Molecular–Serologic Snapshot of a Silent Respiratory Threat: Avian Metapneumovirus Circulation in Unvaccinated Broiler Breeders in Egypt, 2024-2025

Molecular–Serologic Snapshot of a Silent Respiratory Threat: Avian Metapneumovirus Circulation in Unvaccinated Broiler Breeders in Egypt, 2024-2025 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular–Serologic Snapshot of a Silent Respiratory Threat: Avian Metapneumovirus Circulation in Unvaccinated Broiler Breeders in Egypt, 2024-2025 Omar S. Saeed, Mirna A kram Labib, Mahmoud Gamal, Basem M. Ahmed, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8457890/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background Avian metapneumovirus (aMPV) is an economically significant respiratory pathogen of poultry that affects performance, egg production, and fertility of breeder flocks. Despite its impact, aMPV continues to be ill-defined in Egypt with no integrated surveillance studies conducted in breeder flocks. Therefore, the current study was designed to provide the first comprehensive molecular and serological assessment of aMPV circulation in broiler breeders in Egypt. Methods Between 2024 and 2025, 6,000 serum samples and 800 tracheal swabs were collected from 60 unvaccinated broiler breeder flocks across twelve Egyptian governorates. Sera were obtained at 16 weeks (rearing phase) and 35 weeks (production phase) of age and screened for aMPV subtypes A and B by ELISA. Tracheal swabs collected during the production phase were pooled into 80 composite samples and tested for the viral RNA by RT-qPCR. Results Serological analysis revealed widespread aMPV exposure, with flock-level seroprevalence ranging between 94.0% and 97.4%. Antibody titers increased between 16 and 35 weeks, reflecting cumulative viral exposure. Molecular testing detected aMPV RNA in 67 (83.75%) of pooled swabs. Subtype B was the predominant genotype detected solely in 65 (81.25%) sample pools and mixed with subtype A in 2 pools (2.5%). Serological and molecular findings were generally aligned, with flocks positive for aMPV RNA often exhibiting higher antibody titers. Conclusion These findings indicate that aMPV, particularly subtype B, is likely endemic across the Egyptian broiler breeder flocks. The study highlights critical knowledge gaps, emphasizing the need for viral isolation, sequencing, and controlled evaluation of biosecurity and vaccination strategies. Avian metapneumovirus Broiler breeder Egypt RT-qPCR Surveillance Subtype B Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background Avian metapneumovirus (aMPV), formerly known as avian pneumovirus or turkey rhinotracheitis virus, is a major respiratory pathogen of poultry. The virus causes upper respiratory tract disease characterized by sinusitis, facial swelling, swollen head syndrome (SHS), reduced egg production, and increased mortality, with significant economic losses. [ 1 , 2 ]. Its ability to infect multiple avian species including turkeys, chickens, guinea fowl, ducks, and pheasants highlights its broad host range and epidemiologic importance [ 3 , 4 ]. Since its first identification in South African turkeys in 1978 [ 5 , 6 , 7 ] and its subsequent implication in SHS in chickens in England [ 8 ], aMPV has spread widely across Europe [ 9 ], Asia [ 10 , 11 , 12 , 13 ], Africa [ 14 , 15 , 16 , 17 ], the Americas [ 18 , 19 , 20 , 21 , 22 ], and in the Middle East region [ 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. As a typical member of genus Metapneumovirus within the family Pneumoviridae , aMPV is an enveloped virus with helical capsid and a negative single-stranded RNA genome of approximately 13.3–14 kb. Distinct ORFs have been described within the viral genome to encode eight proteins including: nucleoprotein (N), phosphoprotein (P), matrix proteins (M and M2), fusion protein (F), small hydrophobic protein (SH), attachment glycoprotein (G), and the large polymerase (L). N protein is highly conserved and is widely used in serodiagnosis, whereas G protein is highly variable and serves as a major determinant for molecular subtyping [ 30 ]. Four primary aMPV subtypes (A, B, C, and D) have been identified, each exhibiting distinct antigenic and genetic characteristics. Subtypes A and B predominate across Europe, Africa, and most of Asia, subtype C is mainly reported in North America and in some Asian countries, while subtype D has been detected only in France [ 31 ]. While nucleotide sequence is almost identical within subtypes, it varies substantially between subtypes, especially when comparing A/B with C or D [ 32 , 33 , 34 ]. This highlights the importance of targeting conserved antigens, particularly the N protein, for reliable serological screening, cross-subtype detection, and epidemiological monitoring. Recently, additional divergent aMPV-like viruses have been identified in North American wild birds, which indicates further expansion of aMPV genetic diversity [ 35 , 36 ]. As a contributor to the multifactorial respiratory disease complex, aMPV predisposes flocks to secondary infections by Escherichia coli , Mycoplasma gallisepticum , and Ornithobacterium rhinotracheale , with increased morbidity and complicated diagnosis [ 37 ]. Disease severity varies with species and age, with turkeys and broiler chickens often showing more severe outcomes [ 38 , 39 ]. While clinical signs may raise suspicion, definitive diagnosis requires laboratory confirmation. Virus isolation is limited by short shedding periods and sample degradation, whereas molecular assays offer rapid and sensitive detection. Quantitative real-time RT-PCR (qRT-PCR) is considered the most efficient technique for routine surveillance, early detection, and subtype differentiation [ 40 , 41 ]. Serological assays are practical for population-level surveillance, where ELISA kits utilizing the conserved N protein enable reliable detection of natural aMPV exposure. Although some assays are optimized for subtypes A and B, ELISA remains essential for monitoring infection pressure, evaluating flock immunity, and guiding vaccination policies [ 42 , 43 , 44 , 45 , 46 ]. In Egypt, respiratory viral infections continue to challenge poultry production despite widespread vaccination against avian influenza, Newcastle disease, and infectious bronchitis [ 47 , 48 , 49 ]. However, the epidemiology of aMPV remains poorly understood since most studies are either fragmented or geographically limited. Both subtypes A and B have been detected in turkeys, chickens, and ducks, often alongside bacterial co-infections [ 23 , 50 , 51 , 52 , 53 , 54 ]. Nevertheless, no comprehensive surveillance has been conducted in broiler-breeder flocks, leaving gaps in understanding exposure, circulating subtypes, and regional distribution. This study provides the first structured, multi-governorate assessment of aMPV exposure and viral detection in unvaccinated Egyptian broiler-breeder flocks. By combining ELISA-based serology with subtype-specific RT-qPCR, an integrated database was generated to elucidate the current epidemiological situation and to identify the circulating subtypes in order to guide biosecurity measures and vaccination strategies. 2. Materials and Methods 2.1. Study Design and Ethical statement A cross-sectional study was conducted throughout 2025 to investigate the dynamics of aMPV infection in unvaccinated broiler breeder flocks in Egypt. The study combined serological and molecular surveillance to: (i) determine the prevalence and distribution of aMPV specific antibodies, (ii) identify the circulating subtypes and (iii) assess age-related seroconversion and cumulative exposure. All procedures of animal handling and sample collection were approved by the Institutional Animal Care and Use Committee (IACUC), Faculty of Veterinary Medicine, Cairo University (Approval No. Vet CU-301220251281). 2.2. Study Area and Sample Collection A multi-stage sampling design was applied to ensure representative detection of aMPV among broiler breeder flocks in Egypt. The study area was stratified into three major poultry production zones (Delta, Middle Egypt, and Upper Egypt), that represent distinct ecologic and production systems. Within each zone, farms were selected as clusters proportional to flock density and distribution, and individual birds were systematically sampled from multiple houses and sections within each flock to capture overall serological status and minimize house- or pen-level bias. The birds included in this study originated from commercial, privately owned broiler breeder farms distributed across studied Egyptian governorates. The birds were not owned by the authors or by a single institution but belonged to independent poultry production companies and private farm owners operating under routine commercial management. A total of 60 unvaccinated broiler breeder flocks (five per governorate) were included in the study. Flocks were selected based on accessibility, availability of production records, and confirmed absence of prior aMPV vaccination. Although a subset of birds exhibited mild respiratory signs during the rearing phase (16 weeks) and a modest reduction in egg production during the production phase (35 weeks), sampling encompassed both affected and apparently healthy birds to provide a comprehensive overview of flock-level aMPV exposure. On the other hand, 6,000 serum samples (100 per flock) were collected to assess seroconversion dynamics and cumulative exposure during both rearing phase (n = 50 sera/flock), and the production phase (n = 50 sera/flock). This repeated cross-sectional approach allowed evaluation of immunity development over time while maintaining a representative assessment of the flocks. For molecular detection, 800 tracheal swabs were collected during the production period. This included 100 swabs per governorate, with the exception of Giza and Fayoum (40 each) and Beni Suef, Minya, Asyut, and Luxor (30 each). Sterile polyester-tipped swabs were used to collect specimens, which were then immediately placed into 3 mL of viral transport medium (VTM) comprised of Anderson’s modified Hanks Balanced Salt Solution (HBSS) supplemented with 2% heat-inactivated fetal bovine serum (FBS), 100 µg/mL gentamicin, and 0.5 µg/mL amphotericin B (Sigma-Aldrich). Samples were maintained at 2–8°C in insulated ice boxes during transport. Upon arrival at the Virology Laboratory, Faculty of Veterinary Medicine, Cairo University, swab suspensions were vortexed, clarified by brief centrifugation, aliquoted, and stored at − 80°C until use in molecular analysis. 2.3. Serum Preparation and Serological Analysis Approximately 1.5 mL of blood was aseptically collected from the wing vein of randomly selected birds and transferred into sterile tubes. Blood was allowed to clot at room temperature and centrifuged at 3,000 rpm for 10 minutes. The resultant sera were transferred into labeled Eppendorf tubes, identifying flock, governorate, age, and collection date, and stored at − 20°C until testing. Samples showing hemolysis or contamination were excluded from analysis. Serum samples were analyzed using a commercial indirect ELISA kit (IDVET® Laboratories, Grabels, France) for the detection of aMPV subtypes A and B. Samples were diluted 1:500 and incubated in 96-well microtiter plates pre-coated with aMPV antigens. After incubation and washing, anti-chicken horse radish peroxidase (HRP) conjugate was added, followed by TMB substrate for color development. Optical density was measured at 450 nm using an Thermo Scientific™ Multiskan FC™ Photometer as previously described [ 28 , 55 ]. The sample-to-positive (S/P) ratio was calculated as follows: (Sample OD – NC OD) / (PC OD – NC OD). Antibody titers were calculated as Log10(Titer) = 1.09 Log10(S/P) + 3.360. Samples with titers greater than 396 (S/P > 0.2) were considered positive. 2.4. RNA Extraction and Molecular detection by RT-qPCR For each flock, every 10 tracheal swabs were pooled, resulting in a total of 80 pools. Viral RNA was extracted from 200 µL of pooled tracheal swabs using QIAamp Viral Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. All procedures were conducted under RNase-free conditions, and eluted RNA was stored at – 80 ºC for downstream processing. All RNA samples were screened for Mycoplasma gallisepticum , Mycoplasma synoviae , Ornithobacterium rhinotracheale (ORT), Newcastle disease virus (NDV), Infectious Bronchitis Virus (IBV), and avian influenza virus (subtypes H5 and H9) using routine RT-PCR assays. Molecular detection of aMPV was performed using Kylt® aMPV A&B real-time RT-PCR kit (AniCon Labor GmbH, Emstek, Germany) on AB 7500 Fast Real Time PCR system (Thermo Fisher SCientific, Foster City, CA). The assay simultaneously targeted the G gene of subtypes A and B using specific primers and TaqMan probes labeled with FAM (aMPV-A) and Cy5 (aMPV-B), respectively. Beta-actin specific primers and HEX-labeled probe served as an endogenous control as previously reported [ 56 ]. The reaction comprised of 16 µL master mix and 4 µL RNA template, while the thermal profile was adjusted as follows: reverse transcription at 50°C for 10 min, polymerase activation at 95°C for 1 min, 42 cycles of denaturation at 95°C for 10 s, and annealing/extension at 60°C for 1 min with fluorescence detection in FAM, Cy5, and HEX channels. Positive controls included commercial live vaccines for aMPV subtype A (Poulvac® TRT, Zoetis, Parsippany, NJ) and subtype B (Nemovac®, Merial, Lyon, France), while nuclease-free water served as a negative control. Cycle threshold (Ct) values ≥ 37 was considered negative, and differences > 10 cycles between FAM and Cy5 in the same sample were regarded as questionable. 2.5. Statistical Analysis Serological data were initially compared across production phases using the Wilcoxon rank-sum test. Subsequent comparisons among governorates and regions were performed using the Kruskal–Wallis test, followed by pairwise post hoc analyses and p -value adjustment according to the Bonferroni correction. All statistical analyses and data visualizations were conducted in RStudio (version 2025.09.2) [ 57 ] using R programming language (version 4.5.2) [ 58 ]. Data are presented as arithmetic means ± standard deviations, and statistical significance was defined as p < 0.05. 3. Results 3.1 Sero-surveillance of aMPV in broiler breeder flocks A high level of aMPV exposure was detected across all surveyed governorates. Overall, 96.0% of serum samples tested positive for aMPV antibodies, indicating widespread serological evidence of infection at the individual bird level. At the flock level, 100% of sampled broiler breeder flocks were seropositive, confirming that aMPV exposure was present in all investigated production settings. Governorate-level seroprevalence ranged from 94.0% in Beheira to 97.4% in Beni Suef and Giza with minor geographic variation. Antibody titers increased markedly from the rearing phase to the production phase (Fig. 1 ). Antibody levels at 35-week-old flocks were significantly higher than those observed at 16-week-old flocks (Wilcoxon rank-sum test, p < 0.001), which is consistent with the progressive seroconversion during the production cycle. Substantial variability in antibody titers was observed at both ages, indicating heterogeneous immune responses among birds and flocks. At 16 weeks, Middle Egypt exhibited higher antibody titers compared to Delta and Upper Egypt regions (Fig. 2 A). This regional pattern shifted by 35 weeks, when Delta region displayed the highest antibody titers, exceeding those recorded in Middle and Upper Egypt (Fig. 2 B). The spatial maps (Figs. 3 A and 3 B) revealed a persistent north to south gradient in antibody titers across the production phases, with Upper Egypt flocks consistently exhibiting lower antibody levels. At 16 weeks, governorate-level analysis showed higher antibody titers in Beni Suef and Giza, intermediate titers in Fayoum, Qalyubia, Gharbia, Beheira, and Kafr El-Sheikh, and the lowest titers in Asyut, Minya, and Luxor (Fig. 4 A). By 35 weeks, antibody titers increased across all governorates (Fig. 4 B). Delta governorates (Qalyubia, Gharbia, Menoufia, Kafr El-Sheikh, and Beheira) formed a high-titer cluster, while Sharqia and Giza exhibited intermediate responses. Lower antibody levels persisted in Beni Suef, Fayoum, and Upper Egypt governorates, confirming sustained geographic heterogeneity throughout the production cycle. 3.2. Molecular Detection and Subtyping of aMPV by RT-qPCR Among the 80 pools of tracheal swabs, 67 (83.75%) tested positive for aMPV RNA. Subtype B was the predominant genotype that was detected solely in 65 pools of samples (81.25%) and mixed with subtype A in 2 sample pools originated from Beheira governorate (2.5%). No sample pools were exclusively positive for subtype A (Table 1 ). Table 1 RT-qPCR detection of avian metapneumovirus (aMPV) subtypes in pooled tracheal swabs from broiler breeder flocks in Egypt (2024–2025) Governorate Sample Pools (n) Subtype B positive pools (n) A + B co-detection (n) PCR-negative pools (n) Beheira 10 7 2 1 Kafr El-Sheikh 10 9 0 1 Gharbia 10 9 0 1 Sharqia 10 8 0 2 Qalyubia 10 9 0 1 Monufia 10 9 0 1 Giza 4 3 0 1 Fayoum 4 3 0 1 Minya 3 2 0 1 Beni Suef 3 2 0 1 Asyut 3 2 0 1 Luxor 3 2 0 1 Total 80 65 2 13 Positive samples were recognized in all Delta, Middle Egypt, and Upper Egypt governorates (Fig. 5 ), which indicate broad geographic distribution of aMPV. Subtype B was identified in all governorates. High detection rates of subtype B were observed in Delta governorates (80–90%) and slightly decreased while moving south to Lower and Upper Egypt governorates (67.7–75%). Ct values varied among pools with an average range of 18.4 to 32.9 (mean 25.7 ± 3.1), with no obvious geographic pattern. 4. Discussion Using a combined serological and molecular framework, this study elucidates patterns of aMPV circulation in unvaccinated broiler breeder flocks across Egypt. The integration of dual-age antibody profiling with subtype-specific RT-qPCR enabled interpretation of both cumulative exposure and active viral circulation, revealing spatial and temporal heterogeneity in aMPV epidemiology. The uniformly high seroprevalence observed across all surveyed governorates indicates widespread exposure to aMPV in Egyptian broiler breeders. As all flocks were confirmed to be unvaccinated, antibody detection most plausibly reflects natural infection rather than vaccine-induced immunity. Similar high seroprevalence levels have been reported in breeder flocks from Asia and the Middle East, including Bangladesh, Iran, and South Korea [ 12 , 13 , 23 , 26 ]. This supports the concept that long-lived breeder populations experience extended windows of viral exposure compared to the short-cycle broiler flocks In contrast to the earlier studies conducted in Egypt, the current investigation provides more comprehensive understanding of aMPV circulation in broiler breeder flocks. Previous serological surveys often focused on flocks exhibiting clinical signs and typically involved limited sample sizes and/or restricted geographical coverage [ 52 , 53 ]. Molecular studies primarily analyzed mutant strains developed under vaccination pressure, while no studies have targeted unvaccinated populations [ 54 ]. In this study, 60 unvaccinated broiler breeder flocks across twelve governorates were evaluated by combining dual-age serological profiling with subtype-specific RT-qPCR of pooled tracheal swabs. This approach revealed uniformly high seroprevalence (~ 96%) and a predominance of subtype B, with mixed detection of types A and B in Beheira governorate only. The observed difference between our results and the previous reports is likely attributed to variation in host population, sampling strategy, and methodological resolution rather than to temporal change in virus circulation [ 23 , 52 , 53 , 54 ]. These findings reinforce the hypothesis that broiler breeders, due to their longevity and central role in the production pyramid, may serve as reservoirs sustaining aMPV transmission within Egyptian poultry systems. The observed increase in the antibody titers between 16 and 35 weeks of age is consistent with the cumulative immune stimulation over the production cycle. This pattern aligns with reports from breeder flocks exposed to other respiratory viruses, such as infectious bronchitis virus, where repeated or prolonged exposure leads to progressive antibody amplification [ 59 ]. Considerable variability in titers among flocks and governorates further suggests heterogeneous exposure histories, potentially influenced by differences in management practices, biosecurity levels, flock size, and genetic susceptibility, as documented for other avian viral diseases such as Marek’s disease and avian leukosis [ 60 ]. One of the notable findings of this study is the spatial shift in antibody profiles between production stages. At 16 weeks of age, higher mean titers were detected in the governorates of Middle Egypt (Beni Suef, Giza, Fayoum), whereas by 35 weeks, the highest titers clustered in the Nile Delta region. We hypothesize that early exposure during the rearing phase in Middle Egypt may be influenced by regional ecological factors, while later exposure in the Delta is more strongly driven by horizontal transmission within high-density production systems [ 47 , 48 , 49 ]. The early antibody responses detected in Middle Egypt is also coincided with sampling conducted in November 2024 during the migratory season. The proximity of backyard and small-scale breeder operations to wetlands and irrigation canals in Middle Egypt elevates the risk of indirect pathogen transmission from wild birds. By contrast, the dominance of high antibody titers in Delta governorates at 35 weeks of age is likely reflecting sustained within-sector transmission. The Nile Delta is characterized by high poultry density, large flock sizes, and frequent movement of personnel and equipment. These conditions are known to facilitate viral persistence and spread. Similar density-driven amplification for avian pathogens in North African and Middle Eastern production systems [ 39 , 48 , 60 ], further support the interpretation that production intensity becomes a dominant driver of exposure later in the production cycle. RT-qPCR screening of pooled tracheal swabs demonstrated widespread detection of aMPV during the production phase. The predominance of subtype B across all positive governorates, alongside the absence of subtype A as a sole agent, is consistent with regional reports from Morocco, Tunisia, Algeria, Jordan, and Turkey [ 14 , 15 , 38 , 39 ]. These findings suggest shared epidemiological patterns across the Middle East and North Africa, potentially facilitated by poultry trade, shared production practices, and broader ecological connectivity. Co-detection of both A and B subtypes was limited to Beheira governorate. This localized pattern may reflect transient co-circulation events in large breeder flocks, where dense contact networks increase the probability of multiple viral introductions and short-term subtype overlap. Similar phenomena have been described for other avian respiratory pathogens like avian influenza in large commercial populations [ 61 ]. The predominance of subtype B raises the question of whether detected viruses represent circulating field strains or, less likely, vaccine-derived variants. Although all flocks in this study were unvaccinated, indirect exposure to vaccine-related viruses from neighboring farms or environmental persistence cannot be completely excluded. Vaccine-derived reversion events have been documented in Europe, including a subtype A aMPV vaccine strain associated with clinical disease in turkeys despite the absence of recent vaccination of affected farms [ 62 , 63 ]. While there is no direct evidence in the present dataset supporting a vaccine origin, this possibility cannot be resolved without full-genome sequencing. Differentiating field strains from vaccine-related variants remains critical for accurate epidemiological interpretation and informed control strategies. These findings emphasize the importance of implementing routine, integrated serological and molecular surveillance in broiler breeder populations. Future research should incorporate longitudinal sampling, full-genome sequencing, and targeted surveillance at the poultry–wildlife interface, as well as focused investigations into the potential for vertical transmission of aMPV, which has been suggested in previous studies [ 64 , 65 ]. Clarifying the contribution of breeder-to-progeny transmission will be essential to fully understand aMPV epidemiology and to evaluate the suitability of subtype-matched vaccination and control strategies under controlled conditions. 5. Conclusion This study demonstrates that aMPV is likely circulating in the studied Egyptian governorates, predominantly as subtype B. Early exposure in Middle Egypt could potentially be influenced by migratory bird activity, while cumulative transmission in high-density Delta farms likely contributes to later antibody peaks. Serological and molecular results indicate ongoing circulation in unvaccinated flocks, suggesting that breeders may serve as reservoirs. These findings provide a foundation for evidence-based surveillance and biosecurity improvements and suggest that evaluation of vaccination strategies under controlled trials could be considered in the future. Overall, the study contributes to a broader understanding of aMPV epidemiology in the Middle East and North Africa Abbreviations aMPV – Avian metapneumovirus SHS – Swollen Head Syndrome RT-qPCR – Real-time quantitative reverse transcription polymerase chain reaction RNA – Ribonucleic acid ELISA – Enzyme-linked immunosorbent assay ORFs – Open reading frames N – Nucleoprotein P – Phosphoprotein M – Matrix protein M2 – Matrix protein 2 F – Fusion protein SH – Small hydrophobic protein G – Attachment glycoprotein L – Large polymerase protein IACUC – Institutional Animal Care and Use Committee VTM – Viral transport medium HBSS – Hanks’ Balanced Salt Solution FBS – Fetal bovine serum OD – Optical density S/P – Sample-to-positive ratio Ct – Cycle threshold NDV – Newcastle disease virus IBV – Infectious bronchitis virus AIV – Avian influenza virus ORT – Ornithobacterium rhinotracheale MG – Mycoplasma gallisepticum MS – Mycoplasma synoviae HRP – Horseradish peroxidase TMB – 3,3′,5,5′-Tetramethylbenzidine PCR – Polymerase chain reaction RNA – Ribonucleic acid Declarations Ethics approval and consent to participate No euthanasia or sacrifice of birds was performed in this study. All samples were obtained from apparently healthy, live birds during routine farm handling. Blood samples were collected from the wing vein, and tracheal swabs were obtained using sterile polyester-tipped swabs. The birds enrolled in this study were sourced from commercial, privately owned broiler breeder farms and were neither owned nor managed by the authors or by any academic or governmental institution. Before sample collection, permission was formally obtained from the respective farm owners or authorized farm managers to access the farms and to collect samples from their birds for research purposes. All animal handling procedures, farm access, and sample collection from privately owned flocks were conducted in accordance with approved animal welfare guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC), Faculty of Veterinary Medicine, Cairo University (Approval No. Vet CU-301220251281). Consent for publication Not applicable . Availability of data and materials The datasets generated and/or analyzed during the current study are included within the manuscript. Additional data is available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing financial or non-financial interests. Funding This research received no external funding from public, commercial, or non-profit funding agencies. Authors’ contributions Omar S. Saeed: Conceptualization, Study design, Sample collection, Data curation, Formal analysis, Writing original draft, Mirna Akram Labib : Data Curation, review & editing, Mahmoud Gamal: Statistical analysis, Data Curation, Writing—review & editing, Basem M. Ahmed: Supervision, manuscript review and editing, Ayman H. El-Deeb: Supervision, manuscript review and editing, Haitham M. Amer: Supervision, manuscript review and editing. 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1","display":"","copyAsset":false,"role":"figure","size":15381,"visible":true,"origin":"","legend":"\u003cp\u003eELISA antibody titers against avian metapneumovirus (aMPV) in non-vaccinated broiler breeder flocks during rearing and production phases across Egypt (2024–2025). Differences were assessed using the Wilcoxon rank-sum test. Bars represent the arithmetic mean ± standard deviation, and blue dashes indicate the geometric mean.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8457890/v1/d84f40ebdfc43b39a5a61d82.jpg"},{"id":100951359,"identity":"048a1c34-a5f9-4e05-b67f-a1e59eb30194","added_by":"auto","created_at":"2026-01-23 07:10:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36076,"visible":true,"origin":"","legend":"\u003cp\u003eRegional ELISA antibody titers against avian metapneumovirus (aMPV) in non-vaccinated broiler breeder flocks across Egypt (2024–2025). (A) ELISA antibody titers in 16-week-old flocks. (B) ELISA antibody titers in 35-week-old flocks. Differences were assessed using the Kruskal–Wallis test, followed by Bonferroni-corrected pairwise post hoc comparisons. Bars represent the arithmetic mean ± standard deviation, and blue dashes indicate the geometric mean. Statistical significance was set to \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8457890/v1/2fe58b8c22f7dab02cb84104.jpg"},{"id":100951934,"identity":"87bffe4c-0b5d-4524-acd3-ba47067fec80","added_by":"auto","created_at":"2026-01-23 07:11:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59341,"visible":true,"origin":"","legend":"\u003cp\u003eGeographical distribution of ELISA antibody titers against avian metapneumovirus (aMPV) in non-vaccinated broiler breeder flocks across Egyptian governorates (2024–2025). (A) Geographical distribution of ELISA antibody titers in 16-week-old flocks. (B) Geographical distribution of ELISA antibody titers in 35-week-old flocks. Maps are supplemented with a gradient color scale indicating aMPV antibody titer levels. Governorates shown in light grey were not included in the study.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8457890/v1/ed5d7697876abbfea2527cb0.jpg"},{"id":100951632,"identity":"95c9135c-42b5-41b7-baf0-4e159e4d7677","added_by":"auto","created_at":"2026-01-23 07:11:01","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74054,"visible":true,"origin":"","legend":"\u003cp\u003eGovernorate-level ELISA antibody titers against avian metapneumovirus (aMPV) in non-vaccinated broiler breeder flocks across Egypt (2024–2025). (A) ELISA antibody titers in 16-week-old flocks. (B) ELISA antibody titers in 35-week-old flocks. Differences were assessed using the Kruskal–Wallis test, followed by Bonferroni-corrected pairwise post hoc comparisons. Bars represent the arithmetic mean ± standard deviation, and blue dashes indicate the geometric mean. Governorates that do not share letters differ significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8457890/v1/c66df458478a350fa0c42b02.jpg"},{"id":100950487,"identity":"fa168b51-0b57-4a65-bf45-0d6bc36a4695","added_by":"auto","created_at":"2026-01-23 07:08:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26361,"visible":true,"origin":"","legend":"\u003cp\u003eGeographical distribution of avian metapneumovirus (aMPV) subtypes in non-vaccinated broiler breeder flocks across Egyptian governorates (2024–2025). Data are derived from RT-qPCR–positive pooled samples.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8457890/v1/66b87f9e5ea8a9aaf1d59726.jpg"},{"id":100953094,"identity":"a88aec62-d0ee-4819-81d9-2d3516be84ab","added_by":"auto","created_at":"2026-01-23 07:19:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1017667,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8457890/v1/5536de24-253d-4fb8-b030-f817d523bfc5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular–Serologic Snapshot of a Silent Respiratory Threat: Avian Metapneumovirus Circulation in Unvaccinated Broiler Breeders in Egypt, 2024-2025","fulltext":[{"header":"1. Background","content":"\u003cp\u003eAvian metapneumovirus (aMPV), formerly known as avian pneumovirus or turkey rhinotracheitis virus, is a major respiratory pathogen of poultry. The virus causes upper respiratory tract disease characterized by sinusitis, facial swelling, swollen head syndrome (SHS), reduced egg production, and increased mortality, with significant economic losses. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Its ability to infect multiple avian species including turkeys, chickens, guinea fowl, ducks, and pheasants highlights its broad host range and epidemiologic importance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Since its first identification in South African turkeys in 1978 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and its subsequent implication in SHS in chickens in England [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], aMPV has spread widely across Europe [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], Asia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], Africa [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], the Americas [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and in the Middle East region [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs a typical member of genus \u003cem\u003eMetapneumovirus\u003c/em\u003e within the family \u003cem\u003ePneumoviridae\u003c/em\u003e, aMPV is an enveloped virus with helical capsid and a negative single-stranded RNA genome of approximately 13.3\u0026ndash;14 kb. Distinct ORFs have been described within the viral genome to encode eight proteins including: nucleoprotein (N), phosphoprotein (P), matrix proteins (M and M2), fusion protein (F), small hydrophobic protein (SH), attachment glycoprotein (G), and the large polymerase (L). N protein is highly conserved and is widely used in serodiagnosis, whereas G protein is highly variable and serves as a major determinant for molecular subtyping [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Four primary aMPV subtypes (A, B, C, and D) have been identified, each exhibiting distinct antigenic and genetic characteristics. Subtypes A and B predominate across Europe, Africa, and most of Asia, subtype C is mainly reported in North America and in some Asian countries, while subtype D has been detected only in France [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. While nucleotide sequence is almost identical within subtypes, it varies substantially between subtypes, especially when comparing A/B with C or D [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This highlights the importance of targeting conserved antigens, particularly the N protein, for reliable serological screening, cross-subtype detection, and epidemiological monitoring. Recently, additional divergent aMPV-like viruses have been identified in North American wild birds, which indicates further expansion of aMPV genetic diversity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs a contributor to the multifactorial respiratory disease complex, aMPV predisposes flocks to secondary infections by \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eMycoplasma gallisepticum\u003c/em\u003e, and \u003cem\u003eOrnithobacterium rhinotracheale\u003c/em\u003e, with increased morbidity and complicated diagnosis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Disease severity varies with species and age, with turkeys and broiler chickens often showing more severe outcomes [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. While clinical signs may raise suspicion, definitive diagnosis requires laboratory confirmation. Virus isolation is limited by short shedding periods and sample degradation, whereas molecular assays offer rapid and sensitive detection. Quantitative real-time RT-PCR (qRT-PCR) is considered the most efficient technique for routine surveillance, early detection, and subtype differentiation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Serological assays are practical for population-level surveillance, where ELISA kits utilizing the conserved N protein enable reliable detection of natural aMPV exposure. Although some assays are optimized for subtypes A and B, ELISA remains essential for monitoring infection pressure, evaluating flock immunity, and guiding vaccination policies [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn Egypt, respiratory viral infections continue to challenge poultry production despite widespread vaccination against avian influenza, Newcastle disease, and infectious bronchitis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. However, the epidemiology of aMPV remains poorly understood since most studies are either fragmented or geographically limited. Both subtypes A and B have been detected in turkeys, chickens, and ducks, often alongside bacterial co-infections [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Nevertheless, no comprehensive surveillance has been conducted in broiler-breeder flocks, leaving gaps in understanding exposure, circulating subtypes, and regional distribution. This study provides the first structured, multi-governorate assessment of aMPV exposure and viral detection in unvaccinated Egyptian broiler-breeder flocks. By combining ELISA-based serology with subtype-specific RT-qPCR, an integrated database was generated to elucidate the current epidemiological situation and to identify the circulating subtypes in order to guide biosecurity measures and vaccination strategies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Study Design and Ethical statement\u003c/h2\u003e \u003cp\u003eA cross-sectional study was conducted throughout 2025 to investigate the dynamics of aMPV infection in unvaccinated broiler breeder flocks in Egypt. The study combined serological and molecular surveillance to: (i) determine the prevalence and distribution of aMPV specific antibodies, (ii) identify the circulating subtypes and (iii) assess age-related seroconversion and cumulative exposure. All procedures of animal handling and sample collection were approved by the Institutional Animal Care and Use Committee (IACUC), Faculty of Veterinary Medicine, Cairo University (Approval No. Vet CU-301220251281).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Study Area and Sample Collection\u003c/h2\u003e \u003cp\u003eA multi-stage sampling design was applied to ensure representative detection of aMPV among broiler breeder flocks in Egypt. The study area was stratified into three major poultry production zones (Delta, Middle Egypt, and Upper Egypt), that represent distinct ecologic and production systems. Within each zone, farms were selected as clusters proportional to flock density and distribution, and individual birds were systematically sampled from multiple houses and sections within each flock to capture overall serological status and minimize house- or pen-level bias. The birds included in this study originated from commercial, privately owned broiler breeder farms distributed across studied Egyptian governorates. The birds were not owned by the authors or by a single institution but belonged to independent poultry production companies and private farm owners operating under routine commercial management.\u003c/p\u003e \u003cp\u003eA total of 60 unvaccinated broiler breeder flocks (five per governorate) were included in the study. Flocks were selected based on accessibility, availability of production records, and confirmed absence of prior aMPV vaccination. Although a subset of birds exhibited mild respiratory signs during the rearing phase (16 weeks) and a modest reduction in egg production during the production phase (35 weeks), sampling encompassed both affected and apparently healthy birds to provide a comprehensive overview of flock-level aMPV exposure. On the other hand, 6,000 serum samples (100 per flock) were collected to assess seroconversion dynamics and cumulative exposure during both rearing phase (n\u0026thinsp;=\u0026thinsp;50 sera/flock), and the production phase (n\u0026thinsp;=\u0026thinsp;50 sera/flock). This repeated cross-sectional approach allowed evaluation of immunity development over time while maintaining a representative assessment of the flocks.\u003c/p\u003e \u003cp\u003eFor molecular detection, 800 tracheal swabs were collected during the production period. This included 100 swabs per governorate, with the exception of Giza and Fayoum (40 each) and Beni Suef, Minya, Asyut, and Luxor (30 each). Sterile polyester-tipped swabs were used to collect specimens, which were then immediately placed into 3 mL of viral transport medium (VTM) comprised of Anderson\u0026rsquo;s modified Hanks Balanced Salt Solution (HBSS) supplemented with 2% heat-inactivated fetal bovine serum (FBS), 100 \u0026micro;g/mL gentamicin, and 0.5 \u0026micro;g/mL amphotericin B (Sigma-Aldrich). Samples were maintained at 2\u0026ndash;8\u0026deg;C in insulated ice boxes during transport. Upon arrival at the Virology Laboratory, Faculty of Veterinary Medicine, Cairo University, swab suspensions were vortexed, clarified by brief centrifugation, aliquoted, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use in molecular analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Serum Preparation and Serological Analysis\u003c/h2\u003e \u003cp\u003eApproximately 1.5 mL of blood was aseptically collected from the wing vein of randomly selected birds and transferred into sterile tubes. Blood was allowed to clot at room temperature and centrifuged at 3,000 rpm for 10 minutes. The resultant sera were transferred into labeled Eppendorf tubes, identifying flock, governorate, age, and collection date, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until testing. Samples showing hemolysis or contamination were excluded from analysis.\u003c/p\u003e \u003cp\u003eSerum samples were analyzed using a commercial indirect ELISA kit (IDVET\u0026reg; Laboratories, Grabels, France) for the detection of aMPV subtypes A and B. Samples were diluted 1:500 and incubated in 96-well microtiter plates pre-coated with aMPV antigens. After incubation and washing, anti-chicken horse radish peroxidase (HRP) conjugate was added, followed by TMB substrate for color development. Optical density was measured at 450 nm using an Thermo Scientific\u0026trade; Multiskan FC\u0026trade; Photometer as previously described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The sample-to-positive (S/P) ratio was calculated as follows: (Sample OD \u0026ndash; NC OD) / (PC OD \u0026ndash; NC OD).\u003c/p\u003e \u003cp\u003eAntibody titers were calculated as Log10(Titer)\u0026thinsp;=\u0026thinsp;1.09 Log10(S/P)\u0026thinsp;+\u0026thinsp;3.360. Samples with titers greater than 396 (S/P\u0026thinsp;\u0026gt;\u0026thinsp;0.2) were considered positive.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. RNA Extraction and Molecular detection by RT-qPCR\u003c/h2\u003e \u003cp\u003eFor each flock, every 10 tracheal swabs were pooled, resulting in a total of 80 pools. Viral RNA was extracted from 200 \u0026micro;L of pooled tracheal swabs using QIAamp Viral Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer\u0026rsquo;s instructions. All procedures were conducted under RNase-free conditions, and eluted RNA was stored at \u0026ndash; 80 \u0026ordm;C for downstream processing. All RNA samples were screened for \u003cem\u003eMycoplasma gallisepticum\u003c/em\u003e, \u003cem\u003eMycoplasma synoviae\u003c/em\u003e, \u003cem\u003eOrnithobacterium rhinotracheale\u003c/em\u003e (ORT), Newcastle disease virus (NDV), Infectious Bronchitis Virus (IBV), and avian influenza virus (subtypes H5 and H9) using routine RT-PCR assays.\u003c/p\u003e \u003cp\u003eMolecular detection of aMPV was performed using Kylt\u0026reg; aMPV A\u0026amp;B real-time RT-PCR kit (AniCon Labor GmbH, Emstek, Germany) on AB 7500 Fast Real Time PCR system (Thermo Fisher SCientific, Foster City, CA). The assay simultaneously targeted the G gene of subtypes A and B using specific primers and TaqMan probes labeled with FAM (aMPV-A) and Cy5 (aMPV-B), respectively. Beta-actin specific primers and HEX-labeled probe served as an endogenous control as previously reported [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The reaction comprised of 16 \u0026micro;L master mix and 4 \u0026micro;L RNA template, while the thermal profile was adjusted as follows: reverse transcription at 50\u0026deg;C for 10 min, polymerase activation at 95\u0026deg;C for 1 min, 42 cycles of denaturation at 95\u0026deg;C for 10 s, and annealing/extension at 60\u0026deg;C for 1 min with fluorescence detection in FAM, Cy5, and HEX channels. Positive controls included commercial live vaccines for aMPV subtype A (Poulvac\u0026reg; TRT, Zoetis, Parsippany, NJ) and subtype B (Nemovac\u0026reg;, Merial, Lyon, France), while nuclease-free water served as a negative control. Cycle threshold (Ct) values\u0026thinsp;\u0026ge;\u0026thinsp;37 was considered negative, and differences\u0026thinsp;\u0026gt;\u0026thinsp;10 cycles between FAM and Cy5 in the same sample were regarded as questionable.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Statistical Analysis\u003c/h2\u003e \u003cp\u003eSerological data were initially compared across production phases using the Wilcoxon rank-sum test. Subsequent comparisons among governorates and regions were performed using the Kruskal\u0026ndash;Wallis test, followed by pairwise post hoc analyses and \u003cem\u003ep\u003c/em\u003e-value adjustment according to the Bonferroni correction. All statistical analyses and data visualizations were conducted in RStudio (version 2025.09.2) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] using R programming language (version 4.5.2) [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Data are presented as arithmetic means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations, and statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Sero-surveillance of aMPV in broiler breeder flocks\u003c/h2\u003e \u003cp\u003eA high level of aMPV exposure was detected across all surveyed governorates. Overall, 96.0% of serum samples tested positive for aMPV antibodies, indicating widespread serological evidence of infection at the individual bird level. At the flock level, 100% of sampled broiler breeder flocks were seropositive, confirming that aMPV exposure was present in all investigated production settings. Governorate-level seroprevalence ranged from 94.0% in Beheira to 97.4% in Beni Suef and Giza with minor geographic variation.\u003c/p\u003e \u003cp\u003eAntibody titers increased markedly from the rearing phase to the production phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Antibody levels at 35-week-old flocks were significantly higher than those observed at 16-week-old flocks (Wilcoxon rank-sum test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), which is consistent with the progressive seroconversion during the production cycle. Substantial variability in antibody titers was observed at both ages, indicating heterogeneous immune responses among birds and flocks.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt 16 weeks, Middle Egypt exhibited higher antibody titers compared to Delta and Upper Egypt regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This regional pattern shifted by 35 weeks, when Delta region displayed the highest antibody titers, exceeding those recorded in Middle and Upper Egypt (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The spatial maps (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) revealed a persistent north to south gradient in antibody titers across the production phases, with Upper Egypt flocks consistently exhibiting lower antibody levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt 16 weeks, governorate-level analysis showed higher antibody titers in Beni Suef and Giza, intermediate titers in Fayoum, Qalyubia, Gharbia, Beheira, and Kafr El-Sheikh, and the lowest titers in Asyut, Minya, and Luxor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). By 35 weeks, antibody titers increased across all governorates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Delta governorates (Qalyubia, Gharbia, Menoufia, Kafr El-Sheikh, and Beheira) formed a high-titer cluster, while Sharqia and Giza exhibited intermediate responses. Lower antibody levels persisted in Beni Suef, Fayoum, and Upper Egypt governorates, confirming sustained geographic heterogeneity throughout the production cycle.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Molecular Detection and Subtyping of aMPV by RT-qPCR\u003c/h2\u003e \u003cp\u003eAmong the 80 pools of tracheal swabs, 67 (83.75%) tested positive for aMPV RNA. Subtype B was the predominant genotype that was detected solely in 65 pools of samples (81.25%) and mixed with subtype A in 2 sample pools originated from Beheira governorate (2.5%). No sample pools were exclusively positive for subtype A (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRT-qPCR detection of avian metapneumovirus (aMPV) subtypes in pooled tracheal swabs from broiler breeder flocks in Egypt (2024\u0026ndash;2025)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGovernorate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample Pools (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubtype B positive pools (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u003c/p\u003e \u003cp\u003eco-detection (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePCR-negative pools (n)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBeheira\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKafr El-Sheikh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGharbia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSharqia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQalyubia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonufia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGiza\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFayoum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMinya\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBeni Suef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsyut\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuxor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePositive samples were recognized in all Delta, Middle Egypt, and Upper Egypt governorates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which indicate broad geographic distribution of aMPV. Subtype B was identified in all governorates. High detection rates of subtype B were observed in Delta governorates (80\u0026ndash;90%) and slightly decreased while moving south to Lower and Upper Egypt governorates (67.7\u0026ndash;75%). Ct values varied among pools with an average range of 18.4 to 32.9 (mean 25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1), with no obvious geographic pattern.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eUsing a combined serological and molecular framework, this study elucidates patterns of aMPV circulation in unvaccinated broiler breeder flocks across Egypt. The integration of dual-age antibody profiling with subtype-specific RT-qPCR enabled interpretation of both cumulative exposure and active viral circulation, revealing spatial and temporal heterogeneity in aMPV epidemiology.\u003c/p\u003e \u003cp\u003eThe uniformly high seroprevalence observed across all surveyed governorates indicates widespread exposure to aMPV in Egyptian broiler breeders. As all flocks were confirmed to be unvaccinated, antibody detection most plausibly reflects natural infection rather than vaccine-induced immunity. Similar high seroprevalence levels have been reported in breeder flocks from Asia and the Middle East, including Bangladesh, Iran, and South Korea [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This supports the concept that long-lived breeder populations experience extended windows of viral exposure compared to the short-cycle broiler flocks\u003c/p\u003e \u003cp\u003eIn contrast to the earlier studies conducted in Egypt, the current investigation provides more comprehensive understanding of aMPV circulation in broiler breeder flocks. Previous serological surveys often focused on flocks exhibiting clinical signs and typically involved limited sample sizes and/or restricted geographical coverage [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Molecular studies primarily analyzed mutant strains developed under vaccination pressure, while no studies have targeted unvaccinated populations [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In this study, 60 unvaccinated broiler breeder flocks across twelve governorates were evaluated by combining dual-age serological profiling with subtype-specific RT-qPCR of pooled tracheal swabs. This approach revealed uniformly high seroprevalence (~\u0026thinsp;96%) and a predominance of subtype B, with mixed detection of types A and B in Beheira governorate only. The observed difference between our results and the previous reports is likely attributed to variation in host population, sampling strategy, and methodological resolution rather than to temporal change in virus circulation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These findings reinforce the hypothesis that broiler breeders, due to their longevity and central role in the production pyramid, may serve as reservoirs sustaining aMPV transmission within Egyptian poultry systems.\u003c/p\u003e \u003cp\u003eThe observed increase in the antibody titers between 16 and 35 weeks of age is consistent with the cumulative immune stimulation over the production cycle. This pattern aligns with reports from breeder flocks exposed to other respiratory viruses, such as infectious bronchitis virus, where repeated or prolonged exposure leads to progressive antibody amplification [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Considerable variability in titers among flocks and governorates further suggests heterogeneous exposure histories, potentially influenced by differences in management practices, biosecurity levels, flock size, and genetic susceptibility, as documented for other avian viral diseases such as Marek\u0026rsquo;s disease and avian leukosis [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne of the notable findings of this study is the spatial shift in antibody profiles between production stages. At 16 weeks of age, higher mean titers were detected in the governorates of Middle Egypt (Beni Suef, Giza, Fayoum), whereas by 35 weeks, the highest titers clustered in the Nile Delta region. We hypothesize that early exposure during the rearing phase in Middle Egypt may be influenced by regional ecological factors, while later exposure in the Delta is more strongly driven by horizontal transmission within high-density production systems [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The early antibody responses detected in Middle Egypt is also coincided with sampling conducted in November 2024 during the migratory season. The proximity of backyard and small-scale breeder operations to wetlands and irrigation canals in Middle Egypt elevates the risk of indirect pathogen transmission from wild birds. By contrast, the dominance of high antibody titers in Delta governorates at 35 weeks of age is likely reflecting sustained within-sector transmission. The Nile Delta is characterized by high poultry density, large flock sizes, and frequent movement of personnel and equipment. These conditions are known to facilitate viral persistence and spread. Similar density-driven amplification for avian pathogens in North African and Middle Eastern production systems [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], further support the interpretation that production intensity becomes a dominant driver of exposure later in the production cycle.\u003c/p\u003e \u003cp\u003eRT-qPCR screening of pooled tracheal swabs demonstrated widespread detection of aMPV during the production phase. The predominance of subtype B across all positive governorates, alongside the absence of subtype A as a sole agent, is consistent with regional reports from Morocco, Tunisia, Algeria, Jordan, and Turkey [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. These findings suggest shared epidemiological patterns across the Middle East and North Africa, potentially facilitated by poultry trade, shared production practices, and broader ecological connectivity. Co-detection of both A and B subtypes was limited to Beheira governorate. This localized pattern may reflect transient co-circulation events in large breeder flocks, where dense contact networks increase the probability of multiple viral introductions and short-term subtype overlap. Similar phenomena have been described for other avian respiratory pathogens like avian influenza in large commercial populations [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe predominance of subtype B raises the question of whether detected viruses represent circulating field strains or, less likely, vaccine-derived variants. Although all flocks in this study were unvaccinated, indirect exposure to vaccine-related viruses from neighboring farms or environmental persistence cannot be completely excluded. Vaccine-derived reversion events have been documented in Europe, including a subtype A aMPV vaccine strain associated with clinical disease in turkeys despite the absence of recent vaccination of affected farms [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. While there is no direct evidence in the present dataset supporting a vaccine origin, this possibility cannot be resolved without full-genome sequencing. Differentiating field strains from vaccine-related variants remains critical for accurate epidemiological interpretation and informed control strategies.\u003c/p\u003e \u003cp\u003eThese findings emphasize the importance of implementing routine, integrated serological and molecular surveillance in broiler breeder populations. Future research should incorporate longitudinal sampling, full-genome sequencing, and targeted surveillance at the poultry\u0026ndash;wildlife interface, as well as focused investigations into the potential for vertical transmission of aMPV, which has been suggested in previous studies [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Clarifying the contribution of breeder-to-progeny transmission will be essential to fully understand aMPV epidemiology and to evaluate the suitability of subtype-matched vaccination and control strategies under controlled conditions.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrates that aMPV is likely circulating in the studied Egyptian governorates, predominantly as subtype B. Early exposure in Middle Egypt could potentially be influenced by migratory bird activity, while cumulative transmission in high-density Delta farms likely contributes to later antibody peaks. Serological and molecular results indicate ongoing circulation in unvaccinated flocks, suggesting that breeders may serve as reservoirs. These findings provide a foundation for evidence-based surveillance and biosecurity improvements and suggest that evaluation of vaccination strategies under controlled trials could be considered in the future. Overall, the study contributes to a broader understanding of aMPV epidemiology in the Middle East and North Africa\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eaMPV – Avian metapneumovirus\u003c/p\u003e\n\u003cp\u003eSHS – Swollen Head Syndrome\u003c/p\u003e\n\u003cp\u003eRT-qPCR – Real-time quantitative reverse transcription polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eRNA – Ribonucleic acid\u003c/p\u003e\n\u003cp\u003eELISA – Enzyme-linked immunosorbent assay\u003c/p\u003e\n\u003cp\u003eORFs – Open reading frames\u003c/p\u003e\n\u003cp\u003eN – Nucleoprotein\u003c/p\u003e\n\u003cp\u003eP – Phosphoprotein\u003c/p\u003e\n\u003cp\u003eM – Matrix protein\u003c/p\u003e\n\u003cp\u003eM2 – Matrix protein 2\u003c/p\u003e\n\u003cp\u003eF – Fusion protein\u003c/p\u003e\n\u003cp\u003eSH – Small hydrophobic protein\u003c/p\u003e\n\u003cp\u003eG – Attachment glycoprotein\u003c/p\u003e\n\u003cp\u003eL – Large polymerase protein\u003c/p\u003e\n\u003cp\u003eIACUC – Institutional Animal Care and Use Committee\u003c/p\u003e\n\u003cp\u003eVTM – Viral transport medium\u003c/p\u003e\n\u003cp\u003eHBSS – Hanks’ Balanced Salt Solution\u003c/p\u003e\n\u003cp\u003eFBS – Fetal bovine serum\u003c/p\u003e\n\u003cp\u003eOD – Optical density\u003c/p\u003e\n\u003cp\u003eS/P – Sample-to-positive ratio\u003c/p\u003e\n\u003cp\u003eCt – Cycle threshold\u003c/p\u003e\n\u003cp\u003eNDV – Newcastle disease virus\u003c/p\u003e\n\u003cp\u003eIBV – Infectious bronchitis virus\u003c/p\u003e\n\u003cp\u003eAIV – Avian influenza virus\u003c/p\u003e\n\u003cp\u003eORT – Ornithobacterium rhinotracheale\u003c/p\u003e\n\u003cp\u003eMG – Mycoplasma gallisepticum\u003c/p\u003e\n\u003cp\u003eMS – Mycoplasma synoviae\u003c/p\u003e\n\u003cp\u003eHRP – Horseradish peroxidase\u003c/p\u003e\n\u003cp\u003eTMB – 3,3′,5,5′-Tetramethylbenzidine\u003c/p\u003e\n\u003cp\u003ePCR – Polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eRNA – Ribonucleic acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo euthanasia or sacrifice of birds was performed in this study. All samples were obtained from apparently healthy, live birds during routine farm handling. Blood samples were collected from the wing vein, and tracheal swabs were obtained using sterile polyester-tipped swabs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe birds enrolled in this study were sourced from commercial, privately owned broiler breeder farms and were neither owned nor managed by the authors or by any academic or governmental institution. Before sample collection, permission was formally obtained from the respective farm owners or authorized farm managers to access the farms and to collect samples from their birds for research purposes.\u003c/p\u003e\n\u003cp\u003eAll animal handling procedures, farm access, and sample collection from privately owned flocks were conducted in accordance with approved animal welfare guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC), Faculty of Veterinary Medicine, Cairo University \u003cstrong\u003e(Approval No. Vet CU-301220251281).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are included within the manuscript. Additional data is available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial or non-financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding from public, commercial, or non-profit funding agencies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOmar S. Saeed:\u003c/strong\u003e Conceptualization, Study design, Sample collection, Data curation, Formal analysis, Writing original draft, \u003cstrong\u003eMirna Akram Labib\u003c/strong\u003e:\u0026nbsp;Data Curation, review \u0026amp; editing, Mahmoud Gamal: Statistical analysis, Data Curation, Writing—review \u0026amp; editing,\u003cstrong\u003e\u0026nbsp;Basem M. Ahmed:\u003c/strong\u003e Supervision, manuscript review and editing, \u003cstrong\u003eAyman H. El-Deeb:\u003c/strong\u003e Supervision, manuscript review and editing, \u003cstrong\u003eHaitham M. Amer:\u003c/strong\u003e Supervision, manuscript review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the Arab Poultry Breeders Company (OMMAT) for its valuable cooperation and logistical support throughout this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKaboudi K, Lachheb J. Avian metapneumovirus infection in turkeys: A review on turkey rhinotracheitis. Journal of Applied Poultry Research. 2021 Dec 1;30(4):100211. \u003c/li\u003e\n\u003cli\u003eToquin D, B\u0026auml;yon-Auboyer MH, Eterradossi N, Jestin V, Morin H. Isolation of pneumovirus from a Muscovy duck. 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Demonstration of loss of attenuation and extended field persistence of a live avian metapneumovirus vaccine. Vaccine. 2006 Oct 30;24(42-43):6476-82.\u003c/li\u003e\n\u003cli\u003eLupini C, Cecchinato M, Ricchizzi E, Naylor CJ, Catelli E. A turkey rhinotracheitis outbreak caused by the environmental spread of a vaccine-derived avian metapneumovirus. Avian pathology. 2011 Oct 1;40(5):525-30.\u003c/li\u003e\n\u003cli\u003eCook JKA, Chesher J, Orthel F, Woods MA, Orbell SJ, Baxendale W, Huggins MB. Avian pneumovirus infection of laying hens: experimental studies. Avian Pathol. 29:545\u0026ndash;556; 2000. doi:10.1080/03079450020016788.\u003c/li\u003e\n\u003cli\u003eJones RC, Williams RA, Baxterjones C, Savage CE, Wilding GP. Experimental infection of laying turkeys with rhinotracheitis virus: distribution of virus in the tissues and serological response. Avian Pathol. 17:841\u0026ndash; 850; 1988. doi:10.1080/03079458808436506.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Avian metapneumovirus, Broiler breeder, Egypt, RT-qPCR, Surveillance, Subtype B","lastPublishedDoi":"10.21203/rs.3.rs-8457890/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8457890/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAvian metapneumovirus (aMPV) is an economically significant respiratory pathogen of poultry that affects performance, egg production, and fertility of breeder flocks. Despite its impact, aMPV continues to be ill-defined in Egypt with no integrated surveillance studies conducted in breeder flocks. Therefore, the current study was designed to provide the first comprehensive molecular and serological assessment of aMPV circulation in broiler breeders in Egypt.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eBetween 2024 and 2025, 6,000 serum samples and 800 tracheal swabs were collected from 60 unvaccinated broiler breeder flocks across twelve Egyptian governorates. Sera were obtained at 16 weeks (rearing phase) and 35 weeks (production phase) of age and screened for aMPV subtypes A and B by ELISA. Tracheal swabs collected during the production phase were pooled into 80 composite samples and tested for the viral RNA by RT-qPCR.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSerological analysis revealed widespread aMPV exposure, with flock-level seroprevalence ranging between 94.0% and 97.4%. Antibody titers increased between 16 and 35 weeks, reflecting cumulative viral exposure. Molecular testing detected aMPV RNA in 67 (83.75%) of pooled swabs. Subtype B was the predominant genotype detected solely in 65 (81.25%) sample pools and mixed with subtype A in 2 pools (2.5%). Serological and molecular findings were generally aligned, with flocks positive for aMPV RNA often exhibiting higher antibody titers.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings indicate that aMPV, particularly subtype B, is likely endemic across the Egyptian broiler breeder flocks. The study highlights critical knowledge gaps, emphasizing the need for viral isolation, sequencing, and controlled evaluation of biosecurity and vaccination strategies.\u003c/p\u003e","manuscriptTitle":"Molecular–Serologic Snapshot of a Silent Respiratory Threat: Avian Metapneumovirus Circulation in Unvaccinated Broiler Breeders in Egypt, 2024-2025","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 17:58:05","doi":"10.21203/rs.3.rs-8457890/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-24T16:39:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-21T03:49:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-18T04:19:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-17T23:04:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98122953076840367241117086274149164224","date":"2026-01-29T02:51:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259306347584035352030921445853771713325","date":"2026-01-27T22:31:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64903398478513702453649373692130193331","date":"2026-01-27T02:46:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-20T18:42:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-20T14:46:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-20T08:24:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-19T08:21:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2026-01-19T08:10:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d1863a2d-08d6-4298-b1b2-c7e9e370946b","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T12:10:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-22 17:58:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8457890","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8457890","identity":"rs-8457890","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}