A novel species-specific multiplex PCR assay for the differentiation of five Fowl adenovirus species (A to E): Application to field surveillance

Fowl adenovirus (FAdV) is non‐enveloped, icosahedral DNA virus that cause significant economic losses in poultry by inducing inclusion body hepatitis (IBH), hepatitis‐hydropericardium syndrome (HHS), and gizzard erosion (GE) via both horizontal and vertical transmission. FAdV comprises five species (A-E) and twelve serotypes (1-7, 8a, 8b, and 9-11 ) that frequently co‐circulate within flocks, making early and accurate detection essential for effective control. Here, we have developed a single‐tube multiplex PCR assay targeting conserved regions of the fiber and penton base genes, capable of simultaneously detecting all five FAdV species with a sensitivity of 25 copies/tube. When applied to 125 field strains from 79 naturally FAdV-infected cases, the assay achieved 100 % concordance with singleplex PCR and Sanger sequencing. At the infection level, infection profiling showed 53.2 % single‐species cases, 36.7 % dual‐species co‐infections, 8.9 % triple‐species infections, and 1.3 % quadruple‐species infections, with FAdV-D /E co‐infections most prevalent. At the strain level, FAdV‐E (34.4 %) was the most common, followed by D (31.2 %), B (20.0 %), C (8.0 %), and A (6.4 %). Besides, phylogenetic analysis of the complete sequences of the fiber, penton base, and hexon genes confirmed species‐specific clustering perfectly aligned with the multiplex PCR results. Taken together, this rapid, sensitive, specific, and cost‐effective assay represents a powerful tool, providing a foundation for improved management and control of adenoviral diseases in poultry.