Oral vitamin D3 supplementation enhances antiviral immunity in chickens challenged with fowl adenovirus serotype 4.

Vitamin D is increasingly recognized as an important regulator of innate and adaptive immunity, yet its antiviral role in chickens during fowl adenovirus serotype 4 (FAdV-4) infection remains unclear. In this study, we investigated the effects of oral vitamin D3 supplementation on humoral immunity, tissue viral load, and immune-related gene expression in FAdV-4-infected chickens. Ninety 8-day-old specific pathogen-free chickens were randomly divided into two groups (n = 45/group) and fed a basal diet. The experimental group received oral vitamin D3 dissolved in 0.1 mL soybean oil at a dose equivalent to 3000 IU/kg diet daily for 3 weeks, whereas the control group received soybean oil alone. After supplementation, all birds were challenged with FAdV-4. Vitamin D3 supplementation significantly reduced tissue viral loads and increased serum antibody levels compared with the control group. At the transcriptional level, vitamin D3 modulated key innate and adaptive immune markers, including Toll-like receptors (TLRs), avian β-defensins, and cytokines. It downregulated several TLRs and reduced the pro-inflammatory cytokines interleukin (IL)-6 and IL-8, while increasing the antiviral cytokines interferon-γ and IL-1β. Vitamin D3 also increased vitamin D receptor expression, decreased inducible nitric oxide synthase, and reduced the apoptosis regulators B-cell lymphoma-2 (BCL-2) and Bcl2-associated X protein in selected tissues. In conclusion, vitamin D3 supplementation enhanced antiviral defense against FAdV-4 infection, possibly by promoting humoral immunity and modulating innate immune and apoptosis-related responses. These findings provide new insight into the role of vitamin D3 in limiting FAdV-4 infection and support its potential as a nutritional strategy for controlling viral diseases in poultry.