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.