Development of a Non-Invasive Indirect ELISA for Detecting IgY Against a Novel Avian Metapneumovirus Subtype C in Egg Yolks of Laying Ducks

Avian metapneumovirus subtype C (aMPV/C) has re-emerged as a widespread pathogen in China, causing hydrosalpinx fluid syndrome (HFS) in laying ducks and resulting in substantial economic losses, highlighting the urgent need for a reliable immunological detection method. In the present study, a non-invasive indirect enzyme-linked immunosorbent assay (iELISA) was developed and optimized for detecting aMPV/C-specific immunoglobulin Y (IgY) in egg yolks of laying ducks, using purified novel aMPV/C as the coating antigen. Checkerboard titration established the optimal conditions as an antigen concentration of 1.25 μg/mL and an IgY dilution of 1:200. The cut-off value, determined by receiver operating characteristic curve analysis, was 0.3268, enabling a detection limit of 1:3200 for IgY antibodies. The assay showed no cross-reactivity with IgY positive for other duck pathogens, and both intra-assay and inter-assay coefficients of variation were less than 10%, demonstrating good specificity and reproducibility. Clinical evaluation of 151 duck egg yolk samples yielded an overall aMPV/C-specific IgY positivity rate of 58.3% (88/151). The concordance rate between the iELISA and an indirect immunofluorescence assay (IFA) was 97.8% (45/46). These results indicate that the established iELISA for detecting aMPV/C-specific IgY is a reliable and practical tool for large-scale detection of aMPV/C exposure in clinical duck egg yolk samples.