# Immune responses in chicken mucosal lymphoid tissues following oral and eye drop vaccination and revaccination with live modified vaccine of infectious laryngotracheitis virus (ILTV)

## TL;DR

This study compares immune responses in chickens after oral and eye drop vaccination with ILTV, finding that revaccination via an alternate route boosts immune marker expression.

## Contribution

The study reveals that revaccination via an alternate route enhances immune responses more effectively than same-route revaccination in chickens.

## Key findings

- Revaccination via an alternate route upregulates more immune markers in conjunctiva and trachea compared to same-route revaccination.
- Oral and eye drop primary vaccinations increase inflammation-related gene expression in mucosal tissues.
- Eye drop vaccination increases specific immune markers like IL6 and IL2 in the conjunctiva.

## Abstract

Mass vaccination with attenuated infectious laryngotracheitis virus (ILTV) via drinking water is commonly used in commercial chicken farms, although the effectiveness of oral (OR) vaccination is lower than eye drop (ED). The gene expression of a select set of immune markers associated with inflammatory responses (TLR4, TLR7, TLR2-2, IL6, CCL4, CCR5, IFNγ, IL2, IL17C), immune synapse (CD80), T helper cell polarisation (IL4) and effector cell receptors (CD4, CD8α and CD14), following OR and ED primary vaccination and revaccination using the same or alternate route [ED/ED (primary/revaccination), OR/OR, ED/OR and OR/ED] with ILTV SA2 strain in chickens were evaluated. Both ED and OR primary vaccinations were associated with increased gene expression of inflammation-associated markers in the conjunctiva and trachea, with a statistical interaction between application route and profile of immune responses. Revaccination via the alternative route elicited upregulation of more immune markers in the conjunctiva and trachea compared to other methods, while most immune markers were downregulated in the ED/ED group, and close to baseline in the OR/OR group. In conclusion, the immune responses in the conjunctiva and trachea following the ED and OR primary vaccination with ILTV vaccine were tissue-specific, showing increased gene expression of markers related to the inflammatory response. Revaccination using the alternative route elicited upregulation of markers associated with inflammatory responses CCR5 and TLR7 in the conjunctiva and/or trachea. Challenge studies are necessary to confirm the role of these immune responses in protecting vaccinated birds from ILTV.

The online version contains supplementary material available at 10.1007/s00705-026-06560-1.

ED and OR vaccination was associated with increased gene expression of inflammation-associated markers in the conjunctiva and trachea.

ED vaccination was associated with increased gene expression of IL6, IL2, CCR5 in the conjunctiva and CD8α, IL17C in the trachea.

OR vaccination was associated with increased gene expression of CCR5 and TLR7 in the conjunctiva and IL17C in the trachea.

Revaccination with the alternative route was associated with upregulation of more immune markers in the conjunctiva and trachea compared to using the same route.

ED followed by ED revaccination application was associated with downregulation of most immune markers.

The online version contains supplementary material available at 10.1007/s00705-026-06560-1.