Avian Influenza A(H9N2) in Italy: Epidemiology, Surveillance, and Control

Etiology and Virological Characteristics

Avian influenza A(H9N2) is a subtype of the influenza A virus belonging to the family Orthomyxoviridae. The virus possesses a segmented, negative-sense single-stranded RNA genome comprising eight gene segments (PB2, PB1, PA, HA, NP, NA, M, and NS) [1]. The hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins define the H9N2 subtype. H9N2 viruses are classified as low-pathogenicity avian influenza (LPAI) viruses, as they lack the multibasic cleavage site motif in the HA protein that is characteristic of highly pathogenic avian influenza (HPAI) strains [2]. The HA cleavage site of H9N2 viruses typically contains a single basic amino acid (arginine or lysine) at the cleavage site, restricting proteolytic activation to trypsin-like enzymes present in the respiratory and intestinal epithelia of avian hosts [3].

The H9N2 subtype has undergone extensive genetic diversification, with multiple lineages (e.g., G1-like, Y280-like, and Korean-like) circulating globally [4]. The virus exhibits a broad host range, infecting both galliform and anseriform species, and has been isolated from various wild waterfowl and shorebird species [5]. The H9N2 virus is capable of reassortment with other influenza A subtypes, a phenomenon that has generated novel genotypes with altered host tropism and pathogenicity [6].

Epidemiology in Italy

The epidemiology of avian influenza A(H9N2) in Italy is characterized by a complex interplay between domestic poultry populations and wild bird reservoirs. Italy is situated along major migratory flyways of the Palearctic region, specifically the Black Sea/Mediterranean flyway, which facilitates the introduction and dissemination of LPAI viruses from wild waterfowl into domestic poultry holdings [7]. The Lombardy region in northern Italy, a major poultry production area, has been a focal point for surveillance activities [8].

The detection of H9N2 in Italian poultry has been documented through passive and active surveillance programs. The virus is primarily associated with mild or subclinical respiratory infections in gallinaceous birds, particularly in broiler and layer flocks [1]. The clinical presentation of H9N2 infection in poultry is often indistinguishable from other respiratory pathogens, including infectious bronchitis virus (IBV), avian metapneumovirus (aMPV), and Mycoplasma gallisepticum [2]. Coinfections with bacterial agents, such as Escherichia coli and Ornithobacterium rhinotracheale, can exacerbate the clinical severity of H9N2 infection, leading to increased morbidity and mortality in affected flocks [3].

The role of wild birds as reservoirs for H9N2 in Italy is well established. Surveillance studies conducted in the Lombardy region have detected H9N2 viral RNA in fecal samples collected from wild waterfowl, including mallards (Anas platyrhynchos) and Eurasian teal (Anas crecca) [7]. The detection of H9N2 in the African sacred ibis (Threskiornis aethiopicus), an invasive species in Italy, highlights the potential for synanthropic and invasive bird species to act as bridging hosts, facilitating the transmission of LPAI viruses from wild waterfowl to domestic poultry [4]. The African sacred ibis has been identified as a competent host for H9N2, with viral shedding detected in both oropharyngeal and cloacal samples [4].

The spatial distribution of H9N2 risk in Italy has been modeled using spatial multi-criteria decision analysis (SMCDA). This approach integrates environmental, ecological, and anthropogenic risk factors, including waterfowl density, poultry farm density, and land cover type, to generate risk maps for LPAI introduction and spread [6]. The SMCDA model for the Lazio and Toscana regions of central Italy identified areas with high waterfowl abundance and proximity to poultry farms as high-risk zones for H9N2 incursion [6].

Clinical Signs and Pathology

The clinical signs of H9N2 infection in poultry are highly variable and depend on the host species, age, immune status, and the presence of concurrent infections. In broiler chickens, H9N2 infection typically manifests as a mild respiratory disease characterized by sneezing, coughing, nasal discharge, and conjunctivitis [2]. In layer flocks, a transient drop in egg production (5-15%) and an increase in the proportion of shell-less or misshapen eggs may be observed [3]. The clinical signs are often more pronounced in young birds (less than 4 weeks of age) compared to adult birds [4].

Pathological findings in H9N2-infected birds are generally confined to the upper respiratory tract. Gross lesions include mild to moderate catarrhal rhinitis, tracheitis, and airsacculitis [5]. Histopathological examination reveals epithelial cell desquamation, mononuclear cell infiltration in the lamina propria, and hyperplasia of the mucosal glands in the trachea and nasal turbinates [6]. In cases of secondary bacterial infection, fibrinous exudate may be observed in the air sacs and pericardial sac [7].

Surveillance and Diagnostic Approaches

Surveillance for avian influenza A(H9N2) in Italy is conducted under the framework of the European Union (EU) avian influenza surveillance directives and the World Organisation for Animal Health (WOAH) guidelines. The surveillance strategy in Italy is stratified into two components: passive surveillance in wild birds and active surveillance in domestic poultry [5]. Passive surveillance involves the collection of samples (oropharyngeal and cloacal swabs) from dead or moribund wild birds found during mortality events [7]. Active surveillance in domestic poultry is based on the collection of samples from healthy birds at slaughterhouses, live bird markets, and during routine flock health checks [8].

The diagnostic workflow for H9N2 detection follows a hierarchical approach. Initial screening is performed using molecular detection methods, specifically real-time reverse transcription polymerase chain reaction (rRT-PCR) targeting the matrix (M) gene of influenza A virus [2]. Samples that test positive for the M gene are then subjected to subtype-specific rRT-PCR assays targeting the H9 and N2 gene segments [3]. Virus isolation in embryonated chicken eggs (specific-pathogen-free, SPF) is performed for confirmatory diagnosis and for subsequent antigenic and genetic characterization [4].

The following table summarizes the diagnostic methods used for H9N2 detection in Italy:

The Mermaid diagram below illustrates the diagnostic decision tree for H9N2 surveillance in Italy:

graph TD
    A[Sample Collection: Oropharyngeal/Cloacal Swabs], > B[rRT-PCR M Gene Screening]
    B, > C{Positive?}
    C, >|Yes| D[Subtype-specific rRT-PCR H9 and N2]
    C, >|No| E[No Further Action]
    D, > F{Positive for H9N2?}
    F, >|Yes| G[Virus Isolation in Embryonated Eggs]
    F, >|No| H[Report as Unsubtypable LPAI]
    G, > I[Antigenic Characterization: HI Assay]
    G, > J[Genetic Characterization: Sequencing]
    I, > K[Antigenic Cartography]
    J, > L[Phylogenetic Analysis]

Control and Prevention Strategies

The control of avian influenza A(H9N2) in Italy is based on a combination of biosecurity measures, surveillance, and depopulation policies. The primary objective of the control strategy is to prevent the introduction of H9N2 into domestic poultry holdings and to limit the spread of the virus if an incursion occurs [7]. Biosecurity measures are categorized into structural and operational components. Structural biosecurity includes the physical separation of poultry houses from wild bird habitats, the installation of netting to prevent wild bird entry, and the implementation of strict hygiene protocols for personnel and equipment [8]. Operational biosecurity encompasses the management of poultry movement, the quarantine of newly introduced birds, and the disinfection of vehicles and equipment [5].

The characterization of the domestic-wild bird interface is critical for the design of effective biosecurity measures. Camera trap studies conducted in northern Italy have documented the frequency and duration of contact between domestic poultry and wild birds at the perimeter of poultry farms [8]. These studies have identified that wild waterfowl, particularly mallards and teal, are the most frequent visitors to poultry farm perimeters, and that the risk of H9N2 introduction is highest during the autumn migration period [8].

Vaccination against H9N2 is not routinely practiced in Italy, as the virus is classified as LPAI and is not subject to mandatory eradication programs under EU legislation. However, inactivated H9N2 vaccines are available and have been used in some poultry-producing regions outside of Europe to reduce clinical disease and viral shedding [2]. The use of vaccination in Italy is restricted to emergency scenarios and is subject to approval by the competent veterinary authority [3].

Treatment and Therapeutic Interventions

There is no specific antiviral treatment for H9N2 infection in poultry. Supportive therapy, including the provision of clean water, adequate ventilation, and the reduction of environmental stressors, is recommended to minimize clinical signs and reduce secondary bacterial infections [4]. The use of antimicrobial agents is restricted to the treatment of secondary bacterial infections and must be administered under veterinary supervision to comply with EU regulations on antimicrobial use in food-producing animals [5].

Conclusion

The epidemiology of avian influenza A(H9N2) in Italy is characterized by the circulation of the virus in wild waterfowl populations and the sporadic incursion of the virus into domestic poultry holdings. The surveillance infrastructure in Italy, based on a combination of passive and active surveillance, is capable of detecting H9N2 incursions and providing timely information for risk assessment. The control of H9N2 in Italy is dependent on the maintenance of high biosecurity standards and the continued monitoring of the domestic-wild bird interface.

References

[1] Pariani E, Puzelli S, Del Castillo G, et al. Imported case of avian influenza A(H9N2) virus infection in a patient with miliary tuberculosis, Italy, March 2026. Euro Surveill. 2026. Available at: https://pubmed.ncbi.nlm.nih.gov/42141890/

[2] Facchini M, De Marco MA, Piacentini S, et al. Divergent Avian Influenza H10 Viruses from Sympatric Waterbird Species in Italy: Zoonotic Potential Assessment by Molecular Markers. Microorganisms. 2025. Available at: https://pubmed.ncbi.nlm.nih.gov/41304261/

[3] Kadja MC, Bako ABI, Onidje E, et al. Molecular Detection and Genetic Characterization of H9N2 Avian Influenza Virus in Laying Hen and Broiler Farms in Dakar and Thiès Regions, Senegal. Vet Ital. 2025. Available at: https://pubmed.ncbi.nlm.nih.gov/41077876/

[4] Mariacher A, Di Nicola MR, Senese M, et al. Detection of avian influenza virus in the alien invasive African sacred ibis (Threskiornis aethiopicus) in Italy. Front Vet Sci. 2025. Available at: https://pubmed.ncbi.nlm.nih.gov/40989950/

[5] Rapi MC, Martin AMM, Lelli D, et al. Virological Passive Surveillance of Avian Influenza and Arboviruses in Wild Birds: A Two-Year Study (2023-2024) in Lombardy, Italy. Microorganisms. 2025. Available at: https://pubmed.ncbi.nlm.nih.gov/40431131/

[6] Rombolà P, Papa Caminiti LN, Scaramozzino P, et al. A Spatial Multi Criteria Decision Analysis (SMCDA) to map the risk of avian influenza in Lazio and Toscana (central Italy). Vet Ital. 2024. Available at: https://pubmed.ncbi.nlm.nih.gov/39676683/

[7] Trogu T, Bellini S, Canziani S, et al. Surveillance for Avian Influenza in Wild Birds in the Lombardy Region (Italy) in the Period 2022-2024. Viruses. 2024. Available at: https://pubmed.ncbi.nlm.nih.gov/39599782/

[8] Graziosi G, Lupini C, Favera FD, et al. Characterizing the domestic-wild bird interface through camera traps in an area at risk for avian influenza introduction in Northern Italy. Poult Sci. 2024. Available at: https://pubmed.ncbi.nlm.nih.gov/38865769/ *** Disclaimer: This article is for educational and informational purposes only. It is not intended to substitute for professional veterinary advice, diagnosis, treatment, or regulatory guidance. Always consult a licensed veterinarian or qualified specialist regarding animal health, disease diagnosis, and therapeutic decisions.