Two categories of serum samples were used in this study: clinical sera for method establishment and validation, and immune sera for immunogenicity evaluation. Clinical serum samples were collected from 10 commercial meat duck farms (Cherry Valley ducks, n = 210; local breed ducks, n = 80) located in six regions of Jiangsu Province, China (including Xuzhou, Suqian, and Yangzhou) between 2022 and 2024. Ducks were categorized by age: 1–4 weeks (n = 105), 5–8 weeks (n = 123), and >8 weeks (n = 62). Production types included intensive large-scale farms (≥10,000 ducks/farm, n = 4 farms, 165 samples) and small-scale backyard farms (<5,000 ducks/farm, n = 6 farms, 125 samples). Health status was recorded at sampling: clinically healthy ducks (n = 238) and ducks with mild clinical signs (e.g., feather loss, growth retardation, n = 52). Sampling was conducted using a stratified random method: within each farm, ducks were randomly selected from different pens (3–5 pens per farm), with sample size proportional to flock size (10–30 samples per farm). Inclusion criteria: ducks with no recent vaccination against DuCV (within 3 months) and no history of severe infectious diseases (e.g., duck enteritis, hepatitis) in the flock within 2 weeks before sampling. Exclusion criteria: ducks with obvious trauma, systemic inflammation, or incomplete medical records. All samples were transported to the laboratory on ice within 24 h and stored at −20 °C until analysis.

Additionally, laboratory-preserved positive sera against duck enteritis virus (DEV), goose parvovirus (GPV), duck tembusu virus (DTMUV), duck hepatitis virus (DHV), goose circovirus (GoCV) and duck-derived bacteria (riemerella anatipestifer RA, salmonella enteritidis SE, avian pathogenic Escherichia coli APEC) were included to verify the specificity of the iELISA.

Immune sera were prepared by immunizing specific-pathogen-free (SPF) ducks (Health-Tech Laboratory Animal Breeding Co., Shandong, China). A total of 25 healthy 20-day-old SPF ducks were randomly divided into five groups (n = 5) with similar body weights (≤10% variation). Ducks were immunized subcutaneously in the neck. Two groups received low-dose (0.5 μg) and high-dose (2 μg) recombinant DuCV Cap protein, respectively; two groups received the same doses of DuCV VLPs; and the remaining group received phosphate-buffered saline (PBS) as a negative control. Two weeks after immunization, 2 mL of blood was collected from the wing vein of each duck. Sera were obtained by centrifugation (3,000 rpm, 5 min) after standing at 4 °C, aliquoted, and stored at −20 °C until analysis. These samples were used to compare the immunogenicity of Cap and VLPs.

The ORF2 gene encoding DuCV Cap (GenBank ID: EU344805, genotype 2a) was codon-optimized for expression in Escherichia coli and synthesized commercially (Sangon Biotech, Shanghai, China). The optimized gene was cloned into the pCold I vector (Addgene, MA, United States) and transformed into BL21 (DE3) competent cells (Solarbio, Beijing, China). A single colony was inoculated into 5 mL LB medium containing 100 μg/mL ampicillin and cultured overnight at 37 °C with shaking (220 rpm) as a seed culture. The seed culture was then diluted 1:100 into 200 mL LB medium (100 μg/mL ampicillin) and cultured at 37 °C (220 rpm) until OD₆₀₀ reached 0.8 (approximately 3.5 h). Protein expression was induced by adding 0.5 mM isopropyl-β-D-thiogalactoside (IPTG), followed by incubation at 16 °C for 24 h with gentle shaking (180 rpm). Protein solubility was analyzed using SDS-PAGE and Western blotting.

Bacterial pellets were harvested by centrifugation (8,000 × g, 10 min, 4 °C) and resuspended in 30 mL lysis buffer (0.1 M NaH₂PO₄, 20 mM imidazole, 300 mM NaCl, 8 M urea, 0.5% Triton X-100, pH 8.0). After sonication (300 W, 5 s on/5 s off, 30 min) and overnight incubation at 4 °C, lysates were ultracentrifuged (100,000 × g, 30 min, 4 °C) to remove debris. The supernatant was loaded onto a 5 mL Ni Sepharose Fast Flow column (GE Healthcare, IL, United States) pre-equilibrated with lysis buffer. The column was washed with 10 column volumes (CV) of wash buffer (0.1 M NaH₂PO₄, 50 mM imidazole, 300 mM NaCl, 8 M urea, pH 8.0) to remove non-specifically bound proteins. The target protein was eluted with 5 CV of elution buffer (0.1 M NaH₂PO₄, 500 mM imidazole, 300 mM NaCl, 8 M urea, pH 8.0). Protein renaturation was performed by stepwise dialysis in buffer (20 mM NaH₂PO₄, 300 mM NaCl, 2 mM β-mercaptoethanol, 0.4% arginine, 10% glycerol, pH 7.5), gradually reducing urea concentrations (6 M → 4 M → 2 M → 0 M). Finally, the renatured protein was dialyzed in 0.2 M NaH₂PO₄–Na₂HPO₄ buffer (pH 6.0, 4 °C, 24 h) to induce self-assembly into VLPs.

Protein samples were mixed with 5 × loading buffer, boiled for 10 min, and separated on 12% SDS-PAGE gels. Gels were either stained with Coomassie Brilliant Blue R-250 or transferred onto PVDF membranes (Millipore, MA, United States). Membranes were blocked with 5% skim milk for 1 h at room temperature, incubated with anti-His primary antibody (1,5,000, Solarbio), followed by HRP-conjugated goat anti-mouse secondary antibody (1,10,000, Solarbio). After washing with PBST, membranes were developed using enhanced chemiluminescence (ECL) reagent (Millipore).

A 10 μL aliquot of purified VLPs was applied to a 200-mesh copper grid for 10 min at room temperature. Excess liquid was removed with filter paper, and the grid was negatively stained with 2% phosphotungstic acid (Solarbio) for 1 min in the dark. After air-drying, samples were examined using a transmission electron microscope (JEM-2100F, JEOL, Tokyo, Japan).

VLPs were diluted in 50 mM carbonate buffer (pH 9.6) to varying concentrations, and 100 μL per well was coated onto 96-well microplates (Corning, NY, United States) overnight at 4 °C. For the immunogenicity test, Cap or VLPs were coated at 200 ng/well. After coating, plates were washed three times with PBST and blocked with 0.2% bovine serum albumin (BSA) at 37 °C for 60 min. Serum samples were diluted according to the experimental design (typically 1:200) and incubated at 37 °C for 20 min. Plates were then washed three times, incubated with HRP-labeled mouse anti-duck IgY (1:5000, Solarbio) for 20 min at 37 °C, and washed again. The color reaction was developed using 50 μL TMB substrate at 37 °C for 5 min in the dark and stopped with 50 μL of 2 M H₂SO₄. Each assay included blank, negative, and positive controls. For iELISA optimization, checkerboard titration was performed with three technical replicates per condition. Coating concentrations (0.5–10 μg/mL) and serum dilutions (1,100, 1:150, 1:200) were tested, with each combination assayed in triplicate to calculate mean OD₄₅₀ values and P/N ratios (positive serum OD₄₅₀ / negative serum OD₄₅₀).

Forty-five DuCV negative sera were used to determine the ELISA cut-off value. Each sample was tested in triplicate. Each serum sample was tested in three technical replicates, and the signal-to-positive (S/P) ratio was calculated as (sample OD₄₅₀ – blank OD₄₅₀)/(positive control OD₄₅₀ – blank OD₄₅₀). The cut-off value was statistically defined as the mean S/P ratio of negative samples plus 3 standard deviations (SD). Samples with S/p values above the cut-off were considered positive, and those below were negative.

The gold standard for diagnostic performance validation is strictly defined as DuCV-specific real-time quantitative PCR (qPCR) results targeting the conserved region of the DuCV ORF2 gene. A positive gold standard is defined as a qPCR Ct value < 38 with a single melting curve (no non-specific peaks), while a negative gold standard refers to a Ct value ≥ 40 or no amplification curve, with corresponding samples derived from clinically healthy duck flocks confirmed free of DuCV infection via epidemiological surveys.

To assess sensitivity, six positive sera of different reactivity levels were serially diluted (1:100–1:6400) in PBST and tested. The P/N ratio was calculated from OD₄₅₀ values. For specificity testing, positive sera against DEV, GPV, DTMUV, DHV, RA, SE, APEC, and GoCV were diluted 1:200 and analyzed using the optimized protocol. For repeatability and reproducibility, five DuCV positive sera were tested in triplicate within a single plate (intra-assay) and across different plates (inter-assay). The mean S/p value, SD, and coefficient of variation (CV = SD/Mean) were calculated. When CV < 10% was considered acceptable.

All statistical analyses were performed using SPSS 26.0 (IBM Corp., NY, United States) and GraphPad Prism 9.0 (GraphPad Software, CA, USA). Differences in antibody titers between Cap and VLP immunization groups were analyzed using two-way ANOVA. Intra-assay and inter-assay coefficients of variation (CV) were calculated as (SD/mean) × 100%. Temporal trends in seroprevalence (2022–2024) were analyzed using the Cochran-Armitage test for trend. A significance threshold of p < 0.05 was used for all statistical tests.

All animal experiments were conducted in accordance with the Guide for the Care and Use of Animals in Research of the People’s Republic of China. The study protocol was approved by the Committee on the Ethics of Animal Experiments of Jiangsu Agri-Animal Husbandry Vocational College (Approval No. JSAHVC-2023-35).

The full-length DuCV Cap gene was codon-optimized, synthesized, and cloned into the pCold I vector (Figure 1A). Following transformation into E. coli BL21 (DE3) cells, protein expression was induced at low temperature with IPTG. SDS-PAGE analysis revealed a distinct band at approximately 30 kDa, corresponding to the theoretical molecular weight of DuCV Cap (Figure 1B). However, the Cap protein accounted for only about 5% of total cellular protein and was predominantly insoluble.

The target protein was subsequently purified under denaturing conditions (8 M urea) using Ni-affinity chromatography (Figure 1C). After gradual refolding by stepwise dialysis, the purified Cap self-assembled into VLPs. Transmission electron microscopy (TEM) revealed numerous spherical particles approximately 15 nm in diameter, consistent with the morphology and size of native DuCV virions (Figure 1D). These observations confirmed that DuCV Cap successfully self-assembled into VLPs in vitro.

To compare the immunogenicity of Cap monomers and VLPs, ducks were immunized with low (0.5 μg) or high (2 μg) doses of each antigen. Serum samples were collected 2 weeks after immunization and analyzed by ELISA. Both Cap and VLP groups elicited specific antibody responses, whereas the PBS control group remained negative (Figure 2). At equivalent doses, VLPs induced significantly higher antibody titers than Cap monomers. Notably, even the low-dose VLP group produced higher antibody levels than both Cap groups, indicating that VLPs possessed markedly superior immunogenicity.

The positive-to-negative (P/N) ratio was used to determine the optimal antigen coating concentration and serum dilution by checkerboard titration. As shown in Table 1, the highest P/N ratio was obtained when the coating buffer was 50 mM carbonate–bicarbonate (pH 9.6), the antigen concentration was 2.5 μg/mL (equivalent to 250 ng/well), and the serum dilution was 1:200.

Other assay parameters were optimized to further enhance detection performance. The final optimized conditions were as follows: coating overnight at 4 °C; blocking with 0.2% bovine serum albumin (BSA) at 37 °C for 60 min; using HRP-conjugated mouse anti-duck IgY (1:5000) as the secondary antibody; and color development with TMB substrate for 5 min at 37 °C in the dark. The reaction was terminated with 2 M H₂SO₄, and the absorbance at 450 nm (OD₄₅₀) was recorded. This protocol ensured stable color development and reproducible signal intensity.

Forty-five negative serum samples were used to determine the cut-off value of the established ELISA. All serum samples were tested in triplicate to minimize experimental bias. After measuring the OD₄₅₀ values, the mean signal-to-positive (S/P) ratio of all negative samples detected by the DuCV VLP-based ELISA was calculated as 0.156, with a standard deviation (SD) of 0.080 (Figure 3). Accordingly, the cut-off value of this ELISA was defined as 0.396, which was derived from the formula: mean S/P ratio + 3 × SD. Samples with an S/P ratio higher than this cut-off value were classified as positive, while those with an S/P ratio lower than this value were classified as negative.

Six DuCV positive sera with varying reactivity were serially diluted twofold (1:100–1:6400) and tested by the established iELISA. As shown in Figure 4, positive signals were detectable up to a 1:6400 dilution, demonstrating the high sensitivity of the VLP-based assay.

To evaluate assay specificity, positive sera against DEV, GPV, DTMUV, DHV, RA, SE, APEC, and GoCV were tested at a 1:200 dilution (Table 2). The VLP-based iELISA showed no cross-reactivity with any of these pathogens. In contrast, the Cap-based iELISA produced a false-positive reaction with GoCV-positive serum, likely due to partial sequence homology between DuCV and GoCV Cap proteins. These results confirmed that VLPs preserved conformational epitopes unique to DuCV, thereby improving assay specificity.

Five DuCV positive sera were selected to assess intra- and inter-assay variation. The intra-assay CV ranged from 2.1 to 3.5%, and the inter-assay CV ranged from 2.9 to 4.8% (Table 3), both well below the 10% threshold. Additionally, based on the 290 qPCR-verified clinical samples (57 positive and 233 negative), the positive predictive value (PPV) and negative predictive value (NPV) of the VLP-iELISA were calculated as 100.0% (95% confidence interval: 93.5–100.0%) and 100.0% (95% confidence interval: 98.4–100.0%), respectively. These results demonstrated that the VLP-based iELISA exhibited excellent repeatability and reliability.

A total of 290 clinical serum samples collected from duck farms in Jiangsu Province (2022–2024) were tested using the established VLP-iELISA. As summarized in Tables 4, 57 samples were positive and 233 were negative, corresponding to an overall positivity rate of 19.96%. Descriptively, yearly positivity rates showed a gradual numerical increase: 15.29% (13/85) in 2022, 19.05% (20/105) in 2023, and 24.00% (24/100) in 2024. Statistical analysis using the Cochran-Armitage test for trend confirmed that this temporal increase was not statistically significant (p = 0.123). This lack of statistical significance is likely attributed to limited sample size in each year, which reduces the statistical power of the test and limits the ability to detect a true underlying trend. Notably, despite the absence of statistical significance, the consistent numerical upward trend suggests that DuCV infection may be gradually spreading within Jiangsu Province, and this trend warrants verification with expanded sample sizes in future surveillance. Regionally, the highest positivity rate was observed in Yancheng (28.21%), followed by Nantong (25.00%), while Yangzhou (13.95%) had the lowest. These findings suggest that DuCV infection remains prevalent and may be spreading gradually within Jiangsu Province.