Avian Influenza
Etiology
Avian Influenza (AI) [also known as Fowl Plague] is a viral infection affecting wild and domestic birds. These influenza viruses occur naturally in the intestinal tract of many species of wild birds with no disease outcome. However, pathogenic forms of the Avian Influenza virus can result in a highly contagious respiratory infection. Many species of birds are susceptible to AI including chickens, turkeys, guinea fowl, and other domestic birds, as well as a some wild avian species. AI viruses are classified into two general types based on their pathogenicity for chickens: Low Pathogenic (LP) and Highly Pathogenic (HP) types.
AI is caused by viruses that are members of the family Orthomyxoviridae, and the genus Influenzavirus, Type A. Influenza viruses Type B and C are pathogenic for humans but not for birds. Virus classification is based on antigenic differences in the nucleoprotein (NP) and matrix proteins (M1). Further subtyping of the AI virus is made based on the presence of 2 glycoproteins on the surface of the virus, hemagglutinin (HA) and neuraminidase (NA). Each AI virus has 1 of 16 different HA subtype glycoproteins and 1 of 9 different NA subtype glycoproteins (e.g. H5N1).
The HA and NA protein subtypes confer differences in viral pathogenicity. In LP strains, the HA protein can be cleaved by proteases present only in the respiratory and digestive tracts. In HP strains, proteases in most cells of the body can cleave the protein, resulting in a much wider tissue tropism than mildly pathogenic strains.
The HA protein is the major antigen that elicits antibodies which protect against clinical signs and death. Such antibodies are HA subtype specific. The antigenicity of influenza viruses may change gradually by point mutation (antigenic drift) or drastically by genetic reassortment (antigenic shift). Immunological pressure on HA and NA is thought to drive antigenic drift.
AI subtype classification requires viral isolation and characterization. Further subtyping is based on the antigenicity of the two surface glycoproteins, HA and NA. Classification cannot be made through clinical observations alone because the severity of the infection depends not only on the subtype of the virus but also the host species, the age of the bird, the host's immune status, and the presence of any secondary infections or environmental stress.
Due to the significant economic and public health implications of an AI outbreak, the World Organization for Animal Health (OIE) requires notification of any bird that is diagnosed with low pathogenic notifiable AI (LPNAI) or highly pathogenic notifiable AI (HPNAI). For the purposes of international trade the OIE defines notifiable avian influenza (NAI) as an infection of poultry caused by any influenza A virus of the H5 or H7 subtypes or by any AI virus with an intravenous pathogenicity index (IVPI) greater than 1.2 or, as an alternative, if the virus produces at least 75% mortality after inoculation.
HPNAI viruses have an IVPI greater than 1.2 in 6-week-old chickens or, as an alternative, cause at least 75% mortality in 4 to 8-week-old chickens infected intravenously. H5 and H7 virus isolates that do not have an IVPI of 1.2 or greater, nor produce 75% mortality under experimental conditions, but have an amino acid sequence at the cleavage site of the hemagglutinin that is similar to that observed for other HPNAI isolates, should be considered as HPNAI. LPNAI are all influenza A viruses of H5 and H7 subtype that do not display features of HPNAI viruses.
To date, all highly pathogenic strains of AI have been of the H5 or H7 subtypes. Although most H5 and H7 subtypes are in fact low pathogenic strains, since AI is unstable, there is a potential for LPNAI to mutate into HPNAI. Therefore all H5 and H7 subtypes require reporting to the OIE in order to facilitate global virus control measures. Within the United States, these pathotypes are considered exotic diseases and, in addition to being reported to the OIE, they should also be reported to the State and Federal veterinary authorities.
Host range
The AI viruses have been shown to naturally infect a wide variety of wild and domestic birds. The viruses have been isolated from more than 90 avian species however, in free-living birds most natural infections do not produce recognizable disease. The practice of housing birds in captivity and the development of commercial poultry systems have altered the normal epidemiology of the virus, resulting in clinically significant disease within domestic poultry.
In chickens, AI is capable of producing significant disease and economic losses. Ducks and other waterfowl are susceptible to the Asian HPAI H5N1 currently circulating in Asia, but have innate resistance to most other strains of Avian Influenza. Migratory waterfowl can act as carriers and transport viruses between geographic areas, helping to spread AI viruses to other susceptible animals.
In general, mammals are poorly susceptible to AI. However, several infections of highly pathogenic notifiable Avian Influenza (HPNAI) viruses have been reported since 1997 in humans, pigs and other mammals. Most of these cases have been linked to close contact with infected poultry. Although chicken-to-human infections have been rare, the virus is highly unstable and can alter pathotypes by passage in various animal species. The recombination of avian and human influenza viruses in pigs may be a potential source for sporadic pandemics of human influenza.
Epidemiology
AI viruses are distributed worldwide and identified frequently from clinically normal migratory waterfowl, imported birds, and species in live-bird markets. Data shows migratory waterfowl are the reservoir for AI viruses. Migratory birds introduce LPNAI viruses into poultry and, once introduced, the virus adapts to poultry and spreads from flock-to-flock, mostly by human activity. Most outbreaks of HPNAI in poultry arise from mutations in a LPNAI strain.
AI is excreted from the nares, mouth, and cloaca of infected birds. Spread occurs rapidly among birds housed on the floor but is slower in caged birds. Spread is primarily through contaminated feces and aerosol from the respiratory tract. The ingestion of contaminated feed and drinking water is another common source of transmission. The virus may also be transmitted indirectly by fomites such as contaminated equipment, personnel, clothing, foot wear, artificial insemination, and other human activity.
The incubation for AI is usually 1-7 days and depends on the strain, the dose, the species, and the age of the bird. The typical incubation period for an individual bird is approximately 3 days. It may take up to 14 days for an entire flock to become ill.
The morbidity of LPNAI is variable and the mortality is usually low, unless secondary infections are present or the birds are housed under poor management conditions. The morbidity and mortality of HPNAI may be near 100% within 2-12 days after the onset of the first signs of illness.
Clinical Signs
The severity of clinical signs depends on factors such as age, sex, species, concurrent infections, environment, as well as the pathogenicity of the virus.
Low pathogenic AI in wild birds usually produces no clinical signs. In domestic poultry, clinical signs reflect abnormalities in the respiratory, digestive, urinary, and reproductive organs. Generalized signs include depression, decreased activity, huddling, ruffled feathers, decreased feed and water consumption and occasionally greenish diarrhea. Respiratory manifestations include mild to severe coughing, sneezing, rales, rattles, excessive lacrimation, matted eyelids, and nasal discharge. Increased broodiness and a 5-30% decrease in egg production are observed in laying hens and breeders. Turkeys, infected with swine flu, may experience a much larger drop in egg production. Eggs may be thin-shelled and misshapen, but production will usually return to near-normal levels following recovery. Some viral strains may be associated with egg yolk peritonitis.
Some strains of highly pathogenic AI replicate poorly in wild birds and domestic ducks, producing few clinical signs. Ducks, quail, and turkeys may have mild sinusitis. Recent HPAI Asian H5N1 strains have produced more severe clinical signs in ducks and some wild birds.
In chickens, highly pathogenic AI produces clinical signs that reflect widespread viral replication and damage to multiple body systems. The disease may be fulminate, with some birds found dead without exhibiting any clinical signs. Birds that survive 3-7 days post infection may exhibit depression, ruffled feathers, decreased feed and water consumption, and a precipitous drop or total cessation of egg production. Diarrhea is often present. Early in the course of the disease, the feces may be bright green and watery. As the disease progresses, the droppings may become totally white. Birds may show cyanosis and edema of the skin, particularly of the combs, wattles, and periocular areas. Occasionally, edema and ecchymoses of the shanks and feet may also be observed. In some cases neurologic disease develops. Nervous deficits can include head and neck tremor, inability to stand, torticollis, loss of perching reflex and opisthotonus. Respiratory signs are less prominent in HPNAI than with LPNAI but may include coughing, sneezing (with blood tinged discharge), and rales. Most birds will die within 1-2 days following the onset of illness but some birds may survive for as long as a week. Recovery is uncommon.
Post-mortem Lesions
Lesions are variable in distribution and severity, depending greatly on host species, pathogenicity, and secondary infections.
Domestic ducks with LPNAI may have sinusitis, conjunctivitis, and other mild respiratory lesions. In chickens, lesions are more pronounced and are distributed throughout the upper respiratory tract and include catarrhal, fibrinous, serofibrinous, mucopurulent, or fibrinopurulent exudates. The tracheal mucosa may be edematous with congestion and hemorrhages. The infraorbital sinuses may be swollen and mucoid to mucopurulent nasal discharge may be present. Catarrhal to fibrinous inflammation may be observed in the coelomic cavity. The ovaries may be hemorrhagic with ova involution and degeneration. Eggs may be misshapen and fragile. The coelomic cavity may contain ovules or be filled with egg yolk, from ruptured ova, resulting in egg yolk peritonitis. This peritonitis may cause air sacculitis and diffuse coelomic cavity inflammation in birds that survive for 7-10 days post infection. Catarrhal to fibrinous enteritis may also be observed in the ceca and in the intestines.
Birds infected with HPNAI that die peracutely may not show any gross lesions. In chickens infected with the acute to subacute form, significant gross lesions are usually observed. At gross necropsy, one of the most frequent external findings is edema, manifested by swelling of the head (combs, wattles and periocular regions), upper neck, and feet. This edema may be accompanied by petechial and ecchymotic hemorrhages. Additionally, areas of nonfeathered skin such as the wattles and combs, are commonly cyanotic and have dark areas, caused by ecchymotic hemorrhages and necrotic foci. Internally, generalized hemorrhages and edema can be found in the muscles, especially the pectoral muscles, as well as the tissues of the hocks and feet. With most strains of HPNAI, pinpoint petechial hemorrhages are frequently observed along the abdominal fat, pericardium, serosal surfaces and peritoneum. Visceral organs may also be marked by hemorrhages. The glands of the proventriculus may have hemorrhagic lesions, particularly at the junction with the ventriculus (gizzard). The lining of the ventriculus may peel easily, frequently revealing underlying hemorrhages, erosions, and ulcers. The intestinal mucosa may have hemorrhagic areas, especially in the lymphoid tissue of the cecal tonsils and Peyer's patches in the small intestine.
In laying birds, the ovary may be hemorrhagic or degenerated with darkened areas of necrosis. In birds that survive, the peritoneal cavity is frequently filled with yolk from ruptured ova, causing severe airsacculitis and inflammation. The eggs are frequently soft and fragile due to decreased calcium deposition.
The trachea may appear normal, with the exception of excessive mucous accumulation, or it may be severely affected, with extensive hemorrhagic tracheitis. The lungs may be deep red in color, due to congestion and hemorrhage, and may exude edematous fluid when cut. The bursa of Fabricius and thymus are usually atrophic. The kidneys may be swollen, severely congested, and may by plugged with white urate deposits in the tubules. Necrotic foci may be present in the pancreas, spleen, heart, and occasionally the liver and kidneys.
Differential Diagnosis
AI should be differentiated from other respiratory diseases such as: Newcastle disease, infectious bronchitis, infectious laryngotracheitis, avian pneumovirus, and infectious coryza, among others. Other diseases that cause sudden death and septicemias, such as fowl cholera, should also be considered. Particular attention should be given to differentiating AI from mycoplasmosis, chlamydiosis and fowl cholera in young turkeys.
Diagnosis
In cases where HPAI is suspected, it is important to submit clinical samples to a recognized laboratory capable of carrying out AI diagnosis. Currently, there are a number of laboratories in the United States and around the world that have the capabilities to perform molecular testing, such as reverse transcriptase-polymerase chain reaction (RT-PCR) or a simplified faster real time reverse transcriptase polymerase chain reaction (rRT-PCR), to detect the presence of AI from clinical samples. The validated testing procedures performed at these laboratories are very sensitive, accurate, and rapid.
Cases involving respiratory signs and drop in egg production should be investigated for LPNAI. A presumptive diagnosis can be made in the field if compatible clinical signs and gross lesions are accompanied by a positive influenza type A test result, via commercial antigen capture immunoassay. However, because this test does not distinguish between LPAI and LPNAI, and may produce false-positive results, additional laboratory testing will be needed.
HPNAI should be suspected in any flock experiencing peracute death, high mortality, severe depression, inappetence, and a drastic decline in egg production. The presence of compatible gross lesions and the detection of Type A influenza antigen in oropharyngeal or tracheal swabs, supports a field diagnosis of HPNAI. However, these findings should be followed by more specific tests to identify and characterize the virus's antigenic and pathogenic type.
For HPNAI or LPNAI testing, collect and submit tracheal or cloacal swab samples as well as tissue samples taken from several internal organs such as the trachea, lung, spleen, cecal tonsils, or brain. Specimens should be collected from several birds, as it is not unusual for many specimens to fail to yield virus. If large numbers of birds are dead, you can submit up to 5 swab samples in one tube but do not mix swabs taken from different sites.
Blood for serum testing should be collected from several birds using standard serum tubes. If the specimens can be delivered to a laboratory within 24 hours, they should be placed on cold packs for shipping. If delivery will take longer, quick freeze the specimens in dry ice or liquid nitrogen, but do not allow the samples to thaw during transit. Freezing and storage at standard freezer temperature (-20 degrees C) is not recommended.
In the laboratory, virus isolation is usually performed by inoculation of embryonated chicken eggs with organ homogenizates or with tracheal or cloacal swabs. The AI virus is detected if the allantoic fluid of inoculated eggs hemagglutinates chicken red blood cells. The virus can be sub-typed using specific sera against all known HA and NA proteins. Specific hemagglutination inhibition is the basis of the serologic test for influenza antibodies. The ELISA (enzyme-linked immunosorbent assay) and AGID (agar gel immunodiffusion) tests can be used for determining the presence of antibody in the sera.
Any suspected case of Notifiable Avian Influenza (NAI) in the United States should be immediately reported to the State Veterinarian for the State where the case is suspected. A current list of animal health officials (State Veterinarians) contact information is available at http://www.usaha.org/members.shtml#agency . For suspected cases of NAI in other countries, notification should be given to the Chief Veterinary Officer (or OIE Country Delegate) of the country or territory where the case is suspected. A current list of Chief Veterinary Officers is available at http://www.oie.int/eng/OIE/PM/en_PM.htm?e1d1 .
Given the changes in the zoonotic potential recently seen with the emergence of human infections with the HPAI Asian H5N1 strain, it is recommended to treat any future HPAI as a potentially zoonotic disease. Seek the advice of the Public Health authorities in dealing with any outbreak action. A current list of Public Health authorities in the US is available at: www.astho.org or at http://www.statepublichealth.org/. Information on Public Health authorities outside the US is available at: http://www.who.int/countries/en/
Prevention and Control
The recommended strategy for controlling NAI is eradication. This requires 5 components including: 1) inclusion and exclusion biosecurity practices, 2) increasing host resistance through vaccination, 3) diagnostics and surveillance, 4) elimination of infected animals from the flock, and 5) educating personnel in AI control strategies.
1) Implementing biosecurity practices includes controlling human traffic, quarantining birds before introduction, proper cleaning and disinfection of facilities, keeping healthy birds away from contact with sick birds and wild birds, and incubating eggs only from clean flocks. Strict quarantine measures not only reduce the possibility of introducing Avian Influenza to a farm but also to a region or country.
2) Inactivated vaccines have been shown to be effective but are fairly expensive. Recombinant fowl poxvirus, carrying an inserted H5 gene, have shown similar efficacy in chickens. However, no vaccine will provide absolute prevention against infections or control environmental contamination. Other major drawbacks to current vaccines for controlling NAI include the labor associated with administering vaccine injections to individual birds and the need to match the vaccine to the field hemagglutination subtype in order to confer protection.
3) Diagnostics and surveillance play a critical role in controlling NAI. In the field, appropriate diagnostic samples should be collected and submitted to an accredited laboratory, capable of carrying out AI diagnosis. If NAI is suspected, field samples are inoculated into embryonated eggs, and after incubation for several days, allantoic fluid from inoculated eggs are tested for hemagglutinating activity. Upon detection of any hemaglutinating virus, a presumptive diagnosis of NAI is made and an immediate quarantine of the farms under testing should be made. This quarantine is designed to limit the movement of birds, equipment, and personnel to prevent potential viral dissemination. Agents that show hemagglutinating activity are then further identified in the laboratory by a number of techniques including the newer rapid rRT-PCR .
4) If NAI infection is confirmed by laboratory testing, all birds within the flock should be culled, utilizing humane euthanasia techniques, and disposed of properly. This depopulation effort requires the use of proper personal protective equipment (PPE) in accordance with national or international standards.
5) Finally, it is important to have personnel trained in animal disease control measures before a potential outbreak event. The success of disease control activities requires advanced preparation and familiarity with proper biosecurity and emergency management protocols
Due to the significant economic and public health implications of an AI outbreak, all cases of LPNAI and HPNAI must be reported to the State and federal veterinary authorities in order to facilitate global virus control measures.
Selected References
- Charlton, B. R. (ed). 2006. Avian Disease Manual, 6th ed. American Association of Avian Pathologists (AAAP), 953 College Station Road, Athens, Georgia 30602-4875.
- Swayne, D.E. 2008. Avian Influenza. In Foreign Animal Diseases, 7th ed. United States Animal Health Association. C. Brown and A. Torres (ed.).. Boca Publications Group, Inc. Boca Raton, FL. (http://www.usaha.org/pubs/fad.pdf)
- Swayne, D.E. and D.A. Halvorson. 2008. Influenza. In Diseases of Poultry, 12th ed. Y.M. Saif. et al. (ed.). Blackwell Publishing, Ames, Iowa.
- World Organization for Animal Health (OIE) website. 2008. www.oie.int
Thank you to the following individuals for reviewing these materials:
Dennis Senne
Alfonso Torres
Alejandro Banda
Benjamin Lucio-Martinez
Jose Bruzual
Jaime Ruiz
John Coakley
