Development and validation of a recombinant capsid protein-based indirect enzyme-linked immunosorbent assay for serological detection of duck circovirus in commercial flocks.

Background and Aim: Duck circovirus (DuCV) is an immunosuppressive pathogen linked to poor growth, feather abnormalities, and increased susceptibility to co-infections, leading to significant economic losses in duck production. Rapid and large-scale serological screening tools are essential for epidemiological surveillance and biosecurity. This study aimed to develop and validate an indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant capsid (Cap) protein for sensitive and specific detection of antibodies against DuCV. Materials and Methods: The cap gene from a Thai DuCV genotype I isolate was cloned into the pQE-31 vector and expressed in Escherichia coli M15. The 27 kDa recombinant Cap protein was purified under denaturing conditions, and its antigenicity was confirmed by Western blotting. The iELISA was optimized by checkerboard titration to determine the optimal antigen coating concentration and serum dilution. Diagnostic sensitivity, specificity, cross-reactivity, repeatability, reproducibility, and agreement with Western blotting were assessed using 80 positive, 103 negative, and 189 field serum samples. Results: The optimized iELISA used 12 μg/well of antigen and a 1:20 serum dilution, producing the highest positive-to-negative optical density ratio. Receiver operating characteristic curve analysis yielded an area under the curve of 0.996, with 97.5% sensitivity and 98.1% specificity. No cross-reactivity was detected with sera positive for duck Tembusu virus, duck viral enteritis virus, or Riemerella anatipestifer. Intra- and inter-assay coefficients of variation were below 6.5% and 9.1%, respectively. Diagnostic agreement with Western blotting across 189 field sera was 91.0%, with a Cohen's kappa of 0.752, indicating substantial concordance. Conclusion: The developed recombinant Cap-based iELISA provides a reliable, specific, and reproducible tool for large-scale DuCV serosurveillance. Its high diagnostic accuracy and scalability support its application in flock-level monitoring, pre-movement screening, and epidemiological studies, facilitating improved biosecurity and informed disease control strategies within the duck industry.