Genetic Bases for Resistance and Immunity to Avian Diseases

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
315	3299	1090	40%
311	3299	1090	60%

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: In order to reach our target audience of avian immunologist. veterinarians an others interested in the avianimmune system as it pertains to mucosal immunity with a focus on the immune response toinfectious bronchitis virus (IBV) vaccines, three oral presentationsand three poster presentations were delivered at local and international veterinary and immunology meetings. We published one peer review paper, had another accepted and submitted a third. In addition we submitted a book chapter to be published next year. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Over the past year an undergraduate student, a graduate student and a veterinarian student were trained in my lab to do research on viral mucosal immunity in chickens. How have the results been disseminated to communities of interest? As stated our results were presented at local and international meetings as well as publishedas peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals? -We will continue analyzing the avian immune response to the IBV spike protein in the mucosal and systemic immune compartments in order to better understand the contributions of point mutations in the IBV spike protein in the immune escape of an IBV field strain of IBV vaccine induced immune responses. - We will measure IBV -specific T cell and B cells responses in the mucosal and systemic immune compartment after vaccination with Ad5 vector expressing component C2 of the live attenuated IBV vaccine. This C2 component constitute one of the minor component of the live attenuated IBV vaccine an contains specific mutations in the S1 sequence. This C2 population becomes prevalent within days after vaccination and the S1 sequence is stably expressed in an Ad5 vector. In addition, we like to analyze the influence of age on the protective immune response to IBV. -We will develop a novel Ad5-based, redirected avian vaccine vector with the capacity to generate heterotypic immunity to IBV and will measure it ability to induce IBV-specific immunity.

Impacts
What was accomplished under these goals? Cell-mediated immune responses in the head-associated lymphoid tissues to a live-attenuated IBV vaccine. 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-g) 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-g response in the spleen while the cytotoxic effector memory response, as measured by GZMA and perforin mRNA expression, remains associated with HALT after boosting. However the memory cytotoxic response is mainly associated with conjunctiva-associated lymphoid tissues (CALT) rather than the Harderian glands. Coronaviruses continue to generate problems not only for the poultry industry. Thus, it has become increasingly important to better understand vaccine-induced coronavirus-specific immunity and how infectious bronchitis (IBV) field strain coronaviruses are able to escape IBV vaccine-induced immune responses. A decrease in the IBV vaccine-induced humoral response to the IBV field isolate AL/4614/98 is observed when compared to the response to the immunizing Arkansas vaccine strain as measured by ELISPOT and HI assay. A portion of this decrease in the humoral response is comprised of decreased IgA responses to epitopes in the host attachment domain in the field isolate in both lachrymal and plasma antibodies as measured using an overlapping peptide array covering the S1 portion of the spike protein. This decrease in antibody recognition is caused by single amino acid differences in the antibody recognized B cell epitopes. Our results indicate that IgA may play an important role in controlling IBV and that successful field isolates such as AL/4614/98, escape vaccine-induced host attachment domain-binding IgA antibodies through vaccine driven immune selection for point mutations in these B cell epitopes. Consistent with this notion is the observation that most IgA antibodies recognizing linear S1 B cell epitopes outside the host attachment domain seem less affected by changes in these linear B cell epitopes. IBV-Specific Immune Response in the Mucosal and System Immune Compartment: In the Southeastern USA the Ark-serotype is prevalent despite extensive vaccination efforts. Besides the high variability of infectious bronchitis virus (IBV) one other contributing factor for the prevalence of this IBV serotype in the Southeastern USA could be the inability of existing vaccines to induce strong mucosal immune responses, allowing IBV reinfection of epithelial cells after vaccination. To test this, chickens were ocularly vaccinated with a live attenuated IBV Ark-DPI (Arkansas-Delmarva Poultry Industry) vaccine strain. The IBV-specific IgA and IgG ELISPOT demonstrated that the highest IgA spot-forming cell (SFC) response was induced in the Harderian glands (HG) and to a lesser extent in spleen and conjunctiva-associated lymphoid tissues (CALT), while a limited IgG SFC response was observed in either mucosal or systemic lymphoid tissues. Interestingly, the peak SFC response occurred 2 days earlier in spleen than in the head-associated lymphoid tissues (HALT) despite ocular vaccination. Furthermore, IgA IBV-specific antibody levels in tears significantly increased over control levels 3 days earlier than IgG antibodies and 4 days earlier than in plasma. IgA antibody levels were higher than IgG antibodies in the primary response in tears and similar in magnitude with IgG responses in plasma. In addition, a very early increase in IgA antibodies on day 3 post vaccination was observed in tears, which was not observed in plasma. This early increase is consistent with a mucosal T-independent IgA response to IBV. In the secondary response the antibody levels were elevated above controls from day 1and the IgG antibody levels were higher than the IgA antibody levels in both tears and plasma. Thus, after ocular vaccination IgA antibodies prevail in the primary IBV response, while the memory response is dominated by IgG antibodies. This indicates that after the primary response decreased mucosal IgA antibody mediated protection may occur contributing to possible increased vulnerability of the host for re-exposure to IBV.

Publications

Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Gurjar, R.S., S. L. Gulley, and F.W. van Ginkel. 2013. Cell-mediated immune responses in the head-associated lymphoid tissues induced to a live attenuated avian coronavirus vaccine. Dev. Comp. Immunol. 41:715-722, 2013.
Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Toro, H., J.F. Zhang, R.A. Gallardo, V.L. van Santen, F.W. van Ginkel, K.S. Joiner, C. Breedlove. 2013. S1 of Distinct IBV Population Expressed from Recombinant Adenovirus Confers Protection against Challenge. Avian Dis. (in press).
Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Orr, N., S.L. Gulley, H. Toro, R. Gallardo, and F.W. van Ginkel. 2013. Mucosal and Systemic Immune Responses to Infectious Bronchitis Virus after Ocular Vaccination. Avian Dis. (submitted).
Type: Book Chapters Status: Accepted Year Published: 2014 Citation: van Ginkel, F.W. 2013. Ocular vaccination against the avian coronavirus infectious bronchitis virus. Auburn Speaks (in press).
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: R.S. Gurjar, S.L. Gulley, and F.W. van Ginkel Induction of interferon gamma and cytotoxic responses by an Ark-type infectious bronchitis virus vaccine in the mucosal and systemic immune compartments. GSC Research Forum & Symposium, February 26-28, 2013, Auburn AL.
Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: F.W. van Ginkel, R.S. Gurjar, N. Orr, and S.L. Gulley. Ocular infectious bronchitis virus vaccination induces different immune responses in the mucosal and systemic immune compartment. International Congress of Immunology, Milan Italy, August 22-27, 2013.
Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: J. Padgett. Infectious Bronchitis Virus. Merial Veterinary Summer Scholars Program, Auburn, AL, July 26, 2013.
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: J. Padgett, S.L. Gulley, K. Joiner, R.C. Cattley, F.W. van Ginkel. Age-Related Influence on Ocular Live Attenuated IBV Vaccine Induced Immune Response and Immune Protection. Phi Zeta Research Emphasis Day, Auburn AL, October 30, 2013.
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: S.L. Gulley, R.S. Gurjar, N. Orr, F.W. van Ginkel. Mucosal and Systemic Immune Responses Induced after Ocular Avian Coronavirus Vaccination are Evaded by a Field Strain. Phi Zeta Research Emphasis Day, Auburn AL, October 30, 2013.

Progress 10/01/08 to 09/30/13

Outputs
Target Audience: In order to reach our target audience of avian immunologist. veterinarians an others interested in the avian immune system as it pertains to mucosal immunity with a focus on the immune response to infectious bronchitis virus (IBV) vaccines, three oral presentations and three poster presentations were delivered at local and international veterinary and immunology meetings. We published one peer review paper, had another accepted and submitted a third. In addition we submitted a book chapter to be published next year. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Over the past year an undergraduate student, a graduate student and a veterinarian student were trained in my lab to do research on viral mucosal immunity in chickens. How have the results been disseminated to communities of interest? As stated our results were presented at local and international meetings as well as published as peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals? -We will continue analyzing the avian immune response to the IBV spike protein in the mucosal and systemic immune compartments in order to better understand the contributions of point mutations in the IBV spike protein in the immune escape of an IBV field strain of IBV vaccine induced immune responses. - We will measure IBV -specific T cell and B cells responses in the mucosal and systemic immune compartment after vaccination with Ad5 vector expressing component C2 of the live attenuated IBV vaccine. This C2 component constitute one of the minor component of the live attenuated IBV vaccine an contains specific mutations in the S1 sequence. This C2 population becomes prevalent within days after vaccination and the S1 sequence is stably expressed in an Ad5 vector. In addition, we like to analyze the influence of age on the protective immune response to IBV. -We will develop a novel Ad5-based, redirected avian vaccine vector with the capacity to generate heterotypic immunity to IBV and will measure it ability to induce IBV-specific immunity.

Impacts
What was accomplished under these goals? Cell-mediated immune responses in the head-associated lymphoid tissues to a live-attenuated IBV vaccine. 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-g) 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 cytotoxicityassociated 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-g response in the spleen while the cytotoxic effector memory response, as measured by GZMA and perforin mRNA expression, remains associated with HALT after boosting. However the memory cytotoxic response is mainly associated with conjunctiva-associated lymphoid tissues (CALT) rather than the Harderian glands. Coronaviruses continue to generate problems not only for the poultry industry. Thus, it has become increasingly important to better understand vaccine-induced coronavirus-specific immunity and how infectious bronchitis (IBV) field strain coronaviruses are able to escape IBV vaccine-induced immune responses. A decrease in the IBV vaccine-induced humoral response to the IBV field isolate AL/4614/98 is observed when compared to the response to the immunizing Arkansas vaccine strain as measured by ELISPOT and HI assay. A portion of this decrease in the humoral response is comprised of decreased IgA responses to epitopes in the host attachment domain in the field isolate in both lachrymal and plasma antibodies as measured using an overlapping peptide array covering the S1 portion of the spike protein. This decrease in antibodyrecognition is caused by single amino acid differences in the antibody recognized B cell epitopes. Our results indicate that IgA may play an important role in controlling IBV and that successful field isolates such as AL/4614/98, escape vaccineinduced host attachment domain-binding IgA antibodies through vaccine driven immune selection for point mutations in these B cell epitopes. Consistent with this notion is the observation that most IgA antibodies recognizing linear S1 B cell epitopes outside the host attachment domain seem less affected by changes in these linear B cell epitopes. IBV-Specific Immune Response in the Mucosal and System Immune Compartment: In the Southeastern USA the Ark-serotype is prevalent despite extensive vaccination efforts. Besides the high variability of infectious bronchitis virus (IBV) one other contributing factor for the prevalence of this IBV serotype in the Southeastern USAcould be the inability of existing vaccines to induce strong mucosal immune responses, allowing IBV reinfection of epithelial cells after vaccination. To test this, chickens were ocularly vaccinated with a live attenuated IBV Ark-DPI (Arkansas-Delmarva Poultry Industry) vaccine strain. The IBV-specific IgA and IgG ELISPOT demonstrated that the highest IgA spot-forming cell (SFC) response was induced in the Harderian glands (HG) and to a lesser extent in spleen and conjunctiva-associated lymphoid tissues (CALT), while a limited IgG SFC response was observed in either mucosal or systemic lymphoid tissues. Interestingly, the peak SFC response occurred 2 days earlier in spleen than in the head-associated lymphoid tissues (HALT) despite ocular vaccination. Furthermore, IgA IBV-specific antibody levels in tears significantly increased over control levels 3 days earlier than IgG antibodies and 4 days earlier than in plasma. IgA antibody levels were higher than IgG antibodies in the primary response in tears and similar in magnitude with IgG responses in plasma. In addition, a very early increase in IgA antibodies on day 3 post vaccination was observed in tears, which was not observed in plasma. This early increase is consistent with a mucosal T-independent IgA response to IBV. In the secondary response the antibody levels were elevated above controls from day 1and the IgG antibody levels were higher than the IgA antibody levels in both tears and plasma. Thus, after ocular vaccination IgA antibodies prevail in the primary IBV response, while the memory response is dominated by IgG antibodies. This indicates that after the primary response decreased mucosal IgA antibody mediated protection may occur contributing to possible increased vulnerability of the host for re-exposure to IBV.

Publications

Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Gurjar, R.S., S. L. Gulley, and F.W. van Ginkel. 2013. Cell-mediated immune responses in the head-associated lymphoid tissues induced to a live attenuated avian coronavirus vaccine. Dev. Comp. Immunol. 41:715-722, 2013.
Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Toro, H., J.F. Zhang, R.A. Gallardo, V.L. van Santen, F.W. van Ginkel, K.S. Joiner, C. Breedlove. 2013. S1 of Distinct IBV Population Expressed from Recombinant Adenovirus Confers Protection against Challenge. Avian Dis. (in press).
Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Orr, N., S.L. Gulley, H. Toro, R. Gallardo, and F.W. van Ginkel. 2013. Mucosal and Systemic Immune Responses to Infectious Bronchitis Virus after Ocular Vaccination. Avian Dis. (submitted).
Type: Book Chapters Status: Accepted Year Published: 2014 Citation: van Ginkel, F.W. 2013. Ocular vaccination against the avian coronavirus infectious bronchitis virus. Auburn Speaks (in press).
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: R.S. Gurjar, S.L. Gulley, and F.W. van Ginkel Induction of interferon gamma and cytotoxic responses by an Ark-type infectious bronchitis virus vaccine in the mucosal and systemic immune compartments. GSC Research Forum & Symposium, February 26-28, 2013, Auburn AL.
Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: F.W. van Ginkel, R.S. Gurjar, N. Orr, and S.L. Gulley. Ocular infectious bronchitis virus vaccination induces different immune responses in the mucosal and systemic immune compartment. International Congress of Immunology, Milan Italy, August 22-27, 2013.
Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: J. Padgett. Infectious Bronchitis Virus. Merial Veterinary Summer Scholars Program, Auburn, AL, July 26, 2013.
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: J. Padgett, S.L. Gulley, K. Joiner, R.C. Cattley, F.W. van Ginkel. Age-Related Influence on Ocular Live Attenuated IBV Vaccine Induced Immune Response and Immune Protection. Phi Zeta Research Emphasis Day, Auburn AL, October 30, 2013.
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: S.L. Gulley, R.S. Gurjar, N. Orr, F.W. van Ginkel. Mucosal and Systemic Immune Responses Induced after Ocular Avian Coronavirus Vaccination are Evaded by a Field Strain. Phi Zeta Research Emphasis Day, Auburn AL, October 30, 2013.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: I gave four research seminars pertaining to this project one as an invited speaker at Poultry session of the joint Alabama and Georgia Veterinary Medical Association in Sandestine Florida, one at Avian Immunology Research Group meeting in Edinburgh, Scotland, one at the annual meeting of the NE-1034 multi-state meeting in East Lansing Michigan, and one at the School of Veterinary Medicine in Utrecht, the Netherlands. In addition, my graduate student presented a platform presentation at the Phi Zeta conference here in Auburn, Alabama. We also presented in total 6 posters, 4 during the Auburn University Research Week, April 2-5, Auburn, AL, and 2 during the Phi Zeta Research Emphasis Day, November 7, Auburn, AL. We also were involved in 2 publications in Avian Diseases. In part this activity resulted from research projects from both graduate and undergraduate students that were being trained in my lab. PARTICIPANTS: The individuals that work on this project are the PI/PD Dr. F.W. van Ginkel who directs the research, performs the administrative part of the project such as writing progress report, AICUC, BUAs etc. In addition he will also write papers and perform any other form of communization of the data as is appropriate. Stephen Gulley is individual actively involved in the day-to-day research and animal care issues and manages the lab. We are currently training Rucha Gurjar,a graduate student in my lab, in performing anaylsis of the IBV-specific immune response in chickens. In addition, another graduate student this year, Yewanda Fasina, did a research problem on this project looking at lymphocyte proliferation in chickens. Furthermore, 3 undergraduate student did research electives in my lab this past year and they all presented their data at a local meeting. Further training in avian immunology in IBV was provided to a visiting associate professor Feyzullah Beyaz, from the Faculty of Veterinary Medicine, Erciyes University, Kayseri, Turkey, which spend 3 month of training in my lab. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We defined an expansion and contraction phase of the chicken immune responses to ocular IBV vaccination in Harderian glands, CALT and spleen. In the mucosa-associated lymphoid tissues these phases were closely linked to the induction and effector phase of the IBV-specific immune response, based on kinetics of IgA spot forming cells (SFC), IgA levels in tears and plasma, IFN-gamma mRNA expression, and in spleen also with levels of IBV-specific B cells. IFN-gamma mRNA levels induced by ocular IBV are at least biphasic in nature. These two phases seem consistent with induction of a Th1-type immune response and a T cell effector response and seem to coincide with the presence of T memory/effector cells. No defined memory response was observed in the IgA response upon IBV boosting. Initial studies demonstrated the presence of an IFN-gamma memory response in the spleen but not in CALT or Harderian glands. Furthermore, initial analysis of the IBV-specific IgA response in the intestinal tract after ocular and oral vaccination indicated that limited humoral mucosal immunity is generated in the intestinal tract based on IBV-specific IgA levels after exposure to an IBV vaccine strain. These studies are still ongoing. Our studies provide a better understanding of the role of the mucosal versus systemic immune systems and their effect and contributions in controlling Infectious Bronchitis Virus (IBV) in chickens. These studies will provide a foundation to design better vaccines and optimize delivery to induce adaptive immunity to optimize the control of this pathogen. Since the spike protein plays a role in cell attachment and fusion and constitute a highly variable region of IBV, a detailed analysis of IBV-specific immune responses will help to understand the role the variability of the S1 protein plays in immune escape. These studies are still ongoing. In addition, head-associated lymphoid tissues (HALT) have been poorly characterized for their ability to generate pathogen-specific immune responses or for their role in generating mucosal immune responses to avian pathogens. To better understand their role in IBV-specific immunity we will continue to analyze their contributions to both systemic and mucosal immune responses.

Publications

N. Orr, S.L. Gulley, and F.W. van Ginkel. 2012. Mucosal and systemic immune responses to infectious bronchitis virus (IBV) after ocular vaccination. Auburn University Research Week, April 2-5, Auburn, AL.
L. Eads, S.L. Gulley, F.W. van Ginkel. 2012. Ocular and Oral Vaccination to Generate Infectious Bronchitis Virus Specific Immunity. Auburn University Research Week, April 2-5, Auburn, AL.
F.W. van Ginkel, R. Gurjar, N. Orr, H. Toro, and S.L Gulley. 2012. Ocular Infectious Bronchitis Virus Vaccination Induces T and B Cell Responses in Head-Associated Lymphoid Tissues that Differ from that in the Spleen. Avian Immunology Research Group, August 28-31, EdinBurgh, Scotland.
R. Gurjar, S.L. Gulley, and F.W, van Ginkel. 2012. T Cell Response to Ark-type IBV in White Leghorns. Phi Zeta Research Emphasis Day, November 7, Auburn AL.
Peer-reviewed publications: Ndegwa, E.N., K.S. Joiner, H. Toro, F.W. van Ginkel, and V.L. van Santen. 2013. Significance of differences in proportions of specific minor viral subpopulations within Ark-type infectious bronchitis vaccines. Avian Dis. (In press).
Toro, H., D. Pennington, R.A. Gallardo, V.L. van Santen, F.W. van Ginkel, J.F. Zhang, K.S. Joiner. 2012. Infectious bronchitis virus subpopulations in vaccinated chickens after challenge. Avian Dis. 56:501-508.
Abstracts: F.W. van Ginkel. 2012. Novel research on the immune responses of respiratory and digestive tracts of chickens. Poultry session of the GVMA and AVMA. June 1-3, Sandestine, Fl.
R.S. Gurjar, S.L. Gulley, R. Gallardo, H. Toro, and F.W. van Ginkel. 2012. Infectious bronchitis virus-specific effector and memory T cell responses in white leghorns. Auburn University Research Week, April 2-5, Auburn, AL.
M. Lohani, J. Suddeth, D. Shannon, F.W. van Ginkel, and B. Kemppainen. 2012. Effects of American Skullcap on Aflatoxin-Contaminated Feed in Broiler Chickens. Auburn University Research Week, April 2-5, Auburn, AL.
Y.O. Fasina, S.L. Gulley, and F.W. van Ginkel. 2012. Flow Cytometric Analysis of Proliferative Responses of Chicken Peripheral Blood Mononuclear Cells Following Concanavalin A Stimulation. Phi Zeta Research Emphasis Day, November 7, Auburn AL.
R.M. Johnson, S.L. Gulley, H. Toro, and F.W. van Ginkel. 2012. IBV Antibody and S1 Spike Protein Dominant B cell Epitopes Induced After Ocular Immunization with Ad4-S1. Phi Zeta Research Emphasis Day, November 7, Auburn AL.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: For outputs involving Objective 3, i.e., to develop and evaluate methodologies and reagents to assess immune function and disease resistance to enhance production efficiency through genetic selection in poultry, a paper has been accepted in Developmental and Comparative Immunology pertaining to immune responses of chicken conjunctiva-associated lymphoid tissues to infectious bronchitis virus (IBV) as well as two papers pertaining to replication-deficient adenovirus vector induced immune responses and immune protection. Our IBV research was presented as both a poster and a platform presentation and they were published as abstracts in the Proceedings of the 1st International Avian Respiratory Disease Conference, Athens, Georgia. We also presented a seminar for the Scott Ritchey Research Center, Auburn, AL pertaining to the role of head-associated lymphoid tissues in generating antigen-specific immunity as well our research progress at the NE-1034 annual conference in Ithaca, NY. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Mucosal Immunity in the Head-Associated Lymphoid Tissues Adenoviral Vector Induced Immune Response: The Harderian glands and conjunctiva-associated lymphoid tissues (CALT) are both head-associated lymphoid (HALT) structures. Harderian glands and CALT are important mucosal inductive sites producing antigen-specific IgA antibodies and contain increased numbers of cytotoxic T cells and T helper type cells after ocular immunization and contribute to elevated levels of IFN- in tears after ocular immunization. The presence of the pIgR allows active transport of IgA across the epithelium. The prevalence of IgA producing cells in HALT combined with the presence of pIgR demonstrates that they contribute to mucosal immune protection of the eye. Conjunctiva-associated lymphoid tissue's (CALT) role in generating avian mucosal adaptive immunity was further analyzed. We previously reported expression of the polymeric immunoglobulin receptor (pIgR), induction of antigen-specific, IgA-secreting plasma cells, and influence of in ovo Ad5 priming on the CALT immune response after ocular Ad5 boosting. Now, we have demonstrated that CALT contains B cells, gamma-delta T cells, T helper, and cytotoxic T cells, and expresses a T cell alpha-beta and gamma-delta T cell receptor composition, which more resembles Harderian glands than spleen. CALT-derived lymphocytes contain cytokine-producing lymphocytes after ocular Ad5 vaccination. Thus, both mucosal and systemic immunization influenced Ad5-induced IFN-gamma and IL-2 responses in CALT and Harderian glands, which peak on day 8 in CALT and day 7 in Harderian glands after ocular Ad5 boost. IBV-Specific Immune Response in HALT and Spleen: We defined the expansion and contraction phase in the chicken immune responses after ocular IBV vaccination in Harderian glands, CALT and spleen. In the mucosa-associated lymphoid tissues these phases were closely linked to the induction and effector phase of the IBV-specific immune response based on induction of IgA spot forming cells (SFC), IgA levels in tears and plasma, IFN-gamma mRNA expression, and levels of IBV-specific B cells. IFN-gamma mRNA levels induced by ocular IBV are at least biphasic in nature, which is consistent with induction of a Th1 response and a T cell effector phase and seem to coincide with the presence of T memory/effector cells. No defined memory response was observed in the IgA response upon IBV boosting. However, initial studies demonstrated a presumed IFN-gamma memory response in the spleen but not in CALT or Harderian glands after in vitro IBV stimulation. Furthermore, initial analysis of induction of IBV-specific IgA in the intestinal tract after ocular and oral vaccination demonstrated that very limited mucosal immunity is generated in the intestinal tract based on IBV-specific IgA levels after exposure to an IBV vaccine strain. These studies are still ongoing. A better understanding the contributions of the mucosal versus systemic immune systems in controlling IBV in chickens will be important in order to design better vaccines and optimize delivery to induce protective immunity to better control this ever changing pathogen.

Publications

Breedlove, C., J.K. Minc, D.C. Tang, V.L. van Santen, F.W. van Ginkel, and H. Toro. 2012. Avian influenza adenovirus-vectored in ovo vaccination: Target embryo tissues and combination Marek's Disease Vaccine. Avian Dis. (in press)
Abstract 2011
F.W. van Ginkel, S.L. Gulley, A. Lammers, F.J. Hoerr, R. Gurjar, H. Toro. 2011. Conjunctiva-Associated Lymphoid Tissue in Avian Mucosal Immunity. In Proceedings of the 1st International Avian Respiratory Disease Conference, May 15-18, Athens, GA, USA.
H. Toro, R. Gallardo, V. van Santen, F. W. van Ginkel, C. Breedlove, and S. Gulley. 2011. Immune selection during Infectious Bronchitis virus. In Proceedings of the 1st International Avian Respiratory Disease Conference, May 15-18, Athens, GA, USA.
L. Eads, S.L. Gulley, and F.W. van Ginkel. 2011. Mucosal vaccine delivery to generate specific immunity to infectious bronchitis virus. In Proceeding of the Epsilon chapter of Phi Zeta Research Emphasis Day, November 9, Auburn, AL.
N. Orr, S.L. Gulley, and F.W. van Ginkel. 2011. Mucosal and systemic immune responses to infectious bronchitis virus (IBV) after ocular vaccination. In Proceeding of the Epsilon chapter of Phi Zeta Research Emphasis Day, November 9, Auburn, AL.
R.S. Gurjar, S.L. Gulley, R. Gallordo, H. Toro, and F.W. van Ginkel. 2011.Infectious bronchitis virus-specific effector and memory T cell responses in white leghorns. In Proceeding of the Epsilon chapter of Phi Zeta Research Emphasis Day, November 9, Auburn, AL.
Peer-reviewed Publications 2011
van Ginkel, F.W., S.L. Gulley, A. Lammers , F.J. Hoerr, R. Gurjar,H. Toro. 2012. Conjunctiva-associated lymphoid tissue in avian ocular immunity. Dev. Comp. Immunol. doi:10.1016/j.dci.2011.04.012

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: For outputs involving Objective 2, i.e., to identify and characterize environmental, dietary and physiological factors that modulate immune system development, optimal immune function and disease resistance in poultry genetic stocks, we performed a study on the interaction of diet, phytase enzyme, and Eimeria infection in broilers. This study has been submitted to Poultry Science and has been accepted for publication. For outputs involving Objective 3, i.e., to develop and evaluate methodologies and reagents to assess immune function and disease resistance to enhance production efficiency through genetic selection in poultry, we have published a paper in Avian Diseases pertaining to mucosal immunization of chickens to avian influenza using a replication-defective adenovirus vector. This research was also presented as a platform presentation at the American Association of Avian Pathologists (AAAP) meeting in Atlanta, Georgia. We also presented data on the role of head-associated lymphoid tissues in generating antigen-specific mucosal immunity to viral vectors at the Avian Immunology Research Group in Budapest, Hungary and at the Phi Zeta meeting in Auburn, Alabama. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Conjunctiva-associated lymphoid tissue (CALT) together with the Harderian glands are ocular-associated lymphoid structures. We demonstrated previously that CALT contains considerable number of IgA+ B cells and display characteristics of a mucosal inductive site. CALT-derived lymphocytes produced antigen-specific IgA secreting plasma cells. Increased cytokine production is detected at 8 days after immunization with an Ad5 vector. This indicates that both B and T cells antigen-specific immune responses take place in CALT after ocular exposure. The expression of the polymeric immunoglobulin receptor (pIgR) in the epithelium of CALT reiterates the importance of mucosal immune protection by para-ocular lymphoid tissues. These findings are current being composed for publication in Dev. Comp. Immunol. Protective immunity to avian influenza (AI) virus can be elicited in chickens by in ovo or intramuscular (IM) vaccination with replication competent adenovirus (RCA)-free human recombinant adenovirus type 5 (Ad5) encoding AI virus H5 (AdTW68.H5) or H7 (AdCN94.H7) hemagglutinins. We evaluated bivalent in ovo vaccination with AdTW68.H5 and AdCN94.H7. Vaccinated chickens developed robust hemagglutination inhibition (HI) antibody levels to both H5 and H7 AI strains. The immune responses of 1-day-old chickens vaccinated via spray with AdCN94.H7 showed Increased IgA levels in lachrymal fluids and increased interleukin-6 expression in Harderian gland derived lymphocytes. However, specific HI antibodies were not detected in the sera of these birds. These findings were published in Avian Diseases. The effects of a phytase enzyme on broilers undergoing a coccidiosis challenge were analyzed in collaboration with Dr. Blake. Straight-run broiler chicks were placed across 48 floor pens on either fresh or E. acervulina and E. tenella oocysts seeded litter. All birds were fed a corn-soybean meal diet adequate in all nutrients with the exception of Ca and non-phytate P (npP). Treatments were created using a combination of two Ca-npP levels (0.9% Ca, 0.45% npP vs. 0.7% Ca, 0.25% npP and 500 FTU Optiphos), with or without coccidia challenges and/or vaccination on day of hatch Coccivac-B. The duodenum and ceca were obtained for visual and microscopic lesion scoring and to assess immune response via cytokine production on d 10 and 18. Regardless of treatment, visual and microscopic scoring of the duodenum and ceca showed few differences (P>0.05). Expression of IFN-gamma and IL-17 were not up regulated at 10 d of age in either duodenum or cecum. On d 18, IFN-γ gene expression was increased in vaccinated or Eimeria exposed chickens, and IL-17 expression was increased in exposed birds. Phytase supplementation was unable to provide additional benefits to performance or phosphate utilization in birds vaccinated and/or subjected to a coccidiosis infection. Based on cytokine production in the intestinal tract the response to the Eimeria exposure is dominated by production of IFN-gamma and IL-17, but not IL-4 or IL-6. These findings have been accepted for publication in Poultry Science,

Publications

Peer-reviewed Publications Toro, H., Suarez, D.L., Tang, D.C., van Ginkel, F.W. Breedlove, C. 2011. Avian influenza mucosal vaccination in chicken with replication-defective recombinant adenovirus vaccine. Avian Diseases (In press).
A. Shawn, F.W. van Ginkel, K.S. Macklin, J.P. Blake. 2011. Effects of phytase supplementation in broiler diets on a natural Eimeria challenge in nave and vaccinated birds. Poultry Science (In press).
Abstracts 2010
H. Toro, D.C. Tang, D.L. Suarez, F.W. van Ginkel. 2010. Avian Influenza Spray Vaccination with Non-Replicating Adenovirus Vector. AAAP, Atlanta July 31-August 30.
Cassandra Breedlove, Frederik W. van Ginkel, De-chu C. Tang, Haroldo Toro. 2010.Combined In Ovo-Vaccination with Non-Replicating Adenovirus-Vectored Avian Influenza and Marek's Disease Vaccines. AAAP, Atlanta July 31-August 3.
F.W. van Ginkel, S.L. Gulley, A. Lammers , H. Toro. 2010. Adaptive Immunity in Conjunctiva Associated Lymphoid Tissue after Ocular Immunization. AIRG, Budapest, October 7-10.
S.L. Gulley, H. Toro, A. Lammers, R. Gurjar, F.W. van Ginkel. 2010. Adaptive immunity in head-associated lymphoid tissue after ocular immunization. Phi Zeta, epsilon chapter, Research Emphasis Day, November 10, Auburn, AL.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Mucosal Immunity in the Harderian Glands Detection of infectious bronchitis virus (IBV) by quantitative RT-PCR (qRT-PCR) was compared in tears and trachea of IBV-infected chickens. Based on these analyses it was concluded that quantitative detection of IBV RNA in tears is more sensitive than in tracheal homogenates. Furthermore, IBV contained in chicken lachrymal fluid was infectious. 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) was compared. Flow cytometric analyses of lymphocytes in Harderian glands and cecal tonsils demonstrated a reduction of IgM+ B cells in the Harderian glands and cecal tonsils following exposure to IBV in combination with immunosuppressive viruses when 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. ELISPOT assays on cells derived from Harderian glands and cecal tonsils of IBV-infected chickens demonstrated that IBV-specific IgA-secreting cell responses were reduced in chickens infected with CAV, while 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 to IBV. Thus, CAV affects pathogen-specific B cell responses in a mucosal effector site presumably by reducing T helper activity. In Ovo Vaccination with Ad5-H5 against Avian Influenza Chickens were in ovo immunized with three different doses of Ad5-H5. Three weeks after hatching the chickens were ocular boosted with 2 x108 I.U. of Ad5-H5. Tears were collected three days prior to challenge to measure starting values of IFN-gamma levels. No significant differences were observed in IFN-gamma levels in tears. However, the 109 in ovo immunized group had the highest IFN-gamma response in tears, while the responses in both the 107 and 105 groups were somewhat lower. The high IFN-gamma response in the 109 in ovo group coincided with undetectable levels of IFN-gamma in the plasma. No systemic involvement was observed in this group after ocular boosting. Local immunity in the head-associated lymphoid tissue induced by in ovo immunization was sufficient to contain the Ad5 boost. The 105 in ovo group had significantly higher IFN-gamma levels in plasma than either the 107 or 109 in ovo group, while the 107 and 109 in ovo groups did not differ significantly. The activation of the systemic immune response, as measured by IFN-gamma levels in plasma after ocular boosting, seemed proportional to the in ovo immunization dose. Thus, the degree of systemic involvement seems inversely proportional to the in ovo immunization dose. This indicates that better mucosal protection was induced following the higher in ovo immunization doses. These observation have been published in peer-reviewed papers (see below) and presented at local and national venues. PARTICIPANTS: Drs. H. Toro and V.L. vam Santen, Department of Pathobiology, College of Veterinary Medicine, Auburn University. TARGET AUDIENCES: The poultry industry in order to provide a better understanding of mucosal immunity in chickens induced by various vaccines using different vaccine routes and/or adjuvants. Furthermore it may enable us to better evaluate concept of genetic disease resistance in chicken lines by developing approaches to evaluate these parameters. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The exposure to immunosuppressive viruses has consequences for the outcome of induction of specific immunity either after vaccination or exposure to pathogens. To better understand the consequences of exposure to these immunosuppressive viruses on induction of specific immunity either after vaccination or exposure to pathogens may ultimately prevent or allow us to develop strategies to circumvent these problems. It will be important to understand the mucosal immune response and mucosal immune system in chickens and the regulatory events involved, in order to develop better approaches to induce mucosal immunity and protection to avian diseases either through vaccination and/or genetic selection. The finding that mucosal immune protection to mucosal challenge is directly proportional to the in ovo immunization dose may be an important observation to optimize vaccines for protection against pathogens that target the mucosal surfaces associated with the head of chickens.

Publications

van Ginkel, F.W., Toro, H., Tang, D.C., Gulley, S.L. 2008. Induction of Mucosal Immunity in the Avian Harderian Gland with a Replication-Deficient Ad5 vector Expressing Avian Influenza H5. Dev. Comp. Immunol 33:28-34.
van Ginkel, F.W., van Santen, V.L., Gulley, S.L., and Toro, H. 2009. Infectious Bronchitis Virus in the Chicken Harderian Gland and Lachrymal Fluid: Viral Load, Infectivity, Immune Cell Responses, and Effects of Viral Immunodeficiency. Avian Dis. 52: 608-617.

Progress 10/01/08 to 12/31/08

Outputs
OUTPUTS: Infectious Bronchitis Virus in the Chicken Harderian Gland and Lachrymal Fluid: Viral Load, Immune Cell Responses, and Effect of Viral Immunodeficiency Ocular and nasal mucosal surfaces are the port of entry for infectious bronchitis virus (IBV). High concentrations of IBV genomes can be detected in lachrymal fluid of IBV-infected chickens, and we find that quantitative detection of IBV genomes in tears is more sensitive than in tracheal homogenates. Furthermore, we demonstrate that IBV contained in chicken lachrymal fluid is infectious and that tears can be used to infect naive chickens. We monitored the immune responses to IBV in the Harderian gland and cecal tonsils of specific pathogen-free (SPF) chickens infected with IBV and compared these responses in immunocompetent chickens and chickens suffering from viral induced immunodeficiency [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 CAV, but not IBDV, reduced the CD4/CD8 T cell ratios compared to chickens infected with IBV alone. 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. We quantitatively assessed IBV-specific IgA-secreting cells in the Harderian glands and cecal tonsils of IBV-infected chickens by ELISPOT assays. 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. Analyses of these mucosal effector sites indicated that immunosuppressive viruses reduce B cells and T helper cells in the Harderian glands and cecal tonsils, and altered the kinetics or the magnitude of the mucosal immune response against IBV. Immunity to IBDV variant AL2 in young chickens Limited information is available on the effects of the recently emerged infectious bursal disease virus (IBDV) variant AL2. In this study the effects of inoculation with increasing doses IBDV AL2 were characterized in young chickens. IBDV AL2 induced neither overt clinical signs nor mortality. Infected chickens showed reduced bursa weight, reduced bursa indices (BI) as well as bursa lymphocytic depletion as determined by histomorphometry. Both the BI and histomorphometry data indicated a dose-effect. However, even low doses of the virus ultimately resulted in significant damage to the bursa. Infected chickens showed a significant increase of splenic IgM+. Others have shown that cell-mediated immunity is essential for the protection against IBDV. Our results indicate a significant increase (P<0.05) of spleen CD4+ cells on day 8 PI in birds that received higher virus concentrations, indicating a role for these cells in protective immunity, while CD8+ cell counts remained unchanged. We speculate that the changes in splenic CD4+ and IgM+ cell population are associated with protective immune responses against IBDV in the host. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: no modifications

Impacts
It will be important to understand the mucosal immune response and mucosal immune system in chickens and the regulatory events involved to generate protective immunity. This understanding will aid in developing better strategies to generate vaccines and induce mucosal immune protection to avian diseases. In order to better understand the pathogenicity of and the immune response to the IBDV variant AL2 we performed the above outlined study. Since limited information is available on this IBDV variant it may help better understand its pathogenicity.

Publications

van Ginkel, F.W., van Santen, V. L., Gulley, S.L., Toro, H. 2009. Chicken Harderian Gland and Lachryma Fluid: A Source for Infectious Bronchitis Virus and Specific Lymphocytes. Avian Dis. 52: 608-617.
Toro, H., Effler, J.C., Hoerr, F. J., and van Ginkel, F. W. 2009. Effects of Infectious Bursal Disease Virus Variant AL2 on the Immune System in Young Chickens. Avian Dis. (in press).
Toro, H., V. L. van Santen, F. W. van Ginkel, F. J. Hoerr, C. Breedlove, N. Petrenko. 2008. Effects of CAV and IBDV in Commercial Chickens.p71-72. Proceedings of 57th Western Poultry Disease Conference, Puerto Vallarta, Mexico, April 9-12.
Effler, C., C. Breedlove, S. Gulley, F. W. van Ginkel, and H. Toro. 2008. Effects of Infectious Bursal Disease Virus Variant AL2 in Young Chickens. P.59Proceedings of 57th Western Poultry Disease Conference, Puerto Vallarta, Mexico, April 9-12.
F.W. van Ginkel. 2008. Mucosal Immunity in the Harderian Glands after Ocular Immunization with a Human Ad5 Vector Encoding an Avian Influenza H5 Hemagglutinin. 10th Avian Immunology Research Group conference, Australia, June 24-27.