Infectious bronchitis virus (IBV) is a highly contagious coronavirus prevalent in all countries with an extensive poultry industry and continues to cause economic losses. IBV strains of the Ark serotype are highly prevalent in the Southeastern United States despite extensive vaccination. One explanation for this observation is the high genetic variability of IBV. In addition, IBV Ark-type vaccines may induce suboptimal mucosal immune responses, contributing to the prevalence and persistence of the Ark serotype. To test this hypothesis, chickens were ocularly vaccinated with a commercially available live attenuated IBV Ark-Delmarva Poultry Industry vaccine strain and both mucosal and systemic antibody responses were measured. The highest immunoglobulin A (IgA) spot-forming cell (SFC) response was observed in the Harderian glands (HG) and to a lesser extent in the spleen and conjunctiva-associated lymphoid tissues, while a limited IgG SFC response was observed in either the mucosal or systemic immune compartment. Interestingly, the peak IgA SFC response occurred 2 days earlier in spleen than in the head-associated lymphoid tissues despite ocular vaccination. Furthermore, IgA IBV-specific antibody levels significantly increased over controls 3 days earlier in tears and 4 days earlier in plasma than did IgG antibodies. IgA antibody levels were higher than IgG antibody levels throughout the primary response in tears and were similar in magnitude in plasma. In addition, a very early increase in IgA antibodies on day 3 postvaccination was observed in tears; such a response was not observed in plasma. This early increase is consistent with a mucosal T-independent IgA response to IBV. In the secondary response the IBV antibody levels significantly increased over controls starting on day 1 after boosting, and the IgG antibody levels were higher than the IgA antibody levels in both tears and plasma. In summary, ocular vaccination induced higher IgA antibodies in the primary IBV response, while the memory response is dominated by IgG antibodies. Thus, lower mucosal IgA antibody levels are observed upon secondary exposure to IBV, which may contribute to vulnerability of host epithelial cells to infection by IBV and persistence of the Ark serotype.

We compared detection of infectious bronchitis virus (IBV) by quantitative RT-PCR (qRT-PCR) in tears and trachea of IBV-infected chickens and found that quantitative detection of IBV RNA in tears is more sensitive than in tracheal homogenates. Furthermore, we demonstrated that IBV contained in chicken lachrymal fluid is infectious and that tears of IBV-infected chickens can be used to infect naive chickens. We compared the immune responses to IBV in the Harderian gland and cecal tonsils of immunocompetent chickens and chickens infected with chicken anemia virus (CAV) and/or infectious bursal disease virus (IBDV). Flow cytometry analyses of lymphocytes in Harderian glands and cecal tonsils indicated that the relative abundance of IgM+ B cells in the Harderian glands and cecal tonsils following exposure to IBV in combination with immunosuppressive viruses was reduced compared to chickens infected with IBV alone. CAV, but not IBDV, reduced the CD4+/CD8+ T cell ratios compared to chickens infected with IBV alone. Enzyme-linked immuno-spot forming assays on cells in the Harderian glands and cecal tonsils of IBV-infected chickens indicated that maximum IBV-specific IgA-secreting cell responses were reduced in chickens infected with CAV. IBDV co-infected chickens displayed a delayed IgA response to IBV. Thus immunosuppressive viruses reduced B cells and T helper cells in the Harderian glands and cecal tonsils in response to IBV, and slowed the kinetics and/or reduced the magnitude of the mucosal immune response against IBV. We have shown for the first time that CAV affects pathogen-specific B cell responses in a mucosal effector site.

Infectious bronchitis virus (IBV) is an endemic disease of chickens and a major contributor to economic losses for the poultry industry despite vaccination. Recent observations indicated that chicks may have an immature immune system immediately after hatching when vaccinated for IBV. Therefore we hypothesized that early IBV vaccination will generate an immature, poorly protective IBV-specific immune response contributing to immune escape and persistence of IBV. To test this hypothesis the IBV-specific immune response and immune protection were measured in chicks vaccinated at different ages. This demonstrated a delayed production of IgG and IgA plasma antibodies in the 1, 7 and 14-day-old vaccination groups and also lower IgA antibody levels were observed in plasma of the 1-day-old group. Similar observations were made for antibodies in tears. In addition, IgG antibodies from the 1-day-old group had lower avidity indices than day 28 vaccinated birds. The delayed and/or lower antibody response combined with lower IgG avidity indices coincided with increased tracheal inflammation and depletion of tracheal epithelia cells and goblet cells upon IBV field strain challenge. The lack of vaccine-mediated protection was most pronounced in the 1-day-old vaccination group and to a lesser extent the 7-day-old group, while the 14-day-old and older chickens were protected. These data strongly support IBV vaccination after day 7 post hatch.

Humoral immunity is important for controlling viral diseases of poultry, but recent studies have indicated that cytotoxic T cells also play an important role in the immune response to infectious bronchitis virus (IBV). To better understand the cell mediated immune responses to IBV in the mucosal and systemic immune compartments chickens were ocularly vaccinated with IBV. This induced a lymphocyte expansion in head-associated lymphoid tissues (HALT) and to a lesser extent in the spleen, followed by a rapid decline, probably due to homing of lymphocytes out of these organs and contraction of the lymphocyte population. This interpretation was supported by observations that changes in mononuclear cells were mirrored by that in CD3(+)CD44(+) T cell abundance, which presumably represent T effector cells. Increased interferon gamma (IFN-γ) expression was observed in the mucosal immune compartment, i.e., HALT, after primary vaccination, but shifted to the systemic immune compartment after boosting. In contrast, the expression of cytotoxicity-associated genes, i.e., granzyme A (GZMA) and perforin mRNA, remained associated with the HALT after boosting. Thus, an Ark-type IBV ocular vaccine induces a central memory IFN-γ response in the spleen while the cytotoxic effector memory response, as measured by GZMA and perforin mRNA expression, remains associated with CALT after boosting.

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The chicken Harderian gland (HG) plays an important role in adaptive immune responses upon ocular exposure to avian pathogens such as avian influenza (AI). To determine the role of HGs in generating immunity, chickens were immunized ocularly with an adenovirus (Ad5) vector expressing the AI hemagglutinin H5 gene. The Ad5-H5 vector induced H5 transgene expression and induced H5- and Ad5-specific IgA and IgG spot-forming cells (SFCs) in the HGs. The IgA and IgG SFC peaked on day 9 for Ad5 and day 11 for the H5 protein. In addition, Ad5- and H5-specific antibodies were induced in serum. IgA in chicken tears was predominantly dimeric, while in serum monomeric IgA was most abundant. Analysis of HG mRNA confirmed expression of the polymeric immunoglobulin receptor (pIgR). These data demonstrated the importance of HGs to generate mucosal and systemic immunity to AI following ocular Ad5-H5 administration to chickens.

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