Avian Influenza (Bird Flu): Agricultural and Wildlife Considerations

Last updated August 8, 2006

Definition of Avian Influenza
Agent
Hosts
Transmission
Key Outbreaks of HPAI in Domestic Avian Populations
Current Status of H5N1 in Asia and Europe
HPAI As a Biological Weapon
Clinical Features in Domestic Birds
Necropsy Lesions
Differential Diagnosis in Birds
Laboratory Diagnosis in Birds
Treatment
Prevention
Outbreak Control in Poultry
References

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 References: 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 more information.

Agent

Viral Classification and Genetic Composition of Influenza Viruses

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 segments).
- 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 virus.
- 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: Voyles 2002).
- 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 References: Fouchier 2005).
- 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 References: 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 pandemics).
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], A/swine/Iowa/15/30[H1N1]).
- As with other influenza A subtypes, standard nomenclature is used to name avian strains (eg, A/Chicken/HK/5/98 [H5N1]).

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: PHS).
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 findings).
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 compounds

Hosts

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: Horimoto 2001).

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: Kou 2005).

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 News story).

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).

Transmission

Routes of bird-to-bird transmission include:
- Airborne transmission if birds are in close proximity
- 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: OIE 2002)
- 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: Beard 1998)

The disease is highly contagious. One gram of contaminated manure can contain enough HPAI virus to infect 1 million birds (see References: APHIS).

Key Outbreaks of HPAI in Domestic Avian Populations

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.

Major Outbreaks of Avian Influenza*
Year	Subtype	Location	Impact	Comments
1983	H5	Pennsylvania	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.
1994-2003	H5N2	Mexico	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 been identified.
1995-2003	H7N3	Pakistan	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.
1997	H5N1	Hong Kong	Virus was isolated from chickens; avian mortality rates were high. 1.5 million birds were culled in 3 days.	18 human cases with 6 deaths were recognized. Prior to this outbreak, H5N1 was not known to infect humans.
2003	H7N7	The Netherlands	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 involved conjunctivitis.
2003-2006 (ongoing)	H5N1	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, Azerbaijan, Djibouti.
2004	H7N3	British Columbia	Over 19 million birds were culled.	Two human cases were recognized; both patients had conjunctivitis.
2005	H7	North Korea	About 200,000 birds culled as of April 2005.	No human cases have been identified.
Additional outbreaks of HPAI have been identified in a variety of countries. Adapted from Capua 2004 (See References).*

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, and Africa

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 References: 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: Webster 2006).

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 entirely clear.
- 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 (see References: 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.

Countries Affected by H5N1 in Poultry and Wild Birds
East Asia	Europe, Siberia, Central Asia	Africa
Cambodia China Hong Kong Indonesia Japan Laos Malaysia Mongolia South Korea Thailand Vietnam India	Croatia Kazakhstan Romania Russia (Siberia and European Russia) Turkey Iraq Saudi Arabia Cyprus Greece Italy Bulgaria Austria Germany Slovenia Azerbaijan Ukraine France Hungary Georgia (former Soviet republic) Slovakia Bosnia-Herzegovina Serbia Poland Albania Israel Jordan Czech Republic Denmark Scotland Spain	Nigeria Egypt Niger Cameroon Burkina Faso Ivory Coast Djibouti

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 and Outbreak 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 References: FAO 2004). Other alarming features of H5N1 include the following (see References: WHO Influenza pandemic preparedness and response [January 2005]).

Studies comparing virus samples over time indicate that the virus has become progressively more pathogenic for poultry.
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 virus.
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 References: 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 significantly.
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 Birds

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 the virus.

Clinical Features of Highly Pathogenic Avian Influenza in Domestic Birds
Feature	Characteristics
CHICKENS
Incubation Period	3-7 days
Clinical signs	—Sudden death —Severe depression with ruffled feathers —Inappetence —Drastic decline in egg production —Edema of head and neck —Swollen and cyanotic combs and wattles (see Gray Book figure 25 and figure 26 [References: Beard 1998]) —Petechial hemorrhages on internal membrane surfaces —Excessive thirst —Watery diarrhea that begins as bright green and progresses to white —Swollen and congested conjunctiva with occasional hemorrhage —Diffuse hemorrhage between hocks and feet (see Gray Book figure 27 [References: Beard 1998]) —Respiratory signs are dependent on tracheal involvement —Nasal and ocular discharge —Mucus accumulation (varies) —Lack of energy —Coughing/sneezing —Incoordination —Nervous system signs such as paralysis
Complications	—Cessation of egg production, and eggs laid immediately prior to infection often soft-shelled and misshapen —Surviving birds are in poor condition and resume laying only after a period of several weeks
Case-fatality rate	—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
TURKEYS
Incubation period	3-7 days
Clinical signs	—Sudden death —Severe depression with ruffled feathers —Inappetence —Drastic decline in egg production —Edema of the head and neck —Swollen and cyanotic combs and wattles —Petechial hemorrhages on internal membrane surfaces —Excessive thirst and evidence of dehydration —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 involvement —Nasal and ocular discharge —Mucus accumulation (varies) —Lack of energy —Coughing/sneezing —Incoordination —Nervous system signs such as paralysis —Sinusitis —Dehydration
Complications	—Decrease in egg production —Sudden death —Surviving birds are in poor condition and resume laying only after a period of several weeks
Case-fatality rate	—Can be as high as 100% —Most turkeys die within 3 to 10 days
DUCKS AND GEESE
Incubation period	3-7 days
Clinical signs	—Signs of depression, inappetence, and diarrhea similar to those seen in layers —Swollen sinuses —Neurologic signs in younger birds —Sinusitis
Complications	—Decrease in egg production —Sudden death —Surviving birds are in poor condition and resume laying only after a period of several weeks
Case-fatality rate	As high as 100%
Adapted from Aeillo 1998, APHIS, Beard 1998, Capua 2001, OIE 2002, PHS) (see References).

Necropsy Lesions

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 diagnosis.

Lesions Associated With Highly Pathogenic Avian Influenza in Birds
Type of Bird	Characteristics
Chickens	Lesions may be absent in young birds and birds that die from peracute disease Severe congestion of musculature Severe congestion of conjunctivae, sometimes with petechiae Excessive mucous exudates in lumen of trachea Severe hemorrhagic tracheitis Petechiae on 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 gizzard Hemorrhages and erosions of gizzard lining Hemorrhagic foci on lymphoid tissues in intestinal mucosa Ovary may be hemorrhagic or degenerated with darkened areas of necrosis Peritoneal cavity often filled with yolk from ruptured ova
Turkeys	Lesions similar to those in chickens but may not be as severe
Domestic ducks	Lesions may be similar to those seen in chickens though not as marked, or they may be absent altogether
Adapted from Beard 1998, OIE 2002 (see References).

Differential Diagnosis in Birds

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

Sample Collection

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 Animals):

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 7 days.
- 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 (ELISAs)
- 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

Serologic Tests

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 Animals).

AGID
- 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 centrifuged.
- 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 influenzaviruses.
- 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 OIE manual.
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 Reference Laboratory.
Commercial ELISA kits
- These detect antibody against the nucleocapsid protein.
- 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 carefully.

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 expensive.
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 characterized.

Treatment

There is no effective treatment for HPAI in poultry.

Prevention

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 occurred
Segregation of different animal species (eg, chickens, ducks, pigs) and elimination of these animals' intermingling with humans
Incentives for farmers to repo


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