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 pericardial effusion-hepatitis syndrome (HHS). Since 2015, FAdV-4 has spread extensively across major poultry-producing countries, resulting in significant economic losses. Timely and accurate diagnosis of FAdV-4 infection is essential for the effective prevention and control of HHS. In this study, two nonstructural genes from FAdV-4, 100K and 22K, were cloned into the expression vector pET-32a. The recombinant 100K and 22K proteins were expressed and purified, and subsequently used as coating antigens to establish two indirect ELISAs, referred to as 100K-ELISA and 22K-ELISA, respectively. Both ELISAs demonstrated high specificity, showing no cross-reactivity with serum samples positive for other avian diseases. Furthermore, they demonstrate high sensitivity, as FAdV-4 positive serum could still be detected even when diluted to 1:1280 (100K-ELISA) and 1:640 (22K-ELISA). The assays also showed excellent repeatability, with the maximum coefficient of variation between and within batches remaining below 5%. 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 unvaccinated samples. In chickens naturally infected with FAdV-4, the 100K-ELISA and 22K-ELISA showed overall concordance rates of 96.8% and 98.6%, respectively, when compared with a commercial kit coated with whole virions. These findings suggest that 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.