Establishment of an Indirect ELISA Method for Detecting Multiple Virulence Factors from Porcine Diarrhea-Related...

Porcine diarrheagenic Escherichia coli strains exhibit heterogeneous virulence profiles involving multiple factors, complicating broad-spectrum serological diagnosis. Here, we designed a multiepitope fusion antigen, MEAET, containing predicted B-cell epitopes from six major virulence factors, K88/F4, F18, LT, Stx2e, HlyA, and Tir, and developed an indirect ELISA for broad antibody detection. Structural modeling predicted a well-folded conformation with multiple discontinuous B-cell epitopes, and molecular docking suggested favorable interactions between the C-terminal dendritic cell-targeting peptide and the porcine immune receptor SLA1. Immunization of piglets with purified recombinant MEAET generated hyperimmune serum with a titer exceeding 1:102,400, which specifically recognized all six individual antigens by Western blot. The optimized ELISA demonstrated satisfactory repeatability (intra-assay CV ≤ 4.26%) and reproducibility (inter-assay CV ≤ 5.57%), showed no detectable cross-reactivity with antibodies against other tested porcine pathogens, and exhibited good analytical sensitivity, with positive sera detectable at dilutions up to 1:3200. The optimized ELISA showed good repeatability and reproducibility, with intra-assay and inter-assay coefficients of variation of ≤4.26% and ≤5.57%, respectively, no detectable cross-reactivity with the tested heterologous controls, and good analytical sensitivity, with positive sera remaining detectable at dilutions up to 1:3200. The S/P ratio cut-off was 0.203. In field sera, positivity rates were 74.5% (35/47) in diarrheic pigs and 34.0% (36/106) in asymptomatic pigs. These primary results indicate that the MEAET-based indirect ELISA represents a promising tool for serological detection of antibodies against multiple virulence factors of diarrheagenic E. coli in pigs and for field surveillance in swine herds.