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Epidemiology of Avian Influenza

Avian influenza - the disease

Avian influenza (AI) is caused by specified viruses that are members of the family Orthomyxoviridae and placed in the genus influenzavirus A . There are three influenza genera - A, B and C; only influenza A viruses are known to infect birds. Diagnosis is by isolation and characterisation of the virus. This is because infections in birds can give rise to a wide variety of clinical signs that may vary according to the host, strain of virus, the host's immune status, presence of any secondary exacerbating organisms and environmental conditions.

AI viruses are members of the family Orthomyxoviridae. The influenza viruses that constitute this family are classified into types A, B or C based on differences between their nucleoprotein and matrix protein antigens. AI viruses belong to type A. Influenza viruses are further categorised into subtypes according to the antigens of the haemagglutinin (H) and neuraminidase (N) projections on their surfaces. There are 16 haemagglutinin subtypes and 9 neuraminidase subtypes of influenza A viruses, and AI viruses have representatives in all of these subtypes. However, to date all highly pathogenic AI viruses that cause generalised rather than respiratory disease belong to either the H5 or H7 subtypes. For example, the classical fowl plague virus is H7N7 and the virus responsible for the major epidemic in the eastern United States in 1983-84 was H5N2. However, not all H5 and H7 viruses are virulent for poultry.

The pathogenicity of AI viruses is correlated to the ability of trypsin to cleave the haemagglutinin molecule into two subunits. Highly pathogenic strains of H5 and H7 viruses have several amino acid residues at the cleavage site. Trypsin sensitivity and amino acid sequencing can be used diagnostically to determine whether or not an isolated virus is potentially pathogenic.

Natural hosts
Domestic fowl, ducks, geese, turkeys, guinea fowl, quail and pheasants are susceptible. Disease outbreaks occur most frequently in domestic fowl and turkeys. A particular isolate may produce severe disease in turkeys but not in chickens or any other avian species. Therefore, it would be impossible to generalize on the host range for avian influenza, for it will likely vary with the isolate. This assumption is supported by reports of farm outbreaks where only a single avian species of several species present on the farm became infected. Many species of wild birds particularly water birds and seabirds - are also susceptible, but infections in these birds are generally sub-clinical.

World Distribution
AI viruses are probably ubiquitous in wild water birds. Pathogenic strains could emerge and cause disease in domestic poultry in any country at any time without warning. In fact, outbreaks have occurred at irregular intervals on all continents. The most serious outbreaks in recent times have been reported in Hong Kong 1997-1998 and 2003, Chile 2002, The Netherlands 2003 and South East Asia 2004-2006.

The immediate source of infection for domestic poultry can seldom be ascertained, but most outbreaks probably start with direct or indirect contact of domestic poultry with water birds. Many of the strains that circulate in wild birds are either non-pathogenic or mildly pathogenic for poultry. However, a virulent strain may emerge either by genetic mutation or by reassortment of less virulent strains. Scientific evidence indicates that the former mechanism occurred in 1983-1987 in the eastern part of The United States of America.

Swine appear to be important in the epidemiology of infection of turkeys with swine influenza virus when they are in close proximity. Other mammals do not appear to be involved in the epidemiology of HPAI. The infection of humans with an H5 avian influenza virus in Hong Kong in 1997 has resulted in a reconsideration of the role of the avian species in the epidemiology of human influenza.

Once AI is established in domestic poultry, it is a highly contagious disease and wild birds are no longer an essential ingredient for spread. Infected birds excrete virus in high concentration in their faeces and also in nasal and ocular discharges. Once introduced into a flock, the virus is spread from flock to flock by the usual methods involving the movement of infected birds, contaminated equipment, egg flats, feed trucks, and service crews, to mention a few. The disease generally spreads rapidly in a flock by direct contact, but on occasions spread is erratic.

Airborne transmission may occur if birds are in close proximity and with appropriate air movement. Birds are readily infected via instillation of virus into the conjunctival sac, nares, or the trachea.Preliminary field and laboratory evidence indicates that virus can be recovered from the yolk and albumen of eggs laid by hens at the height of the disease. The possibility of vertical transmission is unresolved; however, it is unlikely infected embryos could survive and hatch. Attempts to hatch eggs in disease isolation cabinets from a broiler breeder flock at the height of disease failed to result in any AI-infected chickens. This does not mean that broken contaminated eggs could not be the source of virus to infect chicks after they hatch in the same incubator. The hatching of eggs from a diseased flock would likely be associated with considerable risk.

Incubation Period
The incubation period is 3 to 5 days in general but may be longer. Maximal incubation period is 21 days as defined by the OIE Terrestrial Animal Health Code.

Clinical signs

The clinical signs are very variable and are influenced by factors such as the virulence of the infecting virus, species affected, age, sex, concurrent diseases and environment.

In highly pathogenic avian influenza , the disease appears suddenly in a flock and many birds die either without premonitory signs or with minimal signs of depression, inappetence, ruffled feathers and fever. Other birds show weakness and a staggering gait. Hens may at first lay soft-shelled eggs, but soon stop laying. Sick birds often sit or stand in a semi-comatose state with their heads touching the ground. Combs and wattles are cyanotic and oedematous, and may have petechial or ecchymotic haemorrhages at their tips. Profuse watery diarrhoea is frequently present and birds are excessively thirsty. Respiration may be laboured. Haemorrhages may occur on unfeathered areas of skin. The mortality rate varies from 50 to 100%.

In broilers, the signs of disease are frequently less obvious with severe depression, inappetence, and a marked increase in mortality being the first abnormalities observed. Oedema of the face and neck and neurological signs such as torticollis and ataxia may also be seen. The disease in turkeys is similar to that seen in layers, but it lasts 2 or 3 days longer and is occasionally accompanied by swollen sinuses. In domestic ducks and geese the signs of depression, inappetence, and diarrhea are similar to those in layers, though frequently with swollen sinuses. Younger birds may exhibit neurological signs.

Inactivated quality assured oil-emulsion vaccines have been demonstrated to be effective in reducing mortality, preventing disease, or both, in chickens and turkeys. These vaccines, however, may not prevent infection in some individual birds, and if infected could shed virulent virus. Nevertheless, the amount virus shed is considerable less than that of non-vaccinated and infected birds. It is imperative that the circulating antigenic avian influenza virus is known and the vaccine represent this antigenic strain, since there is no cross-protection among the 15 known HA subtypes. A recombinant fowl pox virus vaccine containing the gene that codes for the production of the H5 antigen has recently been licensed in some countries but is not widely used currently. Homologous inactivated vaccines (H5N1) and heterologous inactivated vaccines (H5Nx) are the most commonly used.

It is imperative that the circulating antigenic avian influenza virus be known and the vaccine represent this antigenic strain, since there is no cross-protection among the 15 known HA subtypes. A recombinant fowl pox virus vaccine containing the gene that codes for the production of the H5 antigen has recently been licensed in some countries but is not widely used currently.

Birds that die of peracute disease may show minimal gross lesions, consisting of dehydration and congestion of viscera and muscles.

In birds that die after a prolonged clinical course, petechial and ecchymotic haemorrhages occur throughout the body, particularly in the larynx, trachea, proventriculus and epicardial fat, and on serosal surfaces adjacent to the sternum. There is extensive subcutaneous oedema, particularly around the head and hocks. The carcase may be dehydrated. Yellow or grey necrotic foci may be present in the spleen, liver, kidneys and lungs. The air sac may contain an exudate. The spleen may be enlarged and haemorrhagic.

Avian influenza is characterised histologically by vascular disturbances leading to oedema, haemorrhages and perivascular cuffing, especially in the myocardium, spleen, lungs, brain and wattles. Necrotic foci are present in the lungs, liver and kidneys. Gliosis, vascular proliferation and neuronal degeneration may be present in the brain.

Differential diagnosis
The following diseases must be considered in the differential diagnosis of virulent AI:

Other diseases causing sudden high mortality
Newcastle disease
infectious laryngotracheitis
duck plague
acute poisonings

Other diseases causing swelling of the combs and wattles:
acute fowl cholera and other septicaemic diseases
bacterial cellulitis of the comb and wattles

Less severe forms of the disease may be confused with, or complicated by, many other diseases with respiratory or enteric signs. AI should be suspected in any disease outbreak in poultry that persists despite the application of preventive and therapeutic measures for other diseases.

Laboratory diagnostic specimens
Specimens required
Specimens should be collected from birds showing signs of acute disease or recently dead (<24 h). Swabs can be taken from the cloacae and oropharynx/trachea of sick and dead birds and then be stored in 3 ml of viral transport medium at low temperature. For dead birds, 3 groups of organs should be separately collected during the necropsy and stored in viral transport medium. These are trachea/lung, brain and digestive organs (pancreas, proventriculus, cecae, intestine). All any obviously abnormal tissue has also to be sampled. Blood should also be collected from live and dead birds (heart blood) for serum testing. Viral transport media can either be prepared locally at a laboratory (it can be isotonic phosphate buffered saline (PBS), pH 7.0-7.4, containing antibiotics, for example 100 g/ml gentamicin sulfate, 2 g/ml amphotericin B) or commercial medium may be purchased. Samples should be taken from several birds in the same suspicion flock.

Transport of specimens
Tissues and swab material should be chilled at 4 °C and forwarded on water ice or with frozen gel packs. If delays of greater than 48 hours are expected in transit, these specimens should be frozen at -80 °C and forwarded with dry ice or liquid nitrogen.

Laboratory procedures
The gold standard of virus detection for avian influenza egg isolation by inoculation of swab material or tissue homogenates into 9-11-day-old embryonated chicken eggs by the allantoic sac route. After 7 days of egg incubation, the presence of hemagglutining virus can be detected in harvested allantoic fluid. The isolate type and subtype can be determined by haemagglutination/neuraminidase inhibition tests using specific antisera. Furthermore, conventional and real-time RT-PCR (Reverse Transcription-Polymerase Chain Reaction) are methods of choice for the rapid detection of AI viruses. Real-time RT-PCR allows the rapid testing of large numbers of samples. Commercial antigen detection Enzyme-Linked Immunosorbent (ELISA) kits allow a rapid and easy diagnosis from tissue homogenates and respiratory swabs but a confirmation of the results by viral isolation and/or RT-PCR is necessary.

Once the sub-type of the isolate is determined, it is necessary to assess its pathogenicity for domestic poultry .Pathogenicity tests are carried out by intravenous inoculation of 4-to-8-week-old chickens with infective allantoic fluid. An intravenous pathogenicity index (IVPI) is determined from the number of healthy, sick, paralysed and dead birds observed each day for 10 days post inoculation. Any virus that has an IVPI greater than 1.2 is considered as a HPAI virus. For H5 and H7 subtypes, pathogenicity is associated with the presence of multiple basic amino acids at the cleavage site of the haemagglutinin. Then, the amino acid sequence of this haemagglutinin cleavage site must be determined for all H5 and H7 viruses of low pathogenicity in chickens. If the sequence is similar to that observed for other highly pathogenic AI isolates, the isolate will be considered highly pathogenic.

Detection of antibodies against type A AI viruses can be performed with AGID (Agar gel immunodiffusion test) or wilth ELISA. Commercial indirect or competitive ELISA kits are available on the market and are convenient for large-scale monitoring. HI and NI tests allow the detection respectively of specific anti-hemagglutinin and anti-neuraminidase antibodies. It is important to note that the use of serology is secondary in HPAI since infected birds will die before secreting antibodies. Then, serology is used for LPAI infections and for the post-vaccination monitoring. Finally, serological surveillance using a DIVA test (Differentiatiing between Infected and Vaccinated Animals) with DIVA vaccines makes possible the mass vaccination instead of massive culling.