Immunopathogenesis of infectious bronchitis virus in chickens: the role of head-associated lymphoid and respiratory tissues

Infectious bronchitis virus (IBV) primarily replicates in the epithelial tissues of the respiratory tract, predominantly trachea. Little information is available on IBV replication and immune responses in head-associated lymphoid tissues (HALT) – Harderian Gland (HG) and choanal cleft, and respiratory (turbinate) tissues. To investigate the role of these tissues in comparison to trachea, 21-days of age commercial broiler chickens were challenged with IBV M41. Lachrymal fluid anti-IBV IgA levels were elevated at 4–5 days post-challenge (dpc). At 5 dpc, the viral load in the turbinate and choanal cleft was higher than those found in the HG, pharyngeal and tracheal tissues. Immunohistochemistry (IHC) confirmed presence of IBV replication in all examined tissues with peak viral antigen score at 3 dpc. There were high levels of TLR3, MDA5, IFN-α, IFN-β and IL-6 gene expression were found in the challenged compared to the unchallenged control group. Findings demonstrated an early innate immune response at 1–5 days after M41 challenge. More specifically, there was a marked up-regulation of TLR3, MDA5, IFN-β and IL-6 mRNA expression in the HG, choanal cleft, turbinate and trachea. With above findings and in an attempt to propose new quantitative parameters for assessment of protection in IBV vaccine efficacy studies, day-old broiler chicks were vaccinated with combined IBV vaccines (Mass and 793B), and at 21 days post- vaccination (dpv) chicks were challenged with IBV M41. Based on ciliary-protection test, vaccinated-unchallenged birds were had protection scores of more than 95%. Vaccinated birds were fully protected against M41 challenge (≥ 93.5 % score). The vaccinated-challenged group showed significant increases in lachrymal IgA levels at 3-5 dpc. A significantly higher viral load was found only in the trachea of vaccinated-challenged birds at 1-4 dpc. For the vaccinated groups, challenged birds had a significantly higher TLR3, MDA5, IFN-α and IL-6 expression in the turbinate, choanal cleft and trachea between 1-2 dpc. Findings from this study for the first time have provided a scientific foundation for the inclusion of quantitative early immune parameters for measurement of protection in IBV vaccination-challenge studies. To cross-compare between IBV vaccine application methods, three strategies were applied [gel, spray, and oculonasal (ON)] in day-old broiler chicks. Birds were then challenged at 21 dpv with M41. All routes of vaccination resulted in an increase of anti-IBV IgA in the lachrymal fluid at 5 dpc compared to the control group. For gel and ON vaccination methods, for 3-21 dpv, the viral load in the turbinate, choanal cleft and trachea was higher than in the spray-vaccinated birds. Only for the oculonasal-challenge (ON-ch) group, TLR3 mRNA expression was significantly up-regulated in all tissues at 1 dpc. At 1 dpc, the mRNA expression of MDA5 and IL-6 in the all vaccinated-challenged groups showed significant up-regulation in all tissues (except for HG) compared to control group. Viral replication and the host gene signatures are likely to be related to the difference in the tissue type and method of vaccine application. It appears that the ON route of vaccination in broiler chicks provided superior protection after challenge with M41 due to higher IgA levels. This study showed that chicks vaccinated (Mass+793B) via gel were equally protected from virulent M41, and this method could be an effective commercial vaccination approach in poultry. To date, the research onto early innate, mucosal and cellular immune responses in layer hens is limited. In this study, HALT, turbinate and tracheal tissues of adult laying hens were evaluated following heterologous vaccine application (H120 or 4/91) via drinking water (DW) or oculonasal (ON). It was found that lachrymal IgA was lower at 7 and 14 dpv compared to IgY in all groups. Viral RNA load in the HG and turbinate was significantly higher in the ON-H120 group compared to DW-H120 and both 4/91-vaccinated groups at 1–3 dpv. Host TLR3, MDA5, and IL-6 mRNA were significantly up-regulated in the HG at 3–5 dpv in the ON-H120 and ON-4/91-vaccinated groups. Expression of CD8α and CD8β indicated high up-regulation at 1–3 dpv in the turbinate and at 3–5 in the trachea. IgA mRNA expression was significantly higher in the ON-H120 and ON-4/91-vaccinated compared to DW-vaccinated groups. Generally, mucosal and cellular immune response was greater in the turbinate than the trachea for both methods of vaccination. Data demonstrated the variable role of innate, mucosal and cellular immunities in relation to vaccine type and route of vaccine administration