Determination of immune responses in mucosal lymphoid tissues following infectious laryngotracheitis virus (ILTV) eye drop and vent brush vaccination of chickens

Eye drop (ED) and vent brush (VB) application of live infectious laryngotracheitis virus (ILTV) vaccine provides protective immunity against disease, but little is known regarding the immune responses following application. This study compared the gene expression of immune markers associated with VB and ED ILTV vaccination in the conjunctiva, trachea and cloaca at 3, 5, 7 and 10 days post-vaccination (dpv) and 3, 5 and 7 days post-revaccination (dprv). The relative gene expression of markers associated with inflammatory responses and their regulation (CCL4, CCR5, IL2, IL4, IL6, IL17C, IFNG), pathogen recognition receptors (TLR2-2, TLR4, TLR7) and cell surface markers (CD4, CD8A, CD14, CD80) was evaluated by RT–PCR after ED or VB vaccination and revaccination using the alternate route (ED/VB (primary/booster), VB/ED). There was increased expression of inflammatory markers at vaccination sites following primary vaccination. In the VB group, there was increased expression of IL6 and IFNG in cloaca (3 dpv) and IL2 in the conjunctiva (7 dpv) compared to the ED group (<i>P</i> &lt; 0.05), while there was increased CCL4 expression in conjunctiva (5 dpv) in the ED group compared to the VB group. ED vaccination was associated with increased expression of IL2 and IL17C in trachea while VB vaccination was associated with increased expression of IL6 and IL17C in trachea. Similarly, ED/VB- and VB/ED-associated increases in IL2, IL6 and IL17C expression were observed at the revaccination sites. In conclusion, immune responses after the first and second ILTV vaccination were site-specific and associated with inflammatory responses in the mucosa of the vaccinated tissues and trachea. <b>RESEARCH HIGHLIGHTS</b>ILTV vaccination elicited inflammatory responses at the vaccination sites (conjunctiva and cloaca).Conjunctival or cloacal vaccination elicited similar inflammatory responses in the trachea after 3 and 5 days, respectively.Revaccination by a different route stimulated similar immune responses in conjunctiva, cloaca and trachea. ILTV vaccination elicited inflammatory responses at the vaccination sites (conjunctiva and cloaca). Conjunctival or cloacal vaccination elicited similar inflammatory responses in the trachea after 3 and 5 days, respectively. Revaccination by a different route stimulated similar immune responses in conjunctiva, cloaca and trachea.

滴眼（Eye drop, ED）与泄殖腔刷拭（vent brush, VB）接种活传染性喉气管炎病毒（infectious laryngotracheitis virus, ILTV）疫苗可诱导针对该病的保护性免疫，但目前对该类接种方式后的免疫应答机制尚不明确。本研究分别在初次接种后3、5、7、10天（post-vaccination, dpv）以及加强免疫后3、5、7天（post-revaccination, dprv），检测了结膜、气管与泄殖腔中与ED、VB途径ILTV疫苗接种相关的免疫标志物基因表达水平并进行对比分析。本研究采用逆转录聚合酶链式反应（RT–PCR），评估了ED或VB途径初次接种及交替途径加强免疫（即ED/VB（初次/加强）、VB/ED）后，炎症应答及其调控相关标志物（CCL4、CCR5、IL2、IL4、IL6、IL17C、IFNG）、病原识别受体（TLR2-2、TLR4、TLR7）以及细胞表面标志物（CD4、CD8A、CD14、CD80）的相对基因表达量。初次免疫后，接种部位的炎症标志物表达量显著上调。与ED组相比，VB组在接种后3天的泄殖腔中IL6与IFNG表达量上调，接种后7天的结膜中IL2表达量上调（*P*<0.05）；而ED组在接种后5天的结膜中CCL4表达量显著高于VB组。ED途径免疫可诱导气管中IL2与IL17C表达上调，而VB途径免疫则可诱导气管中IL6与IL17C表达上调。同样，在加强免疫部位，ED/VB与VB/ED免疫组均观察到IL2、IL6及IL17C表达量升高。综上，ILTV初次与加强免疫后的免疫应答具有组织部位特异性，且与接种组织黏膜及气管的炎症应答密切相关。
**研究亮点（RESEARCH HIGHLIGHTS）**
ILTV疫苗接种可在接种部位（结膜与泄殖腔）诱导炎症应答。
结膜或泄殖腔途径免疫分别在免疫后3天、5天于气管中引发相似的炎症应答。
采用不同途径进行加强免疫，可在结膜、泄殖腔及气管中诱导相似的免疫应答。
ILTV疫苗接种可在接种部位（结膜与泄殖腔）诱导炎症应答。
结膜或泄殖腔途径免疫分别在免疫后3天、5天于气管中引发相似的炎症应答。
采用不同途径进行加强免疫，可在结膜、泄殖腔及气管中诱导相似的免疫应答。