Revista Cienfica, FCV-LUZ / Vol. XXXVI 1 of 7 Received: 08/10/2025 Accepted: 12/03/2026 Published: 28/03/2026 hps://doi.org/10.52973/rcfcv-e362821 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico A novel indirect ELISA for serological surveillance of fowl adenovirus in chickens: ulizing a regionally relevant field strain Una nueva prueba ELISA indirecta para La vigilancia serológica del adenovirus aviar en pollos: Uso de una cepa de campo con relevancia regional Beatriz Padron¹* , Osman Erganis¹ ¹ Selçuk University, Faculty of Veterinary Medicine, Microbiology Department, Konya, Türkiye. *Corresponding author: beatriz_padron_perez@hotmail.com ABSTRACT Fowl adenoviruses are significant avian pathogens that cause considerable economic losses globally, associated with diseases such as inclusion of body hepas, hydropericardium syndrome, and gizzard erosion. There is a crical need for rapid, sensive, and high-throughput diagnosc tools for seroepidemiologic surveillance and evaluang vaccine efficacy, as the tradional diagnosc methods are limited due to the high viral diversity, which oſten hinders the effecveness of group-specific targeted commercial ELISAs, as the angens may not align with regionally prevalent serotypes. This study details the development and validaon of an indirect ELISA designed for the detecon of an-Fowl adenoviruses anbodies in chickens, ulizing an angen derived from a field strain of Fowl adenoviruses from Türkiye and propagated in cell culture. The assay was validated using 109 serum samples, benchmarked against the commercial BioCheck Fowl adenoviruses-1 ELISA CK132 kit. The developed ELISA demonstrated an opmal sample diluon of 1:400 and a cutoff value of 0.125. It achieved a sensivity of 90.20 % and a specificity of 100 %. A Cohen’s kappa index of 0.675 indicated good agreement with the reference method. Furthermore, the assay exhibited good precision, with intra-assay coefficients of variaon (CV %) ranging from 6.24 % to 11.03 % and an inter-assay CV % of 16.20 %, both within acceptable limits. This novel ELISA, employing a regionally relevant field strain, offers a cost-effecve and specific tool for Fowl adenoviruses serological surveillance and vaccine monitoring, parcularly beneficial in regions where circulang strains vary from vaccine strains. Key words: ELISA; fowl adenovirus; validaon; serology; poultry RESUMEN Los adenovirus aviares son importantes patógenos que causan considerables pérdidas económicas a nivel mundial y que están asociados a enfermedades como la hepas por cuerpos de inclusión, el síndrome de hidropericardio y la erosión de molleja. Existe una necesidad críca de herramientas de diagnósco rápidas, sensibles y de alto rendimiento para la vigilancia seroepidemiológica y la evaluación de la eficacia de las vacunas, ya que los métodos de diagnósco tradicionales son limitados debido a la gran diversidad viral, lo que a menudo dificulta la eficacia de los ELISA comerciales específicos para cada grupo, puesto que los angenos pueden no coincidir con los seropos prevalentes en la región. Este estudio detalla el desarrollo y la validación de un ELISA indirecto diseñado para la detección de ancuerpos frente a los adenovirus aviares en pollos, ulizando un angeno derivado de una cepa de campo de adenovirus aviares-E de Turquía y propagada en culvo celular. El ensayo se validó ulizando 109 muestras de suero, comparadas con el kit comercial BioCheck adenovirus aviares-1 ELISA CK132. La prueba desarrollada demostró una dilución óptima de la muestra de 1:400 y un valor de corte de 0,125. Alcanzó una sensibilidad del 90,20 % y una especificidad del 100 %. Un índice kappa de Cohen de 0,675 indicó una buena concordancia con el método de referencia. Además, el ensayo mostró una buena precisión, con coeficientes de variación intraensayo (CV %) que oscilaron entre el 6,24 % y el 11,03 % y un CV % interensayo del 16,20 %, ambos dentro de los límites aceptables. Este novedoso ELISA, que emplea una cepa de campo relevante a nivel regional, ofrece una herramienta específica y eficiente con relación al coste para la vigilancia serológica del adenovirus aviar y el seguimiento de la vacunación, lo que resulta especialmente beneficioso en regiones donde las cepas circulantes varían con respecto a las cepas de las vacunas. Palabras clave: ELISA; adenovirus aviar; validación; serología; avicultura

ELISA for Serological Surveillance of Fowl Adenovirus in Chickens / Padron y Erganis UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Fowl adenoviruses, classified within the Aviadenovirus genus of the Adenoviridae family, are segmented into three groups: Fowl adenoviruses (FAdV)-I, FAdV-II, and FAdV-III [1]. Group I is further subdivided into five species, encompassing twelve serotypes (1–8a, 8b–11) disnguished by their virus-neutralizaon profiles and genomic composion [2 , 3]. Genotype A includes FAdV- 1; Genotype B includes FAdV-5; Genotype C includes FAdV-4; Genotype D groups FAdV-2, FAdV-3, FAdV-9 and FAdV-11; y Genotype E groups FAdV-6, FAdV-7, FAdV-8a and FAdV-8b [4]. Group II includes viruses responsible for hemorrhagic enteris in turkeys and marble spleen disease in pheasants, while Group III is predominantly associated with egg drop syndrome virus [5]. Although FAdVs were historically regarded as non- pathogenic, they have since emerged as significant agents of disease in poultry, contribung to considerable global economic losses through outbreaks of inclusion body hepas (IBH), hydropericardium syndrome (HPS), and gizzard erosion (GE). Specifically, IBH is frequently linked to FAdV-2, FAdV-11, FAdV- 8a, and FAdV-8b serotypes; GE is predominantly caused by FAdV-1; and HPS is aributed to FAdV-4 [6 , 7 , 8]. Adenoviridae family infecons are present worldwide and the prevalence of specific serotypes varies across different geographical regions. For example, FAdV-4 is predominant in Asia, while FAdV-1 and FAdV-8b are more common in Europe and America [9]. In the case of Türkiye, the first cases were reported in 2020 by Şahindokuyucu et al. [10] and Çizmecigil et al. [11] and show that at least serotypes 8b and 11 are circulang; however, further informaon is sll scarce. Diagnosc methodologies for FAdV detecon have undergone considerable advancement. Current angen detecon methods encompass polymerase chain reacon, quantave PCR, loop- mediated isothermal amplificaon, high-resoluon melng analysis, and sandwich enzyme-linked immunosorbent assays [12 , 13 , 14]. Tradional serological assays like agar gel precipitaon and virus neutralizaon tests suffer from limited sensivity and scalability. Consequently, the emergence of rapid, sensive, and high-throughput Enzyme-Linked ImmunoSorbent Assay (ELISA) plaorms is essenal for accurate sero epidemiological surveillance and the assessment of vaccine efficacy, parcularly considering potenally subclinical infecons. Nonetheless, the substanal viral diversity presents a hurdle due to limited cross- protecon among different strains [9 , 15]. Recent advancements include recombinant fiber- and hexon-based ELISAs tailored to specific serotypes, notably FAdV- 4 [9 , 16 , 17]. This research outlines the creaon and validaon of an indirect ELISA employing a cell-cultured FAdV-E field strain from Türkiye. The primary goal was to develop a more appropriate and regionally relevant method for FAdV serological surveillance, by addressing the limitaons of commercial ELISAs, which oſten ulize group-specific angens that may not align with regionally prevalent outbreaks. MATERIALS AND METHODS Serum samples The serum samples ulized in this study were gathered from different poultry (Gallus gallus domescus) farms across Türkiye and sent to the Microbiology Department of the Veterinary Faculty of Selçuk University (Konya, Türkiye), where each blood sample was processed upon arrival and the serum was separated and stored at -40 °C (Ugur, UED 480 DGT D/S R64, Türkiye) unl analysis. In order to assess the new ELISA and evaluate its reliability, only the samples that, independently of this study, were also sent to an external laboratory for diagnosc or control purposes and were analyzed using the commercial FAdV-1 ELISA CK132 kit (Biochek, Ascot, UK) were included in the study; a total of 109 samples. ELISA plate coang The angen used was a strain of FAdV (SÜVF Stock Number 289), which had previously caused an IBH outbreak in southwestern Türkiye. The amplified hexon L1 gene fragment was sequenced directly using Sanger and a BLAST search (hps://blast .ncbi.nlm.nih.gov) was used to compare these sequences with other isolates. SÜVF 289 was found to be related to species E with 100.00 % (e.g. OM858818.1, MK572861.1, MK572859.1) sequence identy. The isolate was then produced in a Vero monolayer cell line. FAdV angen was measured using Qubit Protein Assay Kit (Invitrogen, Q33212, USA), diluted to a concentraon of 5 µg/mL in a 0.05 M carbonate-bicarbonate buffer (pH 9.6) and distributed at a rao of 100 µL/well into a polystyrene 96-well microplate. The plates were then incubated at +4 ºC (Altus, Al 328B, Türkiye) overnight, and at the conclusion of the incubaon phase, they were washed three mes with 300 µL of phosphate-buffered saline (PBS) (pH: 7.2) + 0.05 % Tween 20. To block angen-unbound polystyrene surfaces, 100 µL of PBS (pH: 7.2) containing sterile 5 % skim milk powder (LabM, MC027, England) was distributed into each well and incubated at room temperature for 2 hours (h), followed by three washes with 300 µL of PBS (pH: 7.2) + 0.05 % Tween 20. The plates were then dried and kept at +4 °C (Altus, Al 328B, Türkiye) unl needed [18 , 19 , 20 , 21]. ELISA procedure To achieve the best possible test performance, the opmal sample diluon was determined. To opmize assay performance, a systemac evaluaon of various serum diluons was conducted to idenfy the minimum concentraon yielding opmal signal, thereby ensuring accurate anbody detecon while minimizing sample volume. This process ulized seven posive and one negave serum, whose ters and opcal density values had been previously determined using the afore menoned commercial ELISA kit. Aſterwards, the 109 sera were diluted using PBS buffer (pH 7.2) at a 1:400 concentraon and distributed in duplicate into the coated wells (100 µL each), along with the posive (n = 1) and negave (n = 2) controls. The plates were kept at +4 °C overnight. Aſter a washing step with PBS (pH: 7.2) + 0.05 % Tween 20, 100 µL of rabbit an-chicken IgY (IgG) peroxidase conjugate (Sigma A9046, USA) diluted to 1/20,000 using a 0,05 M carbonate-bicarbonate buffer (pH 9.6) was added to the wells and allowed to incubate for 1 h at room temperature. 2 of 7

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico The plates were washed three mes using the washing buffer, while the substrate, δ-phenylenediamine 98 dihydrochloride (δ-phenylenediamine dihydrochloride tablets, Sigma P8287, USA), was prepared by adding it to a phosphate-citrate soluon (sodium perborate-capped phosphate-citrate buffer, Sigma P4922, USA). 100 µL of substrate was added to each well and incubated in the dark at room temperature for 30 minutes. Upon conclusion of the incubaon period, 50 µL of stop soluon (0.5 M H₂SO₄) was added to each well, and the results were evaluated by assessing the opcal density (OD) at 450 nm with the help of an ELISA microplate reader (BioTek 800TS, USA) [18 , 19 , 20 , 21]. Cutoff value determinaon To determine the opmal cutoff value for the developed ELI- SA test, a Receiver Operang Characterisc (ROC) curve analysis was performed using SPSS 26 soſtware, based on 22 individual measurements. The cutoff point closest to the upper-leſt corner of the ROC curve was selected, as it represents the best trade- off between sensivity and specificity [22]. Definion of the standard A posive and a negave standard were selected from sera idenfied as posive and negave by the commercial FAdV anbody kit. Both standards were distributed in duplicate on each of the tested plates. The inclusion of internal standards on each plate allowed for normalizaon across runs, accounng for inter-assay variability and ensuring the reliability of quantave measurements [23]. FAdV ELISA test validaon The FAdV Ab ELISA developed in this study was validated by comparing its performance with that of the FAdV-1 ELISA CK132 kit, the prevalent serologic test in Türkiye, using 109 serum samples. The sample size ulized in this study aligns with the quanes employed in previous literature for validang ELISA tests [22 , 24 , 25 , 26]. Diagnosc parameters such as diagnosc accuracy, sensivity and specificity offer a comprehensive assessment of the assay’s performance characteriscs, offering insights into its praccal ulity for disease detecon and epidemiological studies [25]. These were calculated using the selected cutoff value, based on the true posives (TP), false posives (FP), false negaves (FN), and true negaves (TN) obtained when compared to the commercial FAdV Ab kit. On the sensivity and specificity parameters, a 95 % confidence interval was applied [22]. True posives, FP, FN and TN Diagnosc accuracy = (TP + TN) / (TP + FP + FN + TN) Sensivity = TP / (TP + FN) * 100 Specificity = TN / (FP + TN) * 100 The precision of the assay was evaluated through both intra- assay and inter-assay variability analyses. Intra-assay variability was assessed using the 109 clinical samples, each tested in duplicate within the same microplate. For each sample, the coefficient of variaon (CV %) was computed. Intra-assay CVs were computed separately for each plate, allowing for plate-specific precision profiling. Inter-assay variability was determined using two reference samples analyzed in duplicate across all four plates (n = 8 measurements per sample) [22]. The CV % for each reference sample was calculated, reflecng reproducibility across independent assay runs. To further minimize variability between assay runs and improve the interpretability of ELISA results, normalizaon techniques based on the reference samples were implemented across different experimental sessions. This methodological approach is designed to migate variaons stemming from procedural differences across assays, thereby improving data consistency and overall dependability [27]. Stascal analysis Receiver Operang Characterisc analysis was performed to determine the cutoff value. SPSS version 26 (SPSS Inc., Chicago, IL, USA) soſtware was used. The remaining calculaons were performed using Microsoſt Excel for Microsoſt 365, version 2401 (Build 16.0.17231.20182), included in Microsoſt Office Professional Plus 2024 (Microsoſt Corporaon, Redmond, WA, USA). In all analyses, P < 0.05 was defined as “significant”. For comparave assays, Cohen’s kappa index was employed to quanfy the degree of agreement, where P0 represents the observed probability of agreement and Pe denotes the probability of agreement occurring by chance [22]. Cohen’s kappa index = (P0 – Pe) / (1 – Pe) RESULTS AND DISCUSSION This work contributes to the development of a novel ELISA test for detecng an-FAdV anbodies in chickens. The developed test was validated against the FAdV-1 ELISA CK132 kit (Biochek, Ascot, UK) as a reference method because it is widely used and accepted in the poultry sector of Türkiye. When benchmarked against the commercial kit, along with a moderate agreement, the assay demonstrated a high specificity. Sample diluon selecon The preliminary ELISA test was conducted using seven posive and one negave samples, diluted from 1:100 to 1:204,800. A good performance was achieved with sera diluted to 1:400 (FIG. 1). As expected, the OD values increased with lower diluons. Nevertheless, the capacity of the assay to perform reliably with small sample volumes is a key advantage for commercial implementaon and roune high-throughput tesng. Cutoff value determinaon and validaon The cutoff threshold for the in-house ELISA was established based on the ROC curve analysis at 0.125. A total of 109 serum samples were analyzed and the assay correctly idenfied 92 of the 102 posive samples (anbody detecon rate: 81.65 %, sensivity: 90.20 %; 95 % CI: 82.7–95.7 %) and all the negave samples (specificity: 100 %; 95 % CI: 59.3–100 %) (FIG. 2). 3 of 7

ELISA for Serological Surveillance of Fowl Adenovirus in Chickens / Padron y Erganis UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 1. Indirect Fowl Adenovirus (FAdV) ELISA showing opcal density at 450 nm (OD450) for serum samples diluted 1:100 to 1:204,800 (two-fold serial diluons). Samples: S3, S4, S11, S15, S21, S14, S23 (posive sera), NC (negave control: negave serum). Colors correspond to sample IDs as labelled. FIGURE 2. Comparison of OD450 values from the developed ELISA versus the reference commercial FAdV ELISA. Each point represents the OD450 value for each individual serum sample numbered 1–109. Red triangles are samples classified as posive by the reference kit but negave by the developed ELISA (discordant false negaves). Horizontal dashed line = posivity cutoff for the developed ELISA, calculated using the Receiver Operang Characterisc (ROC); samples with OD450 below this line were considered negave. OD = opcal density; FAdV = fowl adenovirus; NC = negave control. As the commercial ELISA was used as the standard method, the diagnosc accuracy, as well as the overall agreement between the two methods, was 90.83 % (95% CI: 85.4–96.2 %), with a Cohen’s Kappa index of 0.675, indicang good agreement beyond chance. No FP were observed, while 10 TP samples were not detected by the novel assay. The performance demonstrated, parcularly the high specificity, indicates the assay’s capacity to minimize FP, which is crucial for effecve disease surveillance and management [28]. On the other hand, the occurrence of 10 FN results among the 109 analyzed serum samples suggests that the assay may have reduced sensivity in samples with low anbody ters, variaons in the immune response or infecons by non-E serotypes. This limitaon should be considered, in contexts requiring maximal case detecon, although the assay remains suitable for populaon-level surveillance and vaccine monitoring. Importantly, the present study did not evaluate the ability of the developed ELISA to differenate infecon from vaccinaon. This disncon requires specifically designed experimental infecons or paired serological profiling, which were beyond the scope of the current validaon. Likewise, the assay was not tested in regions with high serotype diversity. The assay should thus be interpreted as a regionally adapted tool opmized for genotype E exposure, rather than a universal replacement for exisng commercial plaorms. These findings are consistent with previous studies that have reported variability in the sensivity of ELISA assays depending on the angen used, the stage of infecon, and the animal species evaluated. In this regard, opmizaon of the ELISA protocol, including the selecon of recombinant angens or cross-validaon with other molecular techniques, could improve its diagnosc performance, as it was demonstrated by Pan et al. [8], Feichtner et al. [9] and Shao et al [29]. The mean intra-assay CV % ranged from 6.24 % to 11.03 % across plates. Although the proporon of samples exceeding an inter- assay variaon threshold of 15 % is higher than ideal (17.43 %), it remains within an acceptable range according to general guidelines [30 , 31 , 32 , 33]. On the other hand, the inter- assay CV % was 16.20 %, which raised some doubts about the use of serum as a control, as it may introduce variability and inconsistencies between assay plates due to the inherent nature of the sample, a phenomenon previously documented in other ELISA methodologies [34], and which could be migated by developing a recombinant protein standard. Interpretaon of discordant results and assay scope The absence of FP underscores the capacity of the assay to minimize erroneous classificaon at the individual-sample level, which is essenal for surveillance and control decisions. The ten FN raise important consideraons: they could reflect low anbody ters in certain samples, variability in host responses, or angenic mismatch between the coang angen and some infecng serotypes. In this instance, the BioChek ELISA’s capacity to detect anbodies against all 12 FAdV serotypes suggests that serum samples negave in this assay may possess anbodies targeng other FAdV serotypes, Importantly, the ELISA in this study was coated with a Turkish field strain of genotype E, so its sensivity is opmized for detecng anbodies against FAdV-E (including 8a/8b) and for measuring vaccine-related responses. Given that previous surveillance has documented circulaon of 8b and 11 [9 , 10], sera negave in this genotype-E coated assay may harbour anbodies induced by infecons with non-E serotypes (e.g., serotype 11) or display anbody profiles shaped by vaccinaon; this paern has been noted in earlier reports [35] and can contribute to discordant results. Assay format, angen choice, and praccal relevance The indirect ELISA format was chosen due to its potenal for higher sensivity compared to compeve and sandwich formats, which target a limited number of epitopes [36 , 37]. A key advantage of this study is the ulizaon of an angen derived from a Turkish field strain of FAdV-E as the coang angen; this regional adaptaon enhances the diagnosc ulity, as current commercial ELISAs oſten target group-specific FAdV angens, which may not always align with the specific serotypes prevalent in a given geographical area [8]; in this case, serotypes belonging to this genotype (especially 8b) have been previously reported in the country [10 , 11]. This correspondence between the angen genotype and the serotypes detected in local outbreaks reinforces the epidemiological relevance of the ELISA developed and its usefulness for regional surveillance This regional angen choice improves epidemiological interpretaon for Egenotype 4 of 7

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico exposure and vaccine monitoring, but it also narrows serotype breadth, reducing sensivity for anbodies primarily directed against Dlineage serotypes such as 11, and thereby explaining some FN. The ELISA design, although with several reagents currently imported, employs a field-derived angen (SÜVF-289), which increases the assay’s epidemiological relevance for local strains and may reduce reliance on imported commercial kits, offering a significant economic advantage. Even though a detailed cost analysis is not available per sample, the use of angens produced in-house and generic reagents allows for reducing operang costs, especially in comparison with imported commercial kits that can be disproporonate in relaon to local purchasing power. This feature makes it a more accessible tool for regional laboratories and low-cost surveillance programs, favoring their large-scale implementaon Limitaons, future opmizaon and recommended applicaon The study presents certain limitaons, such as a limited sample size and the absence of interlaboratory validaon, that warrant further invesgaon. In addion, no informaon was provided about the samples, including vaccinaon status, which could significantly influence the interpretaon of serological results by disnguishing between infecon-induced and vaccine- induced immunity [35], as although heterologous reacons are present, homologous reacons are known to be stronger [38]. A genotypeE coated ELISA helps idenfy FAdV-E exposure and vaccine responses, but should be complemented by assays incorporang genotype D angens or by a mulplex panel that includes both D and E angens to resolve infecons by the other genotypes present in Türkiye. This future opmizaon should also include the usage of recombinant or thoroughly characterized protein standards for plate normalizaon to counteract the higher-than-expected inter-assay variability. In addion, future studies incorporang controlled vaccinaon– challenge designs and broader serotype panels will be essenal to further refine the assay’s diagnosc scope and comparave performance. Overall, the assay’s high specificity, reasonable precision, and regional angen suitability make it a suitable opon for populaon-level serological surveillance and vaccine monitoring. Nonetheless, using it alongside reference methods is advisable in epidemiological research or clinical cases demanding the highest sensivity. CONCLUSIONS AND IMPLICATIONS In summary, the successful creaon and validaon of this indirect ELISA, which uniquely employs a regionally relevant field FAdV-E strain from Southwest Türkiye, establishes it as a robust and highly specific diagnosc instrument tailored to local epidemiological condions. The assay offers cost- effecve and praccal value for populaon-level serological surveillance and vaccine-monitoring programs, parcularly in regions where genotype E is known to circulate. However, its ability to disnguish vaccine-induced from infecon-induced anbodies or to outperform commercial kits in areas with high serotype diversity has not yet been evaluated and future studies incorporang controlled vaccinaon–challenge designs and broader serotype panels will be essenal to further refine the diagnosc scope and comparave performance. This assay significantly enhances FAdV serological surveillance and vaccine monitoring by reflecng the local epidemiological condions within poultry populaons. Moreover, its praccal ulity should be considered in the context of regional disease control programs, where a clear disncon between individual- level diagnosis and populaon-level surveillance is essenal for guiding intervenon strategies and evaluang vaccine coverage. ACKNOWLEDGMENTS The authors thank the Office of Scienfic Research Projects of Selcuk University for the funding under the project number 22212015. Conflict of interest The authors have no conflicts of interest that could be perceived as prejudicing the imparality of the study. BIBLIOGRAPHIC REFERENCES [1] Fitzgerald SD, Rautenschlein S, Mahsoub HM, Pierson FW, Reed WM, Jack SW. Adenovirus infecons. In: Swayne DE, Boulianne M, Logue CM, McDougald LR, Nair V, Suarez DL, Wit S, Grimes T, Johnson D, Kromm M, Prajitno TY, Rubinoff I, Zavala G, editors. 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