subtypes have caused infections in humans
on rare occasions (see References:
CDC: Avian influenza A viruses; NIAID: Timeline of human
- Influenza A nomenclature
- Antigenic strain nomenclature is based on: (1)
host of origin (if other than human), (2) geographic origin, (3)
strain number, (4) year of isolation, and (5) HA and NA types
(eg, for human strains, A/Hong Kong/03/68[H3N2],
- As with other influenza A subtypes, standard
nomenclature is used to name avian strains (eg,
Environmental Survival of Avian Influenza Viruses
- Viruses remain infectious after 24 to 48 hours on
nonporous environmental surfaces and less than 12 hours on porous
surfaces (see References:
Bean 1982). (Note: The importance of fomites in disease
transmission has not been determined.)
- Influenza A virus remains viable at moderate
temperatures for long periods in the environment and can survive
indefinitely in frozen material. It can survive for 4 days in
water at 22:C and for over 30 days at 0:C (see References:
- Recent data from studies of H5N1 in domestic ducks
have shown that H5N1 can survive in the environment for 6 days at
37:C (see References:
WHO: Laboratory study of H5N1 viruses in domestic ducks: main
- Inactivation of the virus occurs under the
following conditions (see
References: OIE 2002, PHS):
- Temperatures of 56:C for 3 hours or 60:C or more
for 30 minutes
- Acidic pH conditions
- Presence of oxidizing agents such as sodium
dodecyl sulfate, lipid solvents, and B-propiolactone
- Exposure to disinfectants: formalin, iodine
Avian influenza A viruses can infect a variety of
domestic and wild avian species (including chickens, turkeys, ducks,
domestic geese, quail, pheasants, partridge, psittacines, gulls,
shorebirds, seabirds, emu, eagles, and others). The clinical
manifestation of infection in birds ranges from asymptomatic
infection to rapidly fatal disease (see References:
Aquatic birds, particularly ducks, shore birds, and
gulls, are considered the natural reservoirs for avian influenza
viruses (see References:
Webster 1992). These waterfowl generally do not develop disease when
infected with avian influenza viruses (see References:
Horimoto 2001); however, an outbreak of H5N1 among migratory geese
and other wild birds in Qinghai province, China, was identified in
May 2005 (see References:
Lui 2005). Recently, HPAI H5N1 viruses were isolated from
asymptomatic tree sparrows in the Henan province of China; the
significance of this finding remains unknown (see References:
Recently, investigators in Asia have shown that
asymptomatically infected domestic ducks are shedding more H5N1
virus for longer periods in 2004 when compared with 2003, which may
be amplifying the spread of H5N1 to domestic poultry (see References:
FAO/OIE/WHO 2004). Another report demonstrated the presence of H5N1
influenza virus in asymptomatic eagles that were smuggled from
Thailand into Belgium in 2004 (see References:
Borm 2005). Just recently H5N1 has been identified in turkeys (see
October 13, 2005, CIDRAP
Certain mammals also are susceptible to influenza.
Influenza A viruses have traditionally been known to also cause
disease in horses, pigs, whales, and seals; however, the range of
several influenza A subtypes is expanding to different mammalian
species. H5N1 influenza A recently has been shown to infect cats,
leopards, tigers, and civets (see References:
Keawcharoen 2004, Thanawongnuwech 2005, Webster 2006). Cases
of canine influenza have been recognized in the United States and
are being caused by H3N8 influenza A, a subtype traditionally
found in horses (see References:
Crawford 2005, Yoon 2005). A recent report involving cats
experimentally infected with H5N1 demonstrated that infected cats
excreted the virus via the respiratory tract and the digestive
tract, suggesting that in addition to the respiratory route, other
routes of transmission may play a role in spread among mammalian
hosts (see References
: Rimmelzwaan 2006).
- Routes of bird-to-bird transmission include:
- Airborne transmission if birds are in close
- Direct contact with contaminated respiratory
secretions or fecal material
- Vertical transmission is not known to occur
- Other factors that contribute to spread within and
between flocks include the following:
- Broken contaminated eggs in incubators infecting
healthy chicks (see References:
- Movement of infected birds between flocks
- Movement of fomites such as contaminated
equipment, egg flats, feed trucks, and clothing and shoes of
employees and service crews (see References:
APHIS, Beard 1998)
- Contact with infected wild birds and waterfowl
- Fecal contamination of drinking water
- Garbage flies (suspected of transmitting the
virus during the 1983-1984 epidemic in Pennsylvania) (see References:
The disease is highly contagious. One gram of
contaminated manure can contain enough HPAI virus to infect 1
million birds (see
Key Outbreaks of HPAI in Domestic Avian
To date, all outbreaks of HPAI in domestic poultry
have been caused by H5 or H7 influenza A subtypes. Until 1999, HPAI
was considered relatively rare, with only 17 outbreaks reported
worldwide between 1959 and 1998; however, since 1999 the number of
outbreaks occurring globally has increased significantly (see References:
Capua 2004). Major outbreaks of avian influenza are highlighted in
the table below.
Caused severe clinical disease and high
mortality rates in chickens, turkeys, and guinea fowl.
17 million birds were culled.
A serologically identical but apparently
mild virus had been circulating in poultry in the area for 6
mo (see References:
Beard 1998). No human cases were identified.
Nearly a billion birds have been affected.
An LPAI virus mutated to an HPAI virus and
caused an outbreak in 1994-1995. The H5N2 strain has continued
to circulate in Mexico since that time. No human cases have
About 3.2 million birds died from avian
influenza during initial outbreak in 1995.
A vaccination campaign apparently ended the
outbreak. No human cases were identified.
Virus was isolated from chickens; avian
mortality rates were high. 1.5 million birds were culled in 3
18 human cases with 6 deaths were
recognized. Prior to this outbreak, H5N1 was not known to
30 million birds out of 100 million birds
in country were killed; 255 flocks were infected. Disease
spread to Belgium but was quite rapidly contained.
Over 80 human cases were reported, and one
veterinarian died (see References:
Fouchier 2004, Stegeman 2005). Most of the human cases
Asia, Europe, Africa
By far the most severe outbreak of avian
influenza ever recognized. An estimated 150 million birds have
died or been culled.
More than 230 human cases have been
recognized, with more than half of them fatal, in Vietnam,
Thailand, Cambodia, Indonesia, China, Turkey, Iraq, Egypt,
Over 19 million birds were culled.
Two human cases were recognized; both
patients had conjunctivitis.
About 200,000 birds culled as of April
No human cases have been identified.
Examples of additional outbreaks of avian influenza
that have occurred in the past include the following (see References:
Horimoto 2001, Capua 2004):
- Australia had outbreaks of HPAI in 1976 (H7N7),
1985 (H7N7), 1992 (H7N3), 1994 (H7N3), and 1997 (H7N4).
- Italy had outbreaks in 1997 (H5N2), 1998 (H5N9),
1999-2001 (H7N1), and 2003-2003 (H7N3).
- The Republic of Ireland had an outbreak in 1998
(H7N7) that spread into Northern Ireland as well.
Current Status of H5N1 in Asia, Europe,
Avian influenza caused by H5N1 in Asia first received
widespread recognition following a 1997 outbreak in poultry in Hong
Kong with subsequent spread of the virus to humans. During that
outbreak, 18 human cases were recognized; six patients died. The
outbreak was stopped when all of the domestic chickens present in
wholesale facilities and vendors in Hong Kong were slaughtered (see
Snacken 1999). Person-to-person transmission of H5N1 was not
recognized at that time (see References:
Uyeki 2002). A precursor to the 1997 H5N1 strain was identified in
Guangdong, China, in 1996, when it caused deaths in geese (see References:
An outbreak of HPAI caused by H5N1 avian influenza
started in Asia in the fall of 2003 and spread in domestic poultry
farms at an historically unprecedented rate. The outbreak tapered
off in spring 2004 but in summer re-emerged in several areas in Asia
and is ongoing. The viruses currently causing outbreaks across Asia
are genetically distinct from the strain isolated from humans in
Hong Kong in 1997.
Beginning in the summer of 2005, H5N1 expanded its
geographic range beyond Asia:
- In late July, outbreaks of H5N1 in poultry were
recognized in Russia, Kazakhstan, and Mongolia (see References:
WHO 2005: Geographical spread of H5N1 avian influenza in birds).
The H5N1 in those areas is being studied to determine whether and
how it is related to the strain in Southeast Asia.
- In October 2005, H5N1 was discovered to have spread
to Turkey and Europe, and the spread across Europe continues.
- In February 2006, H5N1 was confirmed in a commerial
poultry flock in northern Nigeria (see References:
WHO: Avian influenza: situation [birds] in Nigeria), marking the
first reports of the disease in Africa. The source is not clear,
although the country lies along a flight route for birds migrating
from central Asia. Several other African nations have been
affected since then.
- The mechanisms for ongoing spread of H5N1 are not
- Movement of poultry and poultry products has
played an important role in spreading the virus in Asia.
- Free-ranging backyard chickens and ducks
apparently have contributed to spread of the virus in Thailand
Tiensin 2005). Illegal transportation of birds and cockfighting
may also play a role in that country.
- H5N1 has been shown to infect migratory
waterfowl, and spread of the virus via migratory birds is of
ongoing concern (see References:
Chen 2005, Liu 2005, Webster 2006).
As of December 2005, over 140 million domesticated
birds have been killed by the virus or culled to prevent further
spread (see References:
Webster 2006). Areas affected by H5N1 avian influenza in poultry or
migratory birds are shown in the following table.
Europe, Siberia, Central Asia
(Siberia and European Russia)
Since January 2002, the predominant avian H5N1 strain
in southern China has been genotype Z. Since its emergence, this
strain has replaced other genotypes and has become the predominant
genotype circulating in aquatic and terrestrial poultry in the
region (see References:
Li 2004). This strain circulating in Asia appears to be highly
pathogenic for humans, and immunity in the human population is
generally lacking. However, the strain has not yet been shown to be
easily transmitted between humans, and sustained person-to-person
transmission has not occurred.
The unprecedented rapid spread of H5N1 avian influenza
across East Asia and now reaching Europe has been alarming to
international health organizations, and efforts to contain its
spread are ongoing. Control measures currently recommended in Asia
are outlined in the sections below on Prevention
Control in Poultry.
A report from the Food and Agricultural Organization
of the United Nations (FAO) published in September 2004 indicates
that H5 avian influenza viruses have become endemic in parts of
Southeast Asia and that existing reservoirs in ducks, wild birds,
and potentially pigs "pose a serious challenge to eradication" (see
FAO 2004). Other alarming features of H5N1 include the following
WHO Influenza pandemic preparedness and response [January 2005]).
- Studies comparing virus samples over time indicate
that the virus has become progressively more pathogenic for
- The current strain of the virus is now able to
survive several days longer in the environment compared with when
it first emerged.
- The virus appears to be expanding its mammalian
host range, as indicated in the section above on "Hosts."
- The virus has been found increasingly in dead
migratory birds (which are usually not clinically affected by HPAI
viruses); this supports the growing virulence of the current
- Recent genetic sequencing performed on viral
isolates from Turkey demonstrates that the strains contain two
mutations which may make the virus better adapted to humans (see
Butler 2006). These mutations could potentially enhance
transmission from birds to humans and between humans.
An international conference sponsored by the World
Health Organization (WHO) and OIE and held in Ho Chi Minh City in
February 2005 concluded that the agricultural losses from the
current H5N1 avian influenza outbreak as of February 2005 are
estimated at $10 billion. Conference participants also noted that
about $100 million is needed in the region to strengthen animal
health and laboratory diagnostic services.
In addition to the rapid spread of H5N1 in poultry,
more than 230 human cases of H5N1 influenza have been confirmed,
with more than half of them fatal, according to official WHO data.
Cases have occurred in Thailand, Vietnam, Cambodia, Indonesia,
China, Turkey, Iraq, Azerbaijan, Egypt, and Djibouti (see References:
WHO: Cumulative confirmed cases of human H5N1 avian influenza). To
date, sustained person-to-person transmission has not been
recognized, although limited transmission has been suggested in
several family clusters (see References:
Olsen 2005, Ungchusak 2005), most recently in a large cluster in
Indonesia (see May 24, 2006, CIDRAP
News story). Influenza experts are concerned that if the H5N1
virus reassorts with human influenza viruses, a new influenza virus
with pandemic potential could emerge (see References:
Stohr 2005; Monto 2005; WHO: Influenza pandemic preparedness and
response [January 2005]).Another possibility is for an avian strain
to gradually adapt to the human population and develop into a
pandemic strain without genetic reassortment (see References:
Taubenberger 2005). For a pandemic to occur, the new virus would
need to be highly pathogenic for humans and easily transmitted
person-to-person. For more, see the documents "Avian Influenza (Bird
Flu): Implications for Human Disease" and Pandemic Influenza" on
this Web site.
HPAI As a Biological Weapon
HPAI is considered a potential biological weapon
because of the following factors:
- Extremely contagious
- High mortality rate
- Severe economic consequences of an outbreak:
- Large numbers of birds are destroyed or die.
- Control measures disrupt trade of poultry
products from affected areas.
- Prices of retail poultry products may increase
- Virus has a high potential for genetic mutations
and for new strains to arise and affect new species
- The Hong Kong epidemic of 1997 and the associated
human cases demonstrate the ability of the virus to affect
humans and birds.
Clinical Features in Domestic
The clinical signs of HPAI are severe and result in
high mortality rates in many species of birds, especially domestic
fowl. As mentioned above, waterfowl, ratites, and other birds may
not be as susceptible to clinical signs but can act as carriers for
—Severe depression with
—Drastic decline in egg
—Edema of head and neck
—Swollen and cyanotic
combs and wattles (see Gray Book figure
25 and figure
—Petechial hemorrhages on internal membrane
—Watery diarrhea that begins
as bright green and progresses to white
congested conjunctiva with occasional hemorrhage
hemorrhage between hocks and feet (see Gray Book figure
—Respiratory signs are dependent on tracheal
—Nasal and ocular discharge
system signs such as paralysis
—Cessation of egg production, and eggs laid
immediately prior to infection often soft-shelled and
—Surviving birds are in poor condition and resume
laying only after a period of several weeks
—Can be as high as 100%
—Death may occur
prior to any symptoms or as late as a week after symptoms,
though it is frequently within 48 hr
—Severe depression with
—Drastic decline in egg
—Edema of the head and neck
cyanotic combs and wattles
—Petechial hemorrhages on
internal membrane surfaces
—Excessive thirst and evidence
—Watery diarrhea that begins as bright green
and progresses to white
—Swollen and congested conjunctivae
with occasional hemorrhage
—Diffuse hemorrhage between
hocks and feet
—Respiratory signs are dependent on tracheal
—Nasal and ocular discharge
system signs such as paralysis
—Decrease in egg production
—Surviving birds are in poor condition and resume
laying only after a period of several weeks
—Can be as high as 100%
die within 3 to 10 days
DUCKS AND GEESE
—Signs of depression, inappetence, and
diarrhea similar to those seen in layers
—Neurologic signs in younger birds
—Decrease in egg production
—Surviving birds are in poor condition and resume
laying only after a period of several weeks
As high as 100%
HPAI can be recognized by the high mortality rate in
affected flocks as well as by the clinical signs. Characteristic
necropsy lesions, listed in the table below, also can help make the
Type of Bird
Lesions may be absent in young birds and
birds that die from peracute disease
Severe congestion of
Severe congestion of conjunctivae, sometimes
Excessive mucous exudates in lumen of
Severe hemorrhagic tracheitis
inside of sternum
Petechiae on serosal and abdominal fat
and in body cavity
Severe kidney congestion, sometimes with
urate deposits in tubules
Hemorrhages on mucosal surface of
proventriculus, especially at juncture with
Hemorrhages and erosions of gizzard
Hemorrhagic foci on lymphoid tissues in intestinal
Ovary may be hemorrhagic or degenerated with
darkened areas of necrosis
Peritoneal cavity often filled
with yolk from ruptured ova
Lesions similar to those in chickens but
may not be as severe
Lesions may be similar to those seen in
chickens though not as marked, or they may be absent
Differential Diagnosis in
Other diseases to consider when examining birds
suspected of having HPAI include:
- Velogenic (exotic) Newcastle disease
- Infectious laryngotracheitis
- Acute Escherichia coli infections
- Acute fowl cholera (Pasteurella multocida)
- Bacterial sinusitis (ducks)
Laboratory Diagnosis in Birds
According to the OIE Manual of Diagnostic Tests and
Vaccines for Terrestrial Animals (see References),
considerations for sample collection for avian influenza in birds
include the following:
- Samples taken from dead birds should include
intestinal contents (feces) or cloacal swabs and oropharyngeal
swabs. Samples from trachea, lungs, air sacs, intestine, spleen,
kidney, brain, liver, and heart may also be collected and
processed either separately or as a pool.
- Samples from live birds should include both
tracheal and cloacal swabs, although the latter are most likely to
yield virus. Because small, delicate birds may be harmed by
swabbing, the collection of fresh feces may serve as an adequate
alternative. To optimize the chances of virus isolation, it is
recommended that at least 1 gm of feces be processed either as
feces or coating the swab.
- The samples should be placed in isotonic phosphate
buffered saline (PBS), pH 7.0 to 7.4, containing antibiotics.
- The antibiotics can be varied according to local
conditions, but could be, for example, penicillin (2,000
units/mL), streptomycin (2 mg/mL), gentamycin (50 mcg/mL) and
mycostatin (1,000 units/mL) for tissues and tracheal swabs but
at five-fold higher concentrations for feces and cloacal swabs.
It is important to readjust the solution to pH 7.0 to 7.4 after
addition of antibiotics.
- Feces and finely minced tissues should be
prepared as 10% to 20% (w/v) suspensions in the antibiotic
solution. Suspensions should be processed as soon as possible
after incubation for 1 to 2 hours at room temperature.
- When immediate processing is impractical, samples
may be stored at 40C for up to 4 days. For prolonged storage,
diagnostic samples and isolates should be kept at 800C.
Identification of the Agent
Once specimens have been collected and processed, the
OIE Manual recommends the following for identification of avian
influenza (see References:
OIE Manual of Diagnostic Tests and Vaccines for Terrestrial
- The preferred method of growing avian influenza A
viruses is by the inoculation of embryonated specific pathogen
free (SPF) fowl eggs, or specific antibody negative (SAN) eggs of
9 to 11 days' incubation.
- Eggs should be incubated at 35:C to 370C for 4 to
- Eggs containing dead or dying embryos as they
arise, and all eggs remaining at the end of the incubation
period, should first be chilled to 40C and the allantoic fluids
should then be tested for hemagglutination (HA) activity.
- Detection of HA activity indicates a high
probability of the presence of an influenza A virus or of an avian
paramyxovirus. Fluids that give a negative reaction should be
passaged into at least one further batch of eggs.
- Several methods are available to confirm the
presence of influenza A virus; these include:
- Agar gel immunodiffusion (AGID) tests that
demonstrate the presence of the nucleocapsid or matrix antigens
- Various enzyme-linked immunosorbent assays
- Reverse-transcription polymerase chain reaction
(RT-PCR) using nucleoprotein-specific or matrix-specific
conserved primers; the presence of subtype H5 or H7 influenza
virus can be confirmed by using H5- or H7-specific primers
Because sera from infected chickens can yield positive
antibody tests as early as 3 to 4 days after the first signs of
disease appear, serologic tests can be useful to diagnose the
disease. Examples of serologic tests are outlined below (see References:
OIE: Manual of Diagnostic Tests and Vaccines for Terrestrial
- These tests have been widely and routinely used
to detect specific antibodies in chicken and turkey flocks as an
indication of infection.
- They have generally employed
nucleocapsid-enriched preparations made from the chorioallantoic
membranes of embryonated fowl eggs that have been infected at 10
days of age, homogenized, freezethawed three times, and
- The supernatant fluids are inactivated by the
addition of 0.1% formalin or 1% betapropiolactone, recentrifuged
and used as antigen. Not all avian species may produce
precipitating antibodies following infection with
- Concentrated virus preparations contain both
matrix and nucleocapsid antigens; the matrix antigen diffuses
more rapidly than the nucleocapsid antigen.
- Hemagglutination (HA) and
hemagglutination-inhibition (HI) tests
- Variations in the procedures for HA and HI tests
are practiced in different laboratories and are described in the
- Neuraminidase-inhibition test
- This test has been used to identify the avian
influenza neuraminidase type of isolates and to characterize the
antibody in infected birds.
- The procedure requires specialized expertise and
reagents; consequently this testing is usually done in an OIE
- Commercial ELISA kits
- These detect antibody against the nucleocapsid
- Several different test and antigen preparation
methods are in use.
- Such tests have usually been evaluated and
validated by the manufacturer, and it is therefore important
that the instructions specified for their use be followed
Developing Techniques for the Diagnosis
In addition to the tests mentioned above, new
diagnostics have become available in recent years, including:
- Antigen detection tests
- The commercially available Directigen Flu A Kit
(Becton Dickinson Microbiology Systems), which is an
antigen-capture enzyme immunoassay system, has been used for
detecting the presence of influenza A viruses in poultry,
particularly in the United States. The kit uses a monoclonal
antibody against the nucleoprotein and should therefore be able
to detect any influenza A virus.
- The main advantage of the test is that it can
demonstrate the presence of avian influenza within 15 minutes.
- The disadvantages are that it may lack
sensitivity, it has not been validated for different species of
birds, subtype identification is not achieved, and the kits are
- Direct RNA detection
- RT-PCR techniques on clinical specimens could,
with the correctly defined primers, result in rapid detection
and subtype (at least H5 and H7) identification.
- Direct RT-PCR tests may be useful for rapidly
identifying subsequent outbreaks in flocks once the primary
infected premises has been identified and the virus
There is no effective treatment for HPAI in poultry.
Accepted methods for prevention of avian influenza are
outlined below. In response to the situation in Asia at present,
several short-term strategies were put forth by the international
health agencies at a meeting in Malaysia in July 2005 (see References:
FAO/OIE/WHO July 2005). WHO and the Food and Agricultural
Organization of the United Nations (FAO) issued a statement
outlining a multipronged approach that includes:
- Educational efforts focusing on small-scale and
backyard farms, where most cases of human cases of H5N1 have
- Segregation of different animal species (eg,
chickens, ducks, pigs) and elimination of these animals'
intermingling with humans
- Incentives for farmers to report suspected cases of
avian flu and to apply control measures
- Vaccination of poultry flocks
In August 2005, OIE reiterated this approach, calling
for intensification of the measures in view of the spread of H5N1 in
late summer 2005 into Russia and Kazakhstan and urging financial
support from wealthy nations (see References:
OIE: Evolution of the animal health situation with regard to avian
The best way to prevent HPAI from spreading is to
prevent exposure of flocks to the influenza virus. This depends on
the formation of a barrier between farms and the outside
environment. Strategies to create this barrier include the following
APHIS 2002; FAO 2004):
- Avoid contact between domestic poultry and wild
birds, especially waterfowl.
- Open-range operations have a greater risk of
acquiring influenza virus in regions where migratory waterfowl,
sea birds, and shore birds are found.
- Exclude wild waterfowl from ponds that serve as
drinking water for poultry.
- If wild waterfowl cannot be excluded from ponds,
then drinking water obtained from these sources should be
treated (eg, with ultraviolet radiation or chlorination).
- Avoid the introduction of birds of unknown disease
status into a flock.
- Control human traffic.
- Ensure that people with access to the flock wear
proper safety equipment such as boots, coveralls, gloves, face
masks, and headgear.
- Provide clean clothing and disinfection
facilities for employees.
- Follow proper cleaning and disinfection procedures.
- Use an "all-in/all-out" production system.
- Permit only essential workers and vehicles to enter
- Thoroughly clean and disinfect equipment and
vehicles entering and leaving the farm; the tires and
undercarriage of vehicles should be included in the process.
- Do not loan or borrow equipment or vehicles from
- Avoid visiting other poultry farms. If unavoidable
or if visiting a live-bird market, change footwear and clothing
before working with your own flock
- Do not bring birds from slaughter channels,
especially live-bird markets, back to the farm.
Live Market Practices
The 1997 HPAI outbreak in Hong Kong demonstrated the
difficulties of preventing spread of influenza virus in live
markets. Once the virus is established in such a market, it can
easily spread via the movement of birds, crates, or trucks to other
farms and/or markets. It is important to follow biosecurity
protocols at live-bird markets as well as on the farm (see References:
- Use plastic instead of wooden crates for easier
- Keep scales and floors clean of manure, feathers,
and other debris.
- Clean and disinfect all equipment, crates, and
vehicles before returning them to the farm.
- Keep incoming poultry separate from unsold birds,
especially if birds are from different lots.
- Clean and disinfect the marketplace after every day
- Do not return unsold birds to the farm.
Vaccinated birds are less likely to become infected
and are less likely to excrete the virus; therefore, vaccination can
be used either as a tool to support eradication or as a tool to
control the disease and reduce the viral load in the environment.
FAO has described three broad categories of vaccination strategies
- Vaccination in response to an outbreak using a
"ring vaccination" approach or vaccination of only designated
high-risk poultry; this approach should be used in conjunction
with culling of infected poultry.
- Vaccination in response to a "trigger," such as
evidence from surveillance information that a HPAI virus has
entered the area: this approach may be used in situations where
the potential to improve biosecurity is limited.
- Preemptive baseline vaccination, such as
vaccinating poultry during restocking of farms in previously
Two different types of vaccines are currently
available, both of whichare administered by injection (see References:
- Conventional vaccines, which include inactivated
homologous vaccines and inactivated heterologous vaccines
- They involve an inactivated whole avian influenza
virus antigen in oil-based emulsion adjuvant
- These vaccines use a homologous H determinant
(such as H5 for the strain currently circulating in Asia).
- They possess either a homologous (such as N1 for
the strain currently circulating in Asia) or heterologous N
- The use of a heterologous N determinant allows
use of serologic surveillance to detect the circulation of field
virus through the detection of antibodies to the N subtype of
the field virus; this is known as the DIVA approach.
- The DIVA approach was used successfully during an
LPAI outbreak in Italy.
- Recombinant vaccines
- Several recombinant fowlpox virus-vector vaccines
that express the H5 antigen have been developed.
- One vaccine has been licensed and is in use in
A number of additional novel vaccines either have been
developed or are under development. Examples include:
- Subunit vaccines
- DNA vaccines
- Vaccines based on reverse genetics
- Adenovirus-vectored vaccine delivered via drinking
- Newcastle disease-vectored vaccine delivered via
Outbreak Control in Poultry
Several steps should be taken to control an outbreak
of HPAI (see References:
- Controlled movement of birds and products
that may contain virus
- Infected "zones" should be identified, and
movement of items and birds from those zones should be
- Border controls should be instituted as
- Destruction of infected and at-risk poultry
- This should be done as humanely and as quickly as
possible, preferably within 24 hours after infection in the
flock is detected.
- One widely used method is asphyxiation using
- Stringent cleaning and disinfection of the
facilities and equipment should be performed after culling.
- No new birds should be allowed in facilities for
at least 21 days after depopulation and disinfection.
- According to a recent article following a 2003
H7N7 outbreak in the Netherlands, complete depopulation of
infected areas seemed to be the most effective control measure
Stegeman 2005). However, when the HPAI strain becomes endemic
(as in the current situation in Asia with H5N1), efforts to
eradicate the virus from poultry in affected areas are likely to
be less effective.
- Proper disposal of carcasses and all animal
products in contact with the infected flock should be performed in
a biosecure and environmentally acceptable manner.
- Vaccination of flocks may be suitable for control
in some situations or may be used as an adjunct to mass culling
efforts (see References:
FAO 2004). A recent report which evaluated the effectiveness of a
vaccination campaign following an outbreak of HPAI H7N7 in
chickens in the Netherlands demonstrated that vaccination was an
effective strategy to reduce transmission (see References:
van der Goot 2005).
In November 2005, WHO, FAO, and OIE published a
document entitled Global Strategy for the Progressive Control of
Highly Pathogenic Avian Influenza (see References).
It includes recommendations made at the 2nd FAO/OIE Regional Meeting
on Avian Influenza in Asia (Ho Chi Minh City, February 2005) and
applies scientific information presented at the OIE/FAO
International Scientific Conference on Avian Influenza (Paris, April
Strategies for disease control discussed in the
- Effective risk-based surveillance for early
detection, diagnosis, and reporting
- Immediate stamping out of new outbreaks when and
where human life is at risk
- Enhanced biosecurity of poultry farms
- Control of movement of poultry and poultry products
that may harbor virus, including controls at the interface of
infected and uninfected areas
- Rapid, humane culling of infected and "at high
risk" poultry and safe disposal of carcasses
- Strategic vaccination
- Changes to industry practices, such as control of
live bird markets and farm hygiene, to reduce risk
- Separation of poultry species into "compartments"
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