Avian Influenza (Avian Influenza Virus - HPAI & LPAI)

Avian influenza (AI) is a highly contagious viral disease affecting domestic poultry, wild birds, and occasionally mammals. It is caused by type A influenza viruses of the family Orthomyxoviridae, genus Influenzavirus A. The disease manifests in two distinct pathotypes: low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI). LPAI typically causes mild respiratory signs, decreased egg production, and increased mortality, while HPAI is a systemic, often fatal disease with mortality rates approaching 100% in susceptible poultry. The virus is classified based on the antigenicity of its surface glycoproteins, hemagglutinin (H1-H16) and neuraminidase (N1-N9). HPAI is exclusively associated with H5 and H7 subtypes, although not all H5/H7 viruses are highly pathogenic. The disease has significant global economic impact, causing trade restrictions, culling of millions of birds, and posing a zoonotic risk. In commercial poultry, AI affects broilers, layers, breeders, turkeys, ducks, and backyard flocks, with severity varying by species and age. The disease is a notifiable condition under the World Organisation for Animal Health (WOAH) and requires immediate reporting and control measures.

The primary causative agent is influenza A virus, a negative-sense, single-stranded RNA virus with a segmented genome (8 segments). The viral envelope contains two major glycoproteins: hemagglutinin (HA) and neuraminidase (NA). HA is responsible for viral attachment to sialic acid receptors on host cells and fusion of the viral envelope with the endosomal membrane. NA facilitates viral release by cleaving sialic acid residues. The pathogenicity of the virus is determined by the amino acid sequence at the HA cleavage site. HPAI viruses possess multiple basic amino acids at this site, allowing cleavage by ubiquitous host proteases (e.g., furin), leading to systemic infection. LPAI viruses have a single basic amino acid, restricting cleavage to trypsin-like proteases in the respiratory and intestinal tracts. Subtypes of concern include H5N1, H5N2, H5N8, H7N1, H7N3, and H7N9. The virus is enveloped and susceptible to lipid solvents, detergents, and common disinfectants. It is inactivated by heat (56°C for 30 minutes), acidic pH, and ultraviolet radiation. In organic material, the virus can survive for extended periods, especially at low temperatures.

Avian influenza is distributed worldwide, with outbreaks occurring in Asia, Europe, Africa, and North America. The epidemiology is influenced by the poultry sector (broilers, layers, breeders, turkeys, ducks), age of birds, housing system (cages, free-range, deep litter), ventilation, biosecurity level, season, and wild bird vectors. Wild waterfowl, particularly ducks and geese, are the natural reservoir, harboring LPAI viruses without clinical signs. These birds can shed the virus via feces and respiratory secretions, contaminating water and feed. Transmission to domestic poultry occurs through direct contact with infected wild birds, contaminated fomites, vehicles, equipment, and personnel. The disease is more common in temperate seasons, with peaks during migration periods. In commercial flocks, morbidity can reach 100% in HPAI, with mortality ranging from 50% to 100% within 48 hours. LPAI causes lower mortality (usually <5%) but can result in significant production losses, including a 10-50% drop in egg production and increased feed conversion ratio (FCR) by 0.1-0.3. High-density poultry operations, poor biosecurity, and live bird markets are major risk factors. The incubation period is 3-14 days, depending on the strain and dose.

The pathogenesis of avian influenza begins with viral entry via the respiratory or oral route. The virus attaches to sialic acid receptors on epithelial cells, with preference for α-2,3-linked sialic acid in avian species. After endocytosis, the viral RNA is released, and replication occurs in the nucleus. New virions bud from the cell membrane, causing cell lysis and release of progeny viruses. In LPAI, infection is localized to the respiratory and intestinal tracts, causing mild inflammation and necrosis of epithelial cells. In HPAI, the HA cleavage site allows systemic spread via the bloodstream, leading to viremia. The virus replicates in multiple organs, including the vascular endothelium, causing severe endothelial damage, increased vascular permeability, and disseminated intravascular coagulation. This results in widespread hemorrhage, edema, and necrosis in organs such as the comb, wattles, legs, lungs, kidneys, and pancreas. The virus also induces a strong inflammatory response, with release of cytokines and chemokines, contributing to the systemic inflammatory response syndrome (SIRS). Lymphoid tissues, including the bursa of Fabricius and thymus, undergo necrosis and depletion, leading to immunosuppression. The rapid progression of HPAI often results in death before significant antibody response develops.

Intrinsic factors include genetic susceptibility, with turkeys and chickens being highly susceptible to HPAI, while ducks and geese are more resistant. Age plays a role, with younger birds often showing more severe clinical signs. Immune status is critical; birds with prior exposure to LPAI may have partial immunity, but vaccination can mask clinical signs and complicate surveillance. High production stress, such as peak lay, can increase susceptibility. Extrinsic factors include poor biosecurity, allowing contact with wild birds or contaminated fomites. High stocking density and poor ventilation increase the concentration of viral particles and stress. Ammonia accumulation in poultry houses damages the respiratory epithelium, facilitating viral entry. Wet litter and poor sanitation promote viral survival. Feed contamination with wild bird feces can introduce the virus. Vaccination failure due to antigenic mismatch or improper administration can leave flocks vulnerable. In addition, the presence of other immunosuppressive diseases, such as infectious bursal disease (IBD) or chicken anemia virus (CAV), can exacerbate AI severity.

Clinical signs vary by pathotype and species. In LPAI, signs are often mild and may include decreased feed and water consumption, slight depression, ruffled feathers, and a drop in egg production (10-50%) with increased numbers of misshapen, soft-shelled, or shell-less eggs. Respiratory signs such as coughing, sneezing, rales, and sinusitis may be present, especially in turkeys. In HPAI, the onset is sudden, with severe depression, huddling, and a dramatic drop in feed and water intake. Birds may exhibit respiratory distress, with gasping and cyanosis of the comb and wattles. Edema of the head, face, and neck is common. Diarrhea may be present, with greenish or watery feces. Neurological signs include torticollis, ataxia, paralysis, and tremors. Egg production ceases completely in layers. Mortality can reach 100% within 48-72 hours. In ducks and geese, HPAI may cause only mild signs or be asymptomatic, but they can shed the virus. In backyard flocks, the disease may be less severe but still cause significant mortality.

Differential diagnoses include: 1) Newcastle disease (ND): caused by avian paramyxovirus type 1, presents with similar respiratory, neurological, and digestive signs. ND often causes tracheal hemorrhages and intestinal lesions, but lacks the severe edema and cyanosis of HPAI. Laboratory differentiation via virus isolation and RT-PCR is essential. 2) Infectious bronchitis (IB): a coronavirus causing respiratory signs and egg production drops, but mortality is low and no systemic signs. 3) Infectious laryngotracheitis (ILT): a herpesvirus causing severe respiratory distress, gasping, and bloody mucus, but lesions are confined to the upper respiratory tract. 4) Fowl cholera (Pasteurella multocida): a bacterial septicemia causing sudden death, cyanosis, and hemorrhages, but responds to antibiotics and can be cultured. 5) Mycoplasmosis (Mycoplasma gallisepticum): chronic respiratory disease with sinusitis and airsacculitis, but no systemic signs. 6) Avian encephalomyelitis (AE): a picornavirus causing neurological signs in young chicks, but no respiratory or systemic signs. 7) Egg drop syndrome (EDS): an adenovirus causing eggshell abnormalities, but no systemic illness. 8) Highly pathogenic avian influenza must also be differentiated from exotic Newcastle disease (END) and other viral hemorrhagic diseases. Definitive diagnosis requires laboratory confirmation.

The diagnostic algorithm for avian influenza begins with flock history and clinical signs. If HPAI is suspected, immediate notification of veterinary authorities is required. Step 1: Perform a thorough clinical examination, noting the rapid onset, high mortality, and characteristic signs (cyanosis, edema, neurological). Step 2: Conduct a gross necropsy on several birds, looking for lesions such as hemorrhages in the trachea, lungs, proventriculus, and intestines; edema and congestion of the comb and wattles; and necrosis of the pancreas and spleen. Step 3: Collect samples for laboratory testing: oropharyngeal and cloacal swabs from live birds, and tissues (trachea, lung, spleen, kidney, brain) from dead birds. Samples should be placed in viral transport media and kept cold. Step 4: Submit samples to an accredited laboratory for virus isolation in embryonated chicken eggs, which is the gold standard. Step 5: Perform molecular testing using real-time RT-PCR targeting the matrix gene, followed by subtyping for H5 and H7. Step 6: Determine the pathogenicity by sequencing the HA cleavage site or by intravenous pathogenicity index (IVPI) in chickens. Step 7: Conduct serological tests, such as agar gel immunodiffusion (AGID) or ELISA, to detect antibodies in surviving birds. Step 8: Confirm the diagnosis and implement control measures, including quarantine, depopulation, and disinfection.

Serology: ELISA can detect antibodies to the nucleoprotein, but cannot distinguish subtypes. Hemagglutination inhibition (HI) test is used for subtyping, with titers ≥1:16 considered positive. AGID is used for flock screening. Molecular diagnostics: Real-time RT-PCR is the preferred method for rapid detection, with high sensitivity and specificity. It can differentiate HPAI from LPAI by targeting the HA cleavage site. Virus isolation in embryonated eggs is confirmatory, with allantoic fluid tested for hemagglutination activity. Hematology: In HPAI, leukopenia and lymphopenia may be observed. Blood chemistry: Elevated liver enzymes (AST, ALT) and creatinine kinase may be seen due to organ damage. In LPAI, findings are often unremarkable. Coccidiosis lesion scoring is not applicable. Mycotoxin feed assays are not relevant unless concurrent exposure is suspected.

Imaging is not commonly used in avian influenza diagnosis. Radiography may show pulmonary consolidation or airsacculitis in LPAI, but is not specific. Ultrasonography is rarely used in poultry. Gross necropsy photography is essential for documentation and can reveal characteristic lesions: severe edema and cyanosis of the comb and wattles, petechial hemorrhages on the serosal surfaces, and necrotic foci in the pancreas and spleen. In HPAI, the tracheal mucosa may be hemorrhagic, and the lungs may be congested and edematous. The kidneys may be swollen and urate deposits may be present. In layers, the ovary may be hemorrhagic and regressed.

Gross necropsy lesions in HPAI include: severe edema and cyanosis of the comb and wattles, subcutaneous edema of the head and neck, petechial and ecchymotic hemorrhages on the heart, serosal surfaces, and abdominal fat, and hemorrhagic tracheitis. The lungs are congested and edematous. The pancreas may show multifocal necrosis. The spleen is enlarged and mottled. The kidneys are swollen and congested. In LPAI, lesions are limited to the respiratory tract, with mild tracheitis, sinusitis, and airsacculitis. Microscopic histopathology in HPAI reveals severe necrosis of vascular endothelium, leading to hemorrhage and edema. There is necrosis and inflammation in multiple organs, including the heart, lung, kidney, and brain. Lymphoid depletion is evident in the bursa of Fabricius and thymus. In the brain, nonsuppurative encephalitis with perivascular cuffing and neuronal necrosis may be seen. In LPAI, histopathology shows mild to moderate tracheitis with loss of cilia and infiltration of heterophils and lymphocytes.

There is no specific treatment for avian influenza. For LPAI, supportive care may be provided, including increasing ambient temperature, providing electrolytes and vitamins in water, and reducing stress. Antibiotics may be used to control secondary bacterial infections, but are not effective against the virus. Antiviral drugs such as oseltamivir are not approved for poultry and are not practical. For HPAI, the standard control measure is depopulation of infected flocks to prevent spread. This is mandated by regulatory authorities. In some countries, vaccination is used as a control strategy, but it is not a substitute for biosecurity. Vaccination can reduce clinical signs and viral shedding, but may mask infection. Inactivated vaccines are available for H5 and H7 subtypes. In emergency situations, a 'ring vaccination' strategy may be employed around an outbreak. Biosecurity measures include quarantine, movement restrictions, and enhanced disinfection. Supportive therapy for LPAI may include: amoxicillin at 20 mg/kg body weight orally twice daily for 5-7 days, or oxytetracycline at 20 mg/kg IM once, or tylosin at 20 mg/kg IM once, or enrofloxacin at 10 mg/kg IM once (where legal). In drinking water, amoxicillin at 100-200 mg/L for 3-5 days, or oxytetracycline at 200-400 mg/L for 3-5 days, or tylosin at 500 mg/L for 3-5 days. Vitamins and electrolytes can be added to water at label rates.

The prognosis for LPAI is generally good, with recovery in 1-2 weeks, but egg production may take several weeks to return to normal. Mortality is low, but secondary infections can increase losses. The prognosis for HPAI is grave, with mortality approaching 100% in susceptible flocks. Depopulation is usually required, and the flock is lost. In flocks that survive, there may be permanent damage to egg production and increased susceptibility to other diseases. The economic impact is severe, including loss of birds, trade restrictions, and costs of control measures.

After an outbreak, a structured monitoring schedule is essential. For LPAI, flocks should be monitored for clinical signs and production parameters for at least 4 weeks. Serological testing (ELISA or HI) should be repeated 2-4 weeks after recovery to confirm seroconversion. For HPAI, after depopulation, the premises must be cleaned and disinfected thoroughly. Environmental sampling (swabs from surfaces, litter, and equipment) should be conducted to ensure the virus is eliminated. A sentinel flock of susceptible birds may be placed on the premises after 21 days to detect any residual virus. The facility should be restocked only after negative results. Biosecurity audits should be conducted to identify and correct gaps. Regular surveillance of wild birds and neighboring flocks is recommended.

Pearls: 1) In HPAI, the comb and wattles often appear cyanotic (blue-purple) due to vascular damage; this is a red flag. 2) The pancreas often shows multifocal necrosis, which is highly suggestive of HPAI. 3) Sudden high mortality with no prior signs is a hallmark of HPAI. 4) In LPAI, a drop in egg production with misshapen eggs is a common presentation. 5) Always consider AI in any flock with respiratory signs and high mortality. Pitfalls: 1) Do not confuse HPAI with Newcastle disease; laboratory confirmation is essential. 2) Do not delay reporting suspected HPAI; early detection is critical. 3) Avoid vaccinating against AI without proper surveillance, as it can mask infection. 4) Do not rely solely on clinical signs; subclinical LPAI can spread unnoticed. 5) Ensure proper sample collection and shipping to avoid false negatives.

Antibiotics for secondary infections: Amoxicillin: 20 mg/kg PO BID for 5-7 days; in water: 100-200 mg/L for 3-5 days. Oxytetracycline: 20 mg/kg IM once; in water: 200-400 mg/L for 3-5 days. Tylosin: 20 mg/kg IM once; in water: 500 mg/L for 3-5 days. Tilmicosin: 10-20 mg/kg PO once; in water: 75 mg/L for 3 days. Enrofloxacin (where legal): 10 mg/kg IM once; in water: 50-100 mg/L for 3-5 days. Florfenicol: 20 mg/kg IM once; in water: 400 mg/L for 3-5 days. Supportive vitamins: Vitamin A: 10,000 IU/kg feed; Vitamin D3: 2,000 IU/kg feed; Vitamin E: 100 IU/kg feed; Vitamin C: 200 mg/L water; Vitamin K: 5 mg/kg feed. Electrolytes: Provide commercial electrolyte solutions at label rates. Vaccines: Inactivated H5 and H7 vaccines are available; administer SC or IM at 0.5 ml per bird, with a booster after 3-4 weeks. Live vaccines are not recommended for AI. Anticoccidials are not relevant unless coccidiosis is concurrent.

Landmark studies include: 1) Swayne et al. (2013) in 'Diseases of Poultry' provide comprehensive coverage of AI pathogenesis and control. 2) Alexander (2007) reviewed the epidemiology of HPAI in Europe and Asia. 3) Capua and Alexander (2009) discussed the control strategies for AI, including vaccination. 4) A meta-analysis by Elbers et al. (2004) evaluated the diagnostic accuracy of RT-PCR for AI. 5) The AAAP's 'Avian Disease Manual' (2013) provides practical guidelines for diagnosis and management. 6) WOAH (2019) guidelines emphasize biosecurity and rapid response. 7) A study by Spackman et al. (2002) developed the real-time RT-PCR assay for AI detection. 8) Pantin-Jackwood and Swayne (2009) studied the pathogenesis of H5N1 in different poultry species. 9) A field trial by Ellis et al. (2004) in Hong Kong demonstrated the efficacy of vaccination in reducing viral shedding. 10) Consensus recommendations from the WVPA (2018) highlight the importance of surveillance and early detection.