In 2014, Duck Adenovirus type 3 (DAdV-3) emerged in Muscovy ducks and has since spread rapidly across China, causing significant economic losses to the duck industry. Given this situation, the development of reliable diagnostic tools is crucial for effective disease control. In this study, a neutralizing monoclonal antibody (mAb) 2F12 specific to DAdV-3 was generated, which showed a blocking rate of over 70% and a neutralization titer of up to 1:794. A blocking enzyme-linked immunosorbent assay (b-ELISA) was further developed based on mAb 2F12 to efficiently detect neutralizing antibodies against DAdV-3. The cut-off values of percent inhibition (PI) were set based on testing 84 negative duck serum samples, with a value below 16.79% (mean ($\overline{X}$) + 2 standard deviations (SD)) for negative sera and over 21.62% ($\overline{X}$ + 3SD) for positive sera. The b-ELISA exhibited a high specificity, reacting exclusively with DAdV-3 positive serum and showing no cross-reactivity with other representative positive sera tested. Additionally, the b-ELISA showed significantly higher sensitivity than the serum neutralization test (SNT), detecting antibodies 16-fold greater than the endpoint dilution of the SNT. The established b-ELISA, validated with 90 field serum samples from six duck farms, was well-suited for clinical detection of DAdV-3 antibodies and for monitoring post-vaccination antibody levels, representing a significant advancement in DAdV-3 detection and prevention.

Duck hepatitis A virus type 3 (DHAV-3), an avian-specific pathogen primarily impacting ducklings, poses a significant threat to the duck farming industry by causing high mortality rates. A DHAV-3 strain SH52 was isolated from the diseased ducks and phylogenetic analysis of the whole genome revealed that the DHAV-3 SH52 belongs to the prevalent strains in China. The DHAV-3 SH52 strain replicates at high levels in various organs of 8-day-old specific pathogen-free (SPF) shelducks, causing pathological damage and leading to high lethality in 8-day-olds following intramuscular infection. The findings of this study provide an ideal animal challenge model for evaluating the efficacy of DHAV-3 vaccines in the future.