Recombinant Protein-Based ELISA for the Detection and Differentiation of Antibodies Against Fowl Adenovirus Serotype 4 in Infected and Vaccinated Chickens

Fowl adenovirus serotype 4 (FAdV-4) has been identified as the primary pathogen responsible for hydropericardium-hepatitis syndrome (HHS), resulting in significant economic losses in major poultry-producing countries since 2015. Timely and accurate diagnosis of FAdV-4 infection is essential for the effective prevention and control of HHS. In this study, the two nonstructural genes of FAdV-4, 100K and 22K, were inserted into the expression vector pET-32a (+) respectively. The expressed recombinant proteins were used as coating antigens to develop two indirect ELISA methods, designated as 100K-ELISA and 22K-ELISA. Both ELISAs demonstrated high specificity, showing no cross-reactivity with serum samples positive for other avian diseases. Both ELISAs yielded positive results when applied to 50 serum samples from SPF chickens experimentally infected with FAdV-4 and negative results when applied to 50 serum samples from SPF chickens immunized with an inactivated FAdV-4 vaccine. Similarly, the field sample testing results demonstrated a significant ability to distinguish between vaccinated and infected samples. The 100K-ELISA and 22K-ELISA, which are based on nonstructural proteins, may be effective tools for differentiating between FAdV-4 infection and vaccination, offering a promising approach for differentiating infected from vaccinated animals (DIVA) strategies in poultry.