Open Veterinary Journal, (2025), Vol. 15(10): 5361-5367

Research Article

10.5455/OVJ.2025.v15.i10.53

Age-related serological response to H9N2 infection in southwest Tripoli, Libya

Imad Benlashehr¹*, Ahmed Shaban Kassem Agha², Khalid Mohammed Naffati², Salah Abdulhadi Bshina³, Ahmed Abdulmajed Khashkhosha², Abdulatif A. Asheg¹ and Abdulwahab M. Kammon^1,4

¹Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, University of Tripoli, Tripoli, Libya

²Department of Microbiology, Libyan Center for Biotechnology Research, Tripoli, Libya

³Department of Medicine, Faculty of Veterinary Medicine, Azzaytuna University, Tarhuna, Libya

⁴The National Research Center for Tropical and Transboundary Diseases, Zintan, Libya

*Corresponding Author: Imad Benlashehr. Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, University of Tripoli, Tripoli, Libya. Email: i.benlashehr [at] uot.edu.ly

Submitted: 10/05/2025 Revised: 22/08/2025 Accepted: 10/09/2025 Published: 31/10/2025

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Abstract

Background: Avian influenza virus H9N2 is a low-pathogenic subtype that causes respiratory disease and economic losses in poultry and carries zoonotic potential. The virus is endemic across Asia, the Middle East, and North Africa, often exacerbated by co-infections with other pathogens. In Libya, H9N2 was first detected in 2006, but data on age-related immune responses in broilers remain limited.

Aim: This study investigated the age-related serological response to H9N2 in southwest Libyan broiler flocks to provide baseline data for vaccination and control programs.

Methods: A total of 342 blood samples were collected from six unvaccinated broiler flocks. Antibodies against influenza A were detected by indirect enzyme-linked immunosorbent assay, and H9N2-specific antibodies were measured by hemagglutination inhibition assay.

Results: The seropositivity for influenza A increased with age (30%, 52%, and 100% at 10, 24, and 38 days, respectively). The positivity rates for H9N2 were 0%, 22%, and 72%. The progressive increase confirms the active field circulation and endemicity of H9N2. The absence of detectable antibodies at 10 days indicates strong maternal antibody interference.

Conclusion: H9N2 is endemic in Libyan broiler flocks, with continuous exposure reflecting age-dependent seroconversion. Vaccination after the age of 10 days may optimize immune protection by reducing maternal antibody interference. Continuous surveillance and molecular monitoring are essential for guiding effective national control measures and assessing zoonotic risks.

Keywords: H9N2, Seroprevalence, ELISA, HI, Libya.

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Introduction

AIVs belong to the family Orthomyxoviridae, genus Influenza virus A. These viruses cause highly contagious respiratory disease in avian species, particularly aquatic and wild migratory birds (St. Paul et al., 2012; Tong et al., 2013; Swayne et al., 2013). Influenza A viruses consist of 18 hemagglutinin (HA, H1–H18) and 11 neuraminidase (NA, N1–N11) subtypes, with waterfowl and seabirds as their natural reservoirs (Wu et al., 2014). Based on pathogenicity, AIVs are classified as highly pathogenic avian influenza viruses (HPAIVs), including certain H5 and H7 subtypes, which cause severe respiratory symptoms with a high mortality rate. In contrast, low pathogenic avian influenza viruses (LPAIVs), such as H9N2 subtypes, cause mild respiratory symptoms, low mortality rate, and a drop in egg production (Dhama et al., 2005).

H9N2 has become endemic in poultry across Asia, the Middle East, and North Africa since the 1990s, resulting in considerable economic losses in the poultry industry. Once introduced, H9N2 is more difficult to eradicate than HPAI viruses (Sagong et al., 2023; Kareche et al., 2024). The pathogenicity of H9N2 can significantly increase in the presence of live attenuated vaccines or when co-infections occur with other pathogens, such as infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and bacteria, such as E. coli and Mycoplasma (Kammon et al., 2015; Ismail et al., 2018; Ellakany et al., 2018; Wang et al., 2021).

Vaccination programs have been adopted in several countries, such as China, Korea, Morocco, Pakistan, Egypt, and Iran, to control H9N2 infection in poultry farms and reduce the risk of interspecies transmission (Lee and Song, 2013; Bahari et al., 2015; Kilany et al., 2016; Lau et al., 2016). However, these vaccination programs face limitations due to maternally derived antibodies in chicks and antigenic differences between the vaccine and circulating field strains. These factors significantly contribute to the failure of H9N2 vaccination efforts (Kapczynski and Swayne, 2009; Balish et al., 2010; Cattoli et al., 2011; Capua and Cattoli, 2013; Bahari et al., 2015; Alizadeh et al., 2016; Peacock et al., 2019; Cui et al., 2021). Libyan poultry are exposed to multiple pathogens, including IBV, NDV, Escherichia coli, Salmonella, and Mycoplasma, which can cause high mortality and reduced egg production, either alone or through co-infections (Kammon et al., 2013, 2015, 2017 and 2018; Asheg et al., 2023; Agha et al., 2022; Benlashehr et al., 2024).

Serological studies have confirmed the circulation of H9 AIV in broiler and layer flocks in Libya (Fares et al., 2010; Al-Garib et al., 2016). In 2013, field investigations showed that co-infection of H9N2 with NDV was associated with high mortality outbreaks in 2013 (Kammon et al., 2015). More recently, H9N2 was detected in broiler flocks with elevated mortality in southwestern Libya, although the virus alone was unlikely to be the sole cause, suggesting roles for co-infections or management-related stressors (Agha et al., 2023). Despite these findings, information regarding the age-related serological response to H9N2 in broiler flocks in Libya and the monitoring of viral evolution remains limited.

Understanding age-related immunity is essential for evaluating flock susceptibility and guiding preventive strategies. Therefore, the present study was conducted to investigate the age-related serological response to H9N2 in broiler flocks in southwest Libya, providing essential baseline data to inform future vaccination and control strategies.

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Materials and Methods

Field survey

Six broiler farms located in the southwest region of Libya were included in the current study, having sixty thousand broilers. All flocks were obtained from the same parents. A total of 342 blood samples were randomly collected from the six broiler farms at ages of 10, 24, and 38 days (Table 1). Serum samples were separated and used for indirect enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) tests. All flocks were vaccinated against ND, IB, and IBD based on the program approved by the National Center for Animal Health (NCAH) (Table 2). However, the broilers were not vaccinated against AI. The clinical signs and postmortem lesions were recorded.

Table 1. Samples collected from six broiler flocks of different ages.

Table 2. Vaccination program used in the tested farms.

Serological assays

Indirect ELISA to detect antibodies against avian influenza type A was used for the analysis of sera at the NCAH in Tripoli, Libya. The ELISA was conducted according to the manufacturer’s (X-OvO Limited, United Kingdom) instructions. An ELx800 ELISA reader with a 550 nm filter (BIO-TEK Instruments, Inc., Winooski, VT, USA) was used.

The sera were also tested for AIV serotype H9N2 using HI test. HA and HI were performed according to the procedure described by the OIE (2009) using 96 “V-shaped” well microtiter plates, 1% v/v blood cells (RBCs), and 4 HA units of AIV H9N2 antigen (OIE, 2009).

Statistical analysis

Data were managed and analyzed using SPSS, version 27.0 (SPSS Inc., Chicago, IL, USA). Analysis was performed using the chi-square test. Statistical significance was designated for differences with p-values less than or equal to 0.05.

Ethical approval

All samples were collected according to local and international standards for the care and use of research animals.

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Results

Clinical signs and postmortem findings

Broiler flocks included in this study showed mild to moderate respiratory symptoms. The most frequent observations were sneezing, nasal discharge, and mild breathing difficulty. No lesions that could be attributed to a specific respiratory disease were found on postmortem examination. The findings suggest that the signs were nonspecific and could have been influenced by other infections or management-related factors.

ELISA assay for influenza A antibodies

Serum samples from six broiler flocks were tested by indirect ELISA at 10, 24, and 38 days of age (Table 3).

• At 10 days of age, the proportion of positive samples ranged from 16% to 50% (flocks 1–6: 50%, 42%, 25%, 16%, 25%, and 25%). The variation among flocks was not statistically significant.

• Seropositivity increased at 24 days of age, ranging between 25% and 88% (flocks 1–6: 88%, 63%, 40%, 42%, 60%, and 25%). The differences were statistically significant, with flocks 1, 2, and 5 showing higher values (p < 0.05).

• All tested samples were positive (100%) in all flocks by 38 days of age. The trend of increasing seropositivity with age was clear: 30%, 52%, and 100% at 10, 24, and 38 days, respectively.

Table 3. ELISA assay results.

These results confirm that the influenza A virus is circulating in broiler flocks in the study area and that the antibody levels progressively increase as the birds get older.

HI assay for H9N2 antibodies

ELISA-positive samples were further examined for H9N2-specific antibodies using the HI test (Table 4).

• No H9N2 antibodies were detected in any flock at 10 days of age.

• At 24 days of age, antibodies were present at variable levels across the flocks (56%, 16%, 15%, 21%, 15%, and 15%, respectively). This indicates that H9N2 infection started between 2 and 3 weeks of age.

• At 38 days of age, antibody prevalence increased in all flocks, ranging from 60% to 84% (75%, 79%, 65%, 84%, 60%, and 70%). Differences between flocks were statistically significant (p < 0.05).

Table 4. Result of the HI assay.

The absence of detectable antibodies at 10 days, followed by their appearance and sharp increase later, suggests a role for maternal antibody interference and supports the view that broilers were exposed to H9N2 virus during the growing period.

Overall serological trends

Taken together, the ELISA and HI results show that the prevalence of antibodies increased steadily with age. This pattern is consistent with continuous exposure to influenza viruses under field conditions. The results also provide useful information for vaccination programs, indicating that protective immunity may be more effective when vaccination is performed after the decline of maternal antibodies, usually beyond the first week of age.

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Discussion

AIVs cause significant economic losses in poultry and pose public health risks. In this study, all broiler flocks tested positive for AIV type A antibodies by ELISA, confirming natural field exposure. These findings align with previous reports from Libya, Jordan, and Lebanon, demonstrating the endemic circulation of AIVs in poultry populations (Al-Natour and Abo-Shehada, 2005; Barbour et al., 2006; Al-Garib et al., 2007).

HI testing confirmed the presence of H9N2-specific antibodies in several flocks in southwest Libya, indicating continued circulation of this subtype. Comparable findings from Iran, Tunisia, and Egypt highlight its established endemicity in the region (Pazani et al., 2008; Ghaniei et al., 2013; Nagy et al., 2017; Hassan et al., 2016; Naguib et al., 2017). Some ELISA-positive sera tested negative by HI, suggesting the presence of other AIV subtypes or interference from maternally derived antibodies (de Wit et al., 2004; Barbour et al., 2006).

At 10 days of age, no H9N2 antibodies were detected, which is consistent with the high levels of maternal antibodies. By day 24, 22% of birds had detectable antibodies, rising to 72% at day 38. Meanwhile, AIV type A seropositivity increased from 30% to 100% over the same ages. These trends highlight the influence of maternal antibodies on early immune responses and support vaccination after the first week of life to achieve effective seroconversion (Amer et al., 2010; Talat et al., 2020; Pan et al., 2022).

The flocks were not vaccinated against AIV, confirming that the observed seropositivity reflected natural infection. However, they were vaccinated against ND, IB, and IBD. Co-infections with these viruses or bacterial pathogens, such as E. coli, can enhance the susceptibility and severity of H9N2 infection (Kammon et al., 2015; Wang et al., 2021; Huang et al., 2024). Molecular studies have also shown that co-infection with H9N2 can suppress key innate immune responses, promoting viral replication (Huang et al., 2017).

Although our study confirmed H9N2 circulation serologically, ELISA alone cannot identify specific viral variants or monitor genetic changes. Therefore, future studies will employ PCR and sequencing to characterize circulating H9N2 variants and track viral evolution. This approach will complement serological findings and guide targeted vaccination strategies.

Overall, these findings underline the complex ecology of H9N2 in broiler production systems. Effective control requires vaccination strategies timed to the decline of maternal antibodies, improved biosecurity, and ongoing molecular surveillance. Based on age-related serological trends, vaccination after 10 days of age appears optimal for inducing protective immunity while minimizing interference from maternal antibodies.

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Conclusion

Our findings provide clear evidence that the avian influenza virus, particularly H9N2, is endemically circulating in broiler flocks in southwest Libya. In the absence of vaccination, the consistent rise in antibody prevalence with age reflects ongoing natural exposure under field conditions.

The role of maternal antibodies in delaying seroconversion and reducing early vaccine effectiveness was evident. On the basis of these dynamics, vaccination after the first 10 days of life appears to be the most appropriate window for inducing protective immunity.

Co-infections with other respiratory viruses and bacteria remain an important complicating factor, highlighting the need for integrated vaccination strategies alongside strict biosecurity and regular monitoring.

Finally, future work should move beyond serology to include molecular approaches such as polymerase chain reaction (PCR) and sequencing. This will allow the characterization of circulating variants, a better understanding of viral evolution, and informed decision-making for national control programs.

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Acknowledgments

The authors sincerely acknowledge the Libyan Center for Biotechnology Research, the National Center of Animal Health, and the Faculty of Veterinary Medicine, University of Tripoli, for their invaluable support and collaboration. The authors also extend their gratitude to all individuals who contributed directly or provided technical or moral support during sample collection and laboratory work, as their assistance was instrumental in the completion of this study.

Funding

The authors did not receive support from any organization for the submitted work (self-fund).

Authors’ contributions

Imad Benlashehr and Ahmed Shaban Kassem Agha conceptualized the study, designed the experiments, and supervised the project. Ahmed Shaban Kassem Agha and Khalid Mohammed Naffati performed the laboratory experiments and collected the samples. Salah Abdulhadi Bshina and Ahmed Abdulmajed Khashkhosha contributed to the field investigation and sample collection. Khalid Mohammed Naffati and Abdulatif A. Asheg curated and analyzed the data. Imad Benlashehr and Abdulwahab M. Kammon drafted the initial manuscript. Ahmed Shaban Kassem Agha and Khalid Mohammed Naffati reviewed and edited the manuscript. All authors read and approved the final manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Data availability

The data are available upon request.

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