NE1334: Genetic Bases for Resistance and Immunity to Avian Diseases

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[04/09/2015] [04/14/2014] [05/01/2016] [02/16/2017] [02/20/2018]

Date of Annual Report: 04/09/2015

Report Information

Annual Meeting Dates: 10/11/2013 - 10/13/2013
Period the Report Covers: 10/01/2012 - 09/01/2013

Participants

* = written annual station report submitted

Technical Committee Members present:;
Erf, Gisela* (AR);
Klasing, Kirk (CA);
Zhou, Huaijun* (CA);
Gallardo, Rodrigo*(CA);
Keeler, Calvin* (DE);
Parcells, Mark* (DE);
Juul-Madsen, Helle* (DK);
Wakenell, Patricia (IN);
Lamont, Susan* (IA);
Taylor, Robert* (NH);
Ashwell, Chris* (NC);
Selvaraj, Ramesh* (OH);
Rodriguez-Lecompte, Juan (PEI, Chair);
Dalloul, Rami (VA);
Berres, Mark* (WI, Secretary);
Koci, Matthew* (NC);
Schat, Karel (NY-retired);
Qureshi, Muquarrab (NIFA Representative)

Technical Committee Members absent:;
Delany, Mary* (CA);
Dreshsler, Yvonne (WU);
Heidari, Mohammad (USDA);
Miller, Marcia (CH);
van Ginkel, Frederik* (AL);
Sharif, Shayan*(ON);
Beckstead, Robert* (GA)

Collaborators/Guests present:;
Parmentier, Henk*(NL);
van der Poel, Jan* (NL);
Berghof, Thomas (NL; graduate student);
Kaiser, Pete (UK);
Fulton, Janet (Hy-Line International);
Pevzner, Igal (Cobb Vantress);
Poston, Rebecca (Zoetis);
Odemuyiwa, W (Tuskegee University);
Rieger, Mark (Administrative Liaison to ESCOP, University of Delaware);
Song, Jiuzhou (University of Maryland);
Faulkner, Olivia (AR);
Gimeno, Isabel (NC)

Brief Summary of Minutes

8:00 am

The scheduled address by Dr. Muquarrab Qureshi was cancelled due to the federal government
shutdown

8:02 am

Address by Dr. Mark Rieger, Dean, University of Delaware College of Agriculture and Natural
Resources, Townsend Hall, 531 South College Avenue, Newark, Delaware 19716-2103. Phone: (302) 831-2501, email: mrieger@udel.edu

1. Dr. Rieger reminded NE-1034 members about the importance of yearly activity narratives not
only in terms of a basic reporting requirement but also to promote the diverse research and
activities conducted by NE-1034 participants.

2. Dr. Rieger introduced and stressed the importance of impact statements to further the dissemination and promotion of work conducted by NE-1034 participants.

a. Two page format (additional details forthcoming)

b. Intent is to describe in plain language by NE-1034 participants “why should we[government] invest in this research?”

c. In addition to the required yearly activity report, impact statements will be required of all participants in 2014 and each year thereafter

8:15 am

New member presentations

1. Dr. Jiuzhou Song, Ph.D. Associate Professor, Department of Animal and Avian Sciences, University of Maryland, Bldg 142, College Park, Maryland 20742. Phone: 301-405-5943, Fax: 301-405-7980, Email: songj88@umd.edu

a. Presentation title: “Epigenetics Modification Induced by MDV in Inbred Chicken Lines”

b. Presentation focus: Can epigenetic markers predict susceptibility to animal disease?

i. Approach: Study the occurrence and distribution of infection-induced differential methylation regions

2. Dr. Solomon Olawole (Wole) Odemuyiwa, Ph.D. Assistant Professor, Department of Pathobiology, Tuskegee University, A412 Patterson Hall, Tuskegee, AL 36088. Email: sodemuyiwa@mytu.tuskegee.edu

a. Presentation title: “Chicken Macrophages and Dendritic Cells: Influence of Virus Infection on Functional Differentiation.”

b. Presentation focus: How does differentiation of monocytes into M1 and M2
macrophages and dendritic cells occur?

i. Approach: Comparative study of PAMPs and pathogen recognition receptors in ducks (RIG-1,MDA-5) and chickens (MDA-5)

8:45 am

Motion by Dr. Sue Lamont to endorse the membership of Dr. Jiuzhou Song and Dr. Solomon Olawole Odemuyiwa as Technical Committee members of NE-1034. Seconded by Dr. Robert Taylor. Passed by unanimous vote.

8:59 am

Dr. Pat Wakenell announced her upcoming retirement and indicated a desire to host the 2014 NE-1034 meeting at Purdue University

9:01 am

NE-1034 participants openly discussed the venue for meetings over the next four years.

9:04 am

Group consensus established the following upcoming meeting locations for NE-1034:

1. 2014 – Purdue University

2. 2015 – University of California – Davis

3. 2016 – University of Wisconsin – Madison

4. 2017 – University of Prince Edward Island

9:06 am

Dr. Matt Koci acknowledged appreciation for:

1. Organizational efforts of Natasha Dillon

2. Financial contributions from:

a. Cobb-Vantress

b. Zoetis

c. N.C. Agricultural Research Service

d. CALS

e. NCSU

f. Prestage Department of Poultry Science

g. Hy-Line

9:09 am

Dr. Gisela Erf indicated that the new Multi-state organization is to be named NE-1334 for the next 5 years. Dr. Erf thanked her co-writers, Dr. Rami Dalloul, Dr. Kirk Klasing, and Dr. Huaijun Zhou for their critical input in writing the renewal NE-1334 proposal in 2012.

9:11 am

Dr. Kirk Klasing extended appreciation to Dr. Gisela Erf for writing the renewal proposal in 2012.

9:13 am

Dr. Pat Wakenell extended appreciation to Dr. Matt Koci for hosting and organizing the 2013 NE-1034 meeting

9:14 am

Dr. Juan Carlos Rodriguez-Lecompte recognized the organizational efforts of Dr. Matt Koci, Dr. Chris Ashwell, and others in the North Carolina State University group. Dr. Juan Carlos also indicated that Dr. Mark Berres will chair the 2014 meeting. Gisela Erf nominated Ramesh Selvaraj to serve as
secretary in 2014.

9:15 am

Business portion of NE-1034 meeting adjourned

Minutes recorded and prepared by:

Dr. Mark E. Berres
Secretary NE-1034 2013

Accomplishments

The existence of the project has promoted an important amount of research involving both individual as collaborative efforts between groups. Results generated from the support of this project have allowed significant discovery and evaluation of genes and genetic markers participating on the disease resistance in Poultry. Moreover, the group is facing important challenges to use these results in the context of the avian species and the Poultry industry; the work that is underway to identify specific susceptibility genes involved in diseases will be of great value in controlling and modifying possible outbreaks. Our research on chicken line susceptibility in conjunction with mechanisms participating in innate and adaptive immunity has significantly enhanced the genetic bases for resistance and immunity to avian diseases in this group. Subsequent Results of research on aspects such as environmental, physiological and nutritional factors modulating and affecting the avian immune system are inferring interesting potential to modify and optimize the immune response to chickens to important avian pathogens such as Avian Influenza, infectious bursal disease virus, Marek, salmonella among others. Nutritional intervention as a immune modulator is becoming a critical factor to joint industry and research and academia. The ultimate consequences of this collaborative effort will be enhancing disease control, health status and poultry welfare. New tools and strategies have been developed, evaluated and characterized to investigate immune system development, physiology of the immune system and disease control in poultry. Diligent efforts in this field are inferring helpful strategies to evaluate responses in tissue, blood, and organs more quickly and specific. The current and future support of the government in this group will be essential to continue discovering, characterizing and evaluating relevant genes, proteins, environmental factors associates with the resistance to pathogens and how to effectively control disease relevant to the poultry industry and potential zoonotic diseases. Accomplishments: OBJECTIVE 1. Identify and characterize genes and their relationships to disease resistance in poultry with an emphasis on the major histocompatibility complex as well as other genes encoding alloantigens, communication molecules and their receptors and other candidate systems. (AK) Gene network analysis, which represents the intermolecular connections among interacting genes based on functional knowledge inputs, revealed that development of vitiligo appeared to be associated with the interactions among protein kinases (MAPK, ERK1/2, PKC, PRKDC), phosphatase (PPP1CA), ubiquitinylation (UBC) and amyloid production (APP). Together, these studies promise important new information on identifying the specific susceptibility genes responsible for the depigmentation and other abnormalities seen in the SL. CA reported the finalization of the molecular cytogenetic analysis of GGA 16 with regard to genes identified by cCGH; they found in this microchromose: in the small p arm no genes or elements discovered to date other than terminal telomere repeats; in the centromere CNM repeats; and in the q (long) SRCR (cysteine-rich-domain scavenger receptor genes) and OR (olfactory receptor genes) interspersed, the NOR, MHC-Y, GC-rich region (w/PO41 repeats and a secondary constriction), MHC-B, terminal telomere repeats. Also, they did not find evidence of MDV either associated or integrated form on macrophage cell line MQ-NCSU. Using UA04 cell line CA indicated MDV cytogenetic signals in the “associated form” (small punctate signals surround chromosomes), adjacent to the chromosomes with the integrated form upon BrdU exposure (and relative to controls). CA Lung Transcriptome following Avian Influenza Virus Infection in Two Genetically Distinct Chicken Inbred Lines using RNA-seq research showed a total of 2,464 differentially expressed genes, there were 62 genes identified in both Leghorn and Fayoumi birds, which have been reported having antiviral activities, including the chicken MX1 gene. Gene Ontology analysis using DAVID program was conducted (fold enrichment > 2 and FDR < 20%). It is noticed that RIG-I-like receptor-signaling pathway (even though RIG-I does not exist in chickens), which is critical for AIV resistance, was significantly enriched (> 10 fold) in the Fayoumi line. CA reported that the reassortant (vvIBDV segment A + serotype 2 segment B) viruses induces immunodeficiency in broilers and layers. DE reported that avianspecific toll like receptor 15 (TLR15) is functionally equivalent to a group of TLR2 family proteins that the mammalian innate immune system utilizes to recognize a broad spectrum of microbe-associated molecular patterns, including bacterial lipoproteins; Also confirmed that the avian TLR7 receptor recognizes ssRNA, like its mammalian counterpart. DK working on transcription efficiency of different chicken mannose-binding lectin promoter alleles found that the mutation of SNP2 AGGA in A1 to GGGG was expected to increase the transcription activity, but instead the mutation silenced the transcription activity completely. Thus, SNP2 may be used as a diagnostic SNP, but does not appear to be the major functional SNP causing low MBL mRNA expression. DK reported for the first time that MBL expression is present in the lungs of healthy chickens, and that the expression is upregulated at days 3 and 7 after IBV infection. Furthermore, in the liver of infected chickens, the MBL expression was upregulated at day 3 and even more at day 7 after IBV infection, despite the fact that the MBL serum concentrations were decreased below baseline at the latter time point. Thus, these results indicate that MBL is produced locally and may be involved in the regulation of the cellular immune response after an IBV infection. Analyzing host transcriptional response to vaccination and/or infection with avian pathogenic E. coli (APEC) IO has conducted and published microarray analyses of the spleen and peripheral WBC transcriptome evaluated utilizing a chicken 44K Agilent microarray. As a part of chicken breeding program to study disease resistance, the remaining recombinant congenic strains (RCS) provide an opportunity to find genes other than the MHC that impacts immune responses. NH reported in v-src tumor growth research that strains J, R and W were significantly lower than Line 63, strains A, C and L; characterizing antibody response against SRBC they reported that RCS J antibody response was slower to develop whereas RCS N had a more rapid decline. Most interesting, is RCS J, which differed significantly in both v-src TPI and SRBC titer. Working on gene expression in the embryonic immune tissue from high (HAS) and low (LAS) SRBC antibody selected lines exposed to testosterone propionate NH reported that gene expression was consistent with differential immune response between the selected lines. HAS C versus LAS C groups express different genes suggesting diverse B-cell maturation. WI has demonstrated that wild populations of Red Jungle Fowl (the direct ancestor of modern domestic lines) collected in Vietnam have highly diverse and unique alleles not only at Mx and MHC loci, but also presumably in other immunologically active genes. NC working on specific variant within exon 14 of Mx gene (amino acid S631N) which modulates resistance to avian influenza virus infection found that there were significant changes in haplotype frequency in 6 of the 8 lines that were segregating, suggesting some unknown selective advantage for certain haplotypes. UG reported that their result suggest that avian AAV-based vectors can be used for the delivery of shRNA into chicken cells. However, administration of the rAAAV expressing shRNA targeting chicken IFN-? did not seem to abrogate vaccine-induced protection. UG results suggest that erythrocytes constitutively express transcripts for TLRs 2,3,4,5 and 21, as well as for many immunological genes including type I interferons (IFN) and interleukin (IL)-8. Moreover, it was found that treatment with both poly I:C and CpG ODN up-regulated transcripts for type I IFNs, while only poly I:C up-regulated IL-8 transcripts and enhanced the production of nitrite; Also UG has demonstrated that different TLR2 ligands may induce slightly different cytokine responses in chicken splenocytes. GA showed that JetPEI can transfect multiple chick cell types, most notably germline stem cells. They also showed that pairing these two reagents (piggyback (PB) and JetPEI) is a viable and reproducible method for integration of transgenes into the chicken genome; in addition GA, using a protocol for a sperm mediated gene transfer technique reported with high success in fish, they dehydrated sperm with hypotonic diluent and then rehydrated the sperm with hypertonic diluent with plasmid DNA, showing that the plasmid was taken into the sperm and a portion of the plasmid DNA was delivered to the embryos at fertilization, although the GFP gene found in the plasmid was not expressed in the embryo and the process greatly decreased fertility. WD reported that working in a new selection on high or low Natural Antibody (NAb) titers binding KLH they found that for the 2 segregating MHC types there was no indication of an MHC effect on NAb titers; they are working on SNP associations with natural antibody isotypes IgM and IgG binding KLH in laying hens. Objective 2. Identify and characterize environmental, dietary and physiological factors that modulate immune system development, optimal immune function and immune system related disease resistance and welfare in poultry genetic stocks. (AK) Through a time-course study it was confirmed that the signature cytokine profile of SL vitiligo autoimmune disease is a combination of IFN-?, IL-21, and IL-10. In addition AK reported that histopathology of impaired vision in SL vitiligo resembles induced uveitis and other ocular diseases in humans, especially Vogt-Koyanagi-Harada (VKH) syndrome and sympathetic opthalmia (result of penetrating eye injury), both of which are autoimmune diseases associated with vitiligo. CA reported that applying the piggyBac transposon system for establishing stable in vitro chicken cell lines can provide a powerful tool in elucidating the IRF7 function in the host-pathogen interaction in chicken; In CA the obtained results examining pro-inflammatory or anti-inflammatory cytokine expression at the mRNA level in the bursa and spleen, 1 and 4 days post-infection with different infectious bursal disease virus (IBDV) strains including vvIBDV reassortants, reflected differences in the development of the immune system suggesting that the strength of the host responses at transcriptional level maybe a key factor in age dependent immunosuppression. Regarding Marek’s disease virus (MDV) DE research is focused particularly in the functional analysis of MDV genes as they relate to the evolution of MDV virulence Meq and glycoprotein L. DE has found that spliced forms of the Meq oncoprotein, which are expressed during latency, in lymphomas and in cell lines derived from lymphomas have increased affinity for CtBP1, a protein that serves as a scaffold for chromatinremodeling enzymes (histone methyltransferases, histone acetylases, etc.); because MDVinduced tumor composition associated with point mutations in the Meq oncoprotein DE is characterizing lymphomas caused by virulent (v), very virulent (vv) and very virulent + (vv+) MDVs; moreover DE reported on their findings that a glycoprotein L (gL) mutation common to MDV field strains isolated along the East Coast of the US (NH, PA, DE, MD, VA, and NC) since 200 introduced into the RB-1B strain of MDV as an infectious clone (pRB-1B) did not confer increased virulence to this virus in terms of increased replication or ability to cause disease in unvaccinated or HVT-vaccinated chickens. Moreover, it did not appear to confer increased virulence to contactexposed, bivalently-vaccinated chickens. Regarding AI, DE reported that a microarray analysis revealed a core set of 61 genes differentially regulated in response to all three LPAIVs tested and 101, 135, and 628 differentially expressed genes unique to infection with chicken, duck, or turkey origin LPAIV isolates respectively; in addition qRTPCR results revealed significant (p<0.05) induction of IL-1?, IL-2, and IFN? transcription, especially with the chickenorigin isolate. DE has concluded that altering the host immune response using TLR agonists before infection with HPAIV can have beneficial effects as measured by increased survival time. DK reported as MBL ligands co-administered with IBV vaccine induced differences between chicken lines, these results indirectly suggest that MBL is involved in the immune response to IBV vaccination. Furthermore, the higher antibody response in L10H chickens receiving vaccine and FOS makes FOS a potential adjuvant candidate in an IBV vaccine. Within a USDA-AFRI funded Climate Change project (PD: C. Schmidt, UDEL), IO is investigating the interaction of two putative stressors: heat stress and exposure to an inflammation-inducing PAMP (LPS). Working for a central role for Tregs in salmonellosis persistence in chicken, OH has reported that S. enterica might have unique properties that can stimulate Tregs in a host to evade immune responses; they found at 14 d post-S. enterica infection, the percentage and IL-10 mRNA content increased in cecal tonsils. The suppressive properties of cecal tonsils Tregs on CD4+CD25- immune cells increased by 400 % and thus Tregs became supersuppressive. This suggests the role of Tregs in S. enterica persistence. Preliminary studies in NC suggest the production of some as yet unidentified soluble factor in circulation regulates the leukocyte activity. Studies are continuing to isolate and characterize this factor or factors. AL results indicated 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. WD reported to be working on effects of husbandry on immune responsiveness of chickens and immune-modulation of the immune response (Innate immune system) with emphasis on natural antibodies, probiotics and PAMPs. Objective 3. Develop, evaluate and characterize methodologies, reagents and genotypes to assess immune function and disease resistance to enhance production efficiency through genetic selection in poultry. AK continues working on the growing feather as an “in-vivo test-tube” to monitor tissue responses to test-material in vivo in the same individual; they will be examining bioactivity of nanoparticles in vivo using this test-system along with monitoring of the peripheral blood. DE has been working on: (1.) characterizing a vaccine strain that has reverted to virulence; currently they are examining mutations on CVI988-699-2 RV for changes in coding sequences, expression and ultimately, changes in virulence. (2.) developing pathotype-specific infectious clones for use as challenge strains of defined virulence; their plan on generating BACbased infectious clones based on vMDV (GA-22), vvMDV (RB1B) and vv+MDV (TK) field strains, and (3.) developing a targeted set of qPCR primers to define innate sensing, signaling and immune patterning important to MD vaccine protection versus pathogenesis; to evaluate the effect of an immune stimulant on Marek’s disease vaccine efficacy, DE has put together a targeted set of 40+ qPCR primers to evaluate innate sensing, signaling and immune patterning following vaccination. DK has reported a rapid method for quantification of Salmonella spp. in cloacal swab samples that has been developed. The method is based on a short pre-enrichment in buffered peptone water (BPW), DNA extraction and quantification by real-time PCR. In addition, DK reported that assessment of CD107a cell surface expression is potentially a useful tool for CTL studies in chickens. Iowa State University maintains 13 chicken genetic lines. In the past year, they were reproduced in one generation (The ISU genetic lines are of two basic genetic structures: (a) highly inbred lines or (b) advanced intercross lines (AIL)). AL has reported 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

Books and Chapters Cheng, H.H. and Lamont, S.J. 2013 Genetics of disease resistance. Pp. 70-86. In: Diseases of Poultry. 13th ed. D. E. Swayne, J.R. Glisson, L.R. McDougald, V. Nair, L. Nolan, and D.L. Suarez, Eds. Wiley-Blackwell, Ames Delany, M.E. and T.H. O’Hare. 2013. Genetic stocks for immunological research (Appendix I). In Avian Immunology, 2nd Edition (editors: K.A. Schat, B. Kaspers and P. Kaiser). Elsevier: Academic Press, San Diego, CA. ISBN 9780123969651. 456 pp. Erf, G. F. 2013. Autoimmune diseases of poultry. Avian Immunology, 2nd edition. Schat K. A., B. Kaspers, and P. Kaiser, editors. Elsevier, London, San Diego, CA. (in press) Lamont, S.J., Dekkers, J.C.M., and Zhou, H. 2014. Immunogenetics and mapping immunological functions. Pp. 205-221. In: Avian Immunology. F. Davison, B. Kaspars, P. Kaiser, K.A. Schat, Eds., Elsevier, London, San Diego van Ginkel, F.W. 2014. The Scourge of IBV. Auburn Speaks; (in press). Peer-reviewed papers Abernathy, J., C. Corkill, C. Hinojosa, X. Li, H. Zhou. 2013. Deletions in the pyruvate pathway of Salmonella Typhimurium alter SPI1-mediated gene expression and infectivity. Journal of Animal Science and Biotechnology 4:5. DOI: 10.1186/2049-1891-4-5. Anderson, J. L., C. M. Ashwell, S. C. Smith, R. Shine, E. C. Smith, and R. L. Taylor, Jr. 2013. Atherosclerosis-susceptible and atherosclerosis-resistant pigeon aortic cells express different genes in vivo. Poult. Sci. 92:2668-2680 Anderson, J. L., S. C. Smith and R. L. Taylor, Jr. 2013. Atherosclerosis-susceptible and atherosclerosis-resistant pigeon aortic smooth muscle cells express different genes and proteins in vitro. In: Current Trends in Atherogenesis. R. Rezzani, (ed.) InTech, Inc., Rijeka, Croatia (Review) pp. 165-186 Accessed February 27, 2013 DOI: 10.5772/52948 http://www.intechopen.com/articles/show/title/atherosclerosis-susceptible-and-atherosclerosis-resistant-pigeon-aortic-smooth-muscle-cells-express- B.G. de Jong, A. Lammers, L.A.A. Oberendorf, M.G.B. Nieuwland, H.F.J. Savelkoul, H.K. Parmentier. Genetic and phenotypic selection affect natural (auto-) antibody reactivity of chickens. In press, PlosOne, 8 mei 2013. Burks, T. A. and R. L. Taylor, Jr. 2013. Genetic control of Rous sarcoma virus-induced tumor growth in chickens: Role of the major histocompatibility (B) complex. Animal Science Image Gallery. National Agriculture Library http://anscigallery.nal.usda.gov//index.php #5178 in press C. J. Sample, K. E. Hudak, B. E. Barefoot, M. D. Koci, M. S. Wanyonyi, S. Abraham, H. F. Staats, E. A. Ramsburg. A mastoparan-derived peptide has broad-spectrum antiviral activity against enveloped viruses. Peptides. 48:96-105, 2013. Cabral, R., P. Erickson, and R. L. Taylor, Jr. 2013. Processing effects on colostrum quality. Animal Science Image Gallery. National Agriculture Library http://anscigallery.nal.usda.gov//index.php #5181 in press Cheng, H.H., Kaiser, P., and Lamont, S.J. 2013. Integrated genomic approaches to enhance genetic resistance in chickens. Annu. Rev. Anim. Biosci. 2013. 1:239–260 Coble, D.J., E. E. Sandford, T, Ji, J. Abernathy, D. Fleming, H. Zhou, S.J. Lamont. 2012. Impacts of Salmonella enteritidis infection on liver transcriptome in broilers. Genesis DOI: 10.1002/dvg.22351. Coble, D.J., Sandford, E. E., Ji, T., Abernathy, J., Fleming, D., Zhou, H., and Lamont, S.J. 2013. Impacts of Salmonella enteritidis infection on liver transcriptome in broilers. Genesis 51:357–364 E. O. Oviedo-Rondón, N. M. Leandro, R. Ali, M. Koci, V. Moraes, and J. Brake. Broiler breeder feeding programs and trace minerals on maternal antibody transfer and broiler humoral immune response. Journal of Applied Poultry Research. 22:499-510, 2013. Engel, A. T., R. K. Selvaraj, J. P. Kamil, N. Osterrieder, and B. B. Kaufer. 2012. Marek's disease viral interleukin-8 promotes lymphoma formation through targeted recruitment of B cells and CD4+ CD25+ T cells. J Virol 86: 8536-8545. Erf, G.F. 2013. Autoimmune disease: unique research opportunities in the autoimmune vitiligo-prone Smyth line of chicken. World J. Immunol. (in press) Erf., G.F. 2013. Laboratory Update - Gisela Erf. PASPCR Newsletter 21 (2):15-17. G. den Hartog, R.P.M.A. Crooijmans, H.K. Parmentier, H.F.J. Savelkoul, N.A. Bos, A. Lammers. Ontogeny of the avian intestinal immunoglobulin repertoire: modification in CDR3 length and conserved VH-pseudogene usage. Molecular Immunology in press Gadde, U., T. Rathinam, G. F. Erf, and H. D. Chapman. 2013. Cellular immune responses to infection with the protozoan parasite Eimeria adenoeides in turkey poults. Poult. Sci. (in press) Gerritsen, G.J. Klaassen, G. Schuttert, S.M.G. Rouwers, H.K. Parmentier, F. Molist. The effect of a mixture of dairy-based feet ingredients, vegetable fats, and yest cell walls on performance and innate immunity of weaned piglets. J. Anim. Sci. 90: 269-271. 2012. 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. H.K. Parmentier, L.P.M. Verhofstad, G. De Vries Reilingh, M.G.B. Nieuwland. Breeding for high specific immune reactivity affects sensitivity to the environment and is negatively associated with egg production in layers. Poultry Science 91: 3044-3051, 2012. H.T.L. Lai, M.G.B. Nieuwland, A.J.A. Aarnink, B. Kemp, H.K. Parmentier. Effects of two size classes of intratracheally administered airborne dust particles on primary and secondary specific antibody responses and body weight gain of broilers: a pilot study on the effects of naturally occurring dust. Poultry Science 91: 604-615, 2012. Haq K, Schat KA, Sharif S. Immunity to Marek's disease: Where are we now? Dev Comp Immunol. 2013 Nov;41(3):439-46. Haq K, Wootton SK, Barjesteh N, St Paul M, Golovan S, Bendall AJ, Sharif S. Small interfering RNA-mediated knockdown of chicken interferon-? expression. J Interferon Cytokine Res. 2013 Jun;33(6):319-27. J. Groffen, H.K. Parmentier, W.A.C. Van de Ven, M. Van Weerd. Effect of different rearing strategies and age on levels of natural antibodies in saliva of Philippine crocodiles. Asian Herpetological Research: 4: 22-24, 2013. Jia X., H. Zhou, D. Li, W. Liu, N. Yang. 2013. Copy number variations identified in the chicken using a 60K SNP BeadChip. Animal Genetics 44:276-84. DOI:10.1111/. Kjærup, R.M., Skjødt K., Dalgaard T.S., and Juul-Madsen H.R. (2013). Chicken mannose-binding lectin (MBL) gene variants with influence on MBL serum concentration. Immunogenetics 65; 461-471. DOI: 10.1007/s00251-013-0689-6. Kumar S, Kunec D, Buza JJ, Chiang HI, Zhou H, Subramaniam S, Pendarvis K, Cheng HH, Burgess SC. 2012. Nuclear Factor kappa B is central to Marek's disease herpesvirus induced neoplastic transformation of CD30 expressing lymphocytes in-vivo. BMC Syst Biol. 6:123. doi: 10.1186/1752-0509-6-123. Maughan, MN, Dougherty LS, Preskenis LA, Ladman BS, Gelb, J., Jr., Spackman, EV, Keeler CL, Jr. 2013. Transcriptional analysis of the innate immune response of ducks to different species-of-origin low pathogenic H7 avian influenza virus. Virology Journal 10:94 Miller, M.M., C.M. Robinson, J. Abernathy, R.M. Goto, M. Hamilton, H. Zhou, and M.E. Delany Mapping of genes for olfactory receptors, cysteine-rich-domain containing scavenger receptors and other proteins to GGA 16, the chicken MHC microchromosome. In review. Norup L.R., Dalgaard T.S.,Pleidrup J., Permin A., Schou T.W., Jungersen G., Fink D.R., and Juul-Madsen H.R. (2013). Comparison of parasite-specific immunoglobulin levels in two chicken lines during sustained infection with Ascaridia galli. Vet Parasitology 191: 187 - 190. DOI.org/10.1016/j.vetpar.2012.07.031. Oven I, Resman K, Dušani? D, Ben?ina D, Keeler CL , Narat M. 2013. Diacylated lipopeptide from Mycoplasma†synoviae†mediates TLR15 induced innate immune responses. Veterinary Research (in press) Pleidrup J.A.,Norup L.R., Dalgaard T.S., Rothwell L., Kaiser P., Permin A., Schou T.W., Fink D.R., Jungersen G., Sorensen P., and Juul-Madsen H.R. (2013). No protection in chickens immunised by the oral or intra-muscular immunisation route with Ascaridia galli soluble antigen. Avian Pathol. 2013 Jun;42(3):276-82. doi: 10.1080/03079457.2013.783199 Robinson, C.M., H.H. Cheng, and M.E. Delany. Marek's disease herpesvirus and chicken host genome interactions: Viral genome integration occurs early in infection and over a timeframe associated with latency, yet integration alone is not sufficient for cellular transformation. In review. Shaikh SA, Katneni UK, Dong H, Gaddamanugu S, Tavlarides-Hontz P, Jarosinski KW, Osterrieder N, Parcells MS. 2013. A deletion in the glycoprotein L (gL) gene of U.S. Marek's disease virus (MDV) field strains is insufficient to conferincreased pathogenicity to the bacterial artificial chromosome (BAC)-based strain, RB-1B. Avian Dis. 2013 :509-18. Shanmugasundaram, R., and R. K. Selvaraj. 2012. Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on regulatory T cell depletion and CD4+CD25- T cell properties in chickens. Develop. Comp. Immunol. 36: 578-583. Shanmugasundaram, R., and R. K. Selvaraj. 2012. In vivo lipopolysaccharide injection alters CD4+CD25+ cell properties in chickens. J. Anim. Sci. 90: 2498-2504. St Paul M, Barjesteh N, Paolucci S, Pei Y, Sharif S. Toll-like receptor ligands induce the expression of interferon-gamma and interleukin-17 in chicken CD4+ T cells. BMC Res Notes. 2012 Nov 1;5:616. St Paul M, Brisbin JT, Abdul-Careem MF, Sharif S. Immunostimulatory properties of Toll-like receptor ligands in chickens. Vet Immunol Immunopathol. 2013 Apr 15;152(3-4):191-9. St Paul M, Paolucci S, Barjesteh N, Wood RD, Sharif S. Chicken erythrocytes respond to Toll-like receptor ligands by up-regulating cytokine transcripts. Res Vet Sci. 2013 Aug;95(1):87-91. St Paul M, Paolucci S, Read LR, Sharif S. Characterization of responses elicited by Toll-like receptor agonists in cells of the bursa of Fabricius in chickens. Vet Immunol Immunopathol. 2012 Oct 15;149(3-4):237-44. St Paul M, Paolucci S, Sharif S. Characterization of response initiated by different Toll-like receptor 2 ligands in chicken spleen cells. Res Vet Sci. 2013 Jul 30. doi:pii: S0034-5288(13)00224-5. Sun Y.Y., E.D. Ellen, J.J. van der Poel, H.K. Parmentier, P. Bijma. Modelling of feather pecking behaviour in beak trimmed and non-beak trimmed crossbred laying hens: variance component and trait-based approach. Submitted to Poultry Science, May 2013 T. Berghof, H.K. Parmentier, A. Lammers. Transgenerational epigenetic effects on innate immunity in broilers, an underestimated field to be explored. In press, Poultry Science 2013 T.V.L. Berghof, H.T.L. Lai, A. Lammers, G. de Vries Reilingh, M.G.B. Nieuwland, A.J.A. Aarnink, H.K. Parmentier. Localization and (semi-) quantification of fluorescent beads of two sizes in chicken over time after simultaneous intratracheal and cloacal administration. Poultry Science 92: 1186-1194, 2013. Wang Y, Brahmakshatriya V, Lupiani B, Reddy S, Okimoto R, Li X, Chiang H, Zhou H. 2012. Associations of chicken Mx1 polymorphism with antiviral responses in avian influenza virus infected embryos and broilers. Poult Sci. 91(12):3019-24. doi: 10.3382/ps.2012-02471. Wideman, R. F., D. D. Rhoads, G. F. Erf, and N. B. Anthony. 2013. Pulmonary Hypertension Syndrome (PHS, Ascites Syndrome) in Broilers: A Review. Poult. Sci. 92:64-83. Y. Sun, E.D. Ellen, H.K. Parmentier, J.J. van der Poel. Genetic parameters of natural antibody isotypes and survival analysis in beak trimmed and non-beak trimmed crossbred laying hens. In press, Poultry Science 2013. Y. Sun, F. Biscarini, H. Bovenhuis, H.K. Parmentier, J.J. van der Poel. Genetic parameters and across-line SNP associations differ for natural antibody isotypes IgM and IgG in laying hens. Animal Genetics: In press. DOI: 10.1111/age.12014. Zhang, H. F. Guo, H. Zhou and G. Zhu. 2012. Transcriptome Analysis Reveals Unique Metabolic Features in the Cryptosporidium parvum Oocysts Associated with Environmental Survival and Stresses. BMC Genomics.2012, 13:647. ABSTRACTS & PROCEEDINGS A. L. Ballou, R. Qiu, R. A. Ali, W. J. Croom, and M. D. Koci. Direct fed microbial supplementation affects host immune function and energy consumption. May 2013. Experimental Biology 2013. Boston, MA. Abernathy J, X. Li, X. Jia, C. L. Swaggerty, M. H. Kogut, I. Pevzner, K. Drake, H. Zhou. 2013. A systems biology approach using Dynamic Bayesian Networks reveals bio-signatures of broiler chicken bursa response to Campylobacter jejuni inoculation. Plant & Animal Genome XXI, San Diego, CA. Barrios MA, Jordan BJ, and Beckstead RB. (2013) Sperm-mediated gene transfer in chicken using a dehydration/rehydration protocol. Southeastern Society for Developmental Biology Meeting. Beckstead RB and Jordan BJ. (2012) New methods to produce transgenic chickens. 109th Annual Meeting of Southern Association of Agricultural Scientists Bed'Hom B., Fulton J., Juul-Madsen H., and Chazara O. 2013. Large scale analysis of MHC variability in chicken using a dedicated SNP panel. 10th International Veterinary Immunology Symposium - IVIS 2013, Milan, italy Byrne, K. A.*, D. Falcon*, O. Alaamri*, R. L Dienglewicz, and G. F. Erf. 2013. Autoimmune vitiligo in Smyth line chickens is preceded by altered blood leukocyte profiles and responses to bacterial cell wall components. Poult. Sci. (E-Suppl. 1) 92:54. Dalgaard, TS, Norup, LR, Pleidrup, J, Kjærup, RM, Wattrang, E & Juul-Madsen, HR 2013, 'Evauluation of an Easy Flow-Cytometric Assay for Detection of Specific Cell Mediated Immunity in Activated Whole Blood or PBMC from Chickens: SSI13-1108' Scandinavian Journal of Immunology, vol 77, nr. 4, s. 294-294. Demeure, O, Hamzic, E, Juin, H, Naciri, M, Juul-Madsen, HR, Okimoto, R, Pinard-van der Laan, MH & Bed'Hom, B 2013, 'Investigation of Immune Response to Eimeria maxima in Broilers: SSI13-1106' Scandinavian Journal of Immunology, vol 77, nr. 4, s. 276-276. Dong, L.*, R. L. Dienglewicz, and G. F. Erf. 2012. Divergent gene-expression profiles in 4-TBP-injected growing feathers of vitiligo-prone Smyth- and control line of chickens. Arkansas Biosciences Institute, Fall Research Symposium, Scientific Session II. P 12. Erf, G. F. 2012. Biomedical research opportunities in the chicken model. Arkansas Biosciences Institute. Fall Research Symposium, Fayetteville, AR, Scientific Session I. P 2. Erf, G. F., L. Dong*, F. Shi*, and R.L Dienglewicz. Multifactorial, non-communicable diseases: new insights from the Smyth line chicken model for autoimmune vitiligo. Poult. Sci. (E-Suppl. 1) 92:54. 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. Gadde, U., T. Rathinam, G. F. Erf, and H. D. Chapman, 2013. Acquisition of immunity to the protozoan parasite E. adenoeides in turkey poults and cellular responses to infection. Poult. Sci. (E-Suppl. 1) 92:110. Gallardo, R. Characterization of the immune response and immunosuppression in chickens challenged with very virulent infectious bursal disease virus reassortants. AVMA American Association of Avian Pathologists (AAAP) annual meeting. Chicago, IL, July 2013. Gregory D., M. Golding, and H. Zhou. 2013. Lentivirus-Mediated RNAi Knockdown of Chicken ATF3 and MYD88 genes in the HD-11 Cell Line. Proc. Plant & Animal Genome XXI, San Diego, CA. H. Zhou, Y. Wang, S.J. Lamont, P. Ross. 2013. Re-annotation of Chicken Genome using RNA-seq Data. 102th Annual Poultry Science meeting, San Diego, CA H. Zhou. Abernathy J, X. Li, X. Jia, C. L. Swaggerty, M. H. Kogut, I. Pevzner, K. Drake. 2013. Systems biology analysis of the ceca and bursa revealed unique innate immune responses to Campylobacter jejuni in two genetically distinct lines of chickens has been selected for a poster presentation. American Association of Immunology Meeting, Honolulu, HI Huff, S. and G. F. Erf. 2013. Autoimmune vitiligo in the Smyth line chicken model: altered immune cell profiles in spleens from hens without active vitiligo lesions. Annual Biomedical Research Conference for Minority Students. Nov 16, 2013. J. Rojas-Amortegui. 2013. Comparison of a BAC-based Marek’s Disease Virus, Strain CVI988 that has reverted to virulence to its parental vaccine strain. 85th NECAD, Sept. 18, 2013. J.E. Fulton, J. Arango, M. Koci, and C.M. Ashwell. Variation in the MX gene in commercial egg layer elite lines. September 2013. 8th European Symposium on Poultry Genetics. Venice, Italy. Jordan BJ, Stark MR, Beckstead RB. (2012) Novel method of producing chimeric chicks using piggyBac and JetPEI, International Poultry Scientific Forum Jordan BJ*, Stark MR, Stark MR, and Beckstead RB. (2012) Utilizing piggyBac in transgenic chick strategies. Annual Meeting of the Poultry Science Association Juul-Madsen, HR, Norup, LR, Sørensen, P & Dalgaard, TS 2013, 'Ontogenic Development of Lymphocyte Subsets in Two Selected Chicken Lines that Differ in Mannose-Binding Lectin (MBL) Serum Concentration Under Conventional Rearing: SSI13-1030' Scandinavian Journal of Immunology, vol 77, nr. 4, s. 268-268. Kjærup R. M., Dalgaard T.S., Norup L.R. Goti R.M., Miller M.M., and Juul-Madsen H.R. Effect of polymorphism in chicken mannose-binding lectin promoter on the transcription efficiency. 12th Avian Immunology Research Group Meeting 28 – 31. August 2012, Edinburgh, UK. Kjærup R.M., Dalgaard T.S., Norup L.R., and Juul-Madsen H.R. Polymorphism in the chicken Mannose-Binding Lectin 2 gene. Annual Meeting in Danish Society of Immunology, 24th May Aarhus, Denmark. Kjærup, RM, Dalgaard, TS, Norup, LR, Sørensen P & Juul-Madsen, HR 2013, ’Can Vaccines be improved in Chickens by Adding Mannose-Bonding Lectin (MBL) Ligands?: SSI13-1101’ Scandinavian Journal of Immunology, vol 77, nr. 4, s. 320-320. Krishnamoorthy, S., A. Al-Rubaye, S. Dey, K. Al-Zahrani, G. F. Erf, R. F. Wideman, N.B. Anthony, and D. Rhoads. 2012. Pulmonary arterial hypertension in the chicken: genetic similarities to humans. Arkansas Biosciences Institute, Fall Research Symposium, Scientific Session II. P 33. Li J., Q. Li, R. Li, L. Li, Y. Wang, V. Brahmakshatriya, B. Lupiani, S. Reddy, X. Hu, S. Lamont, S. Hu, H. Zhou and N. Li. 2013. Single base resolution DNA methylome between avian influenza virus resistant and susceptible chickens. Plant & Animal Genome XXI, San Diego, CA. Miller M. M., C. Robinson, J. Abernathy, R. M. Goto, H. Zhou and M. E. Delany. 2013. FISH and Trisomy Mapping Locate Olfactory Receptor and CD163 Genes on GGA16. Plant & Animal Genome XXI, San Diego, CA. Milonczyk, A. and M. E. Berres (2012) Genetic modification of chickens to confer resistance to avian influenza. Introductory Biology 152 Undergraduate Research Symposium. University of Wisconsin – Madison N. Siano, S. Nath Neerukonda, S. P. Golovan, M. S. Parcells. 2013. Pro-tumorigenic reprogramming of energy metabolism by Marek’s disease virus (MDV) in T-cell lymphomas. 85th NECAD, Sept. 18, 2013. Norup, LR, Dalgaard, TS, Pleidrup, J, Schou, TW, Permin, A, Fink, DR, Jungersen, G & Juul-Madsen, HR 2013, 'Presence of Recently Activated B Lymphocytes in Chicken Gut Associated Lymphoid Tissue after an Ascaridia galli Infection: SSI13-1110' Scandinavian Journal of Immunology, vol 77, nr. 4, s. 283-283. Payne J. and Beckstead RB. (2013) MACS sorting as a means of isolating primordial germ cells from stage X embryos. Southeastern Society for Developmental Biology Meeting. Pleidrup A. J., Norup L.R., Dalgaard T.S., Kaiser P., Permin A., Schou T.W., Jungersen G., Sørensen P., and Juul-Madsen H.R. The effect of A. galli infection on vaccine-induced immunity to NDV in chickens. 12th Avian Immunology Research Group Meeting 28 – 31. August 2012, Edinburgh, UK. Pleidrup J., Norup L.R., Dalgaard T.S., and Juul-Madsen H.R. Chicken immune responses after immunisation with Ascaridia galli antigens and protectivity after challenge infection 2012. Annual Meeting in Danish Society of Immunology, 24th May Aarhus, Denmark R. A. Gallardo, H. Zhou, P. Woolcock, F. J. Hoerr. 2013. Characterization of the Immune Response and Immunosuppression in Commercial Chickens Challenged with Very Virulent Infectious Bursal Disease Virus Reassortants. American Association of Avian Pathologists, Chicago, IL. 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. 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. 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. 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. Research week, April 2-4, 2013, Auburn AL. Rhoads, D. D., S. Krishnamoorthy, A. Al-Rubaye S. Dey, K. Al-Zahrani, G. F. Erf, .R. F. Wideman, N. B. Anthony. 2013. Mapping ascites QTLs in broilers. Plant and Animal Genomics :W642 S. Nath Neerukonda, S. P. Golovan, and M. S. Parcells. 2013. Targeted transcriptome analysis of immune signatures in the chickens in†ovo† vaccinated with bivalent HVT/SB-1. 85th NECAD, Sept. 18, 2013. Sorrick, J.*, R. L. Dienglewicz, and G. F. Erf. 2013. Uveitis and blindness in Smyth line chickens with autoimmune vitiligo: immunopathology associated with melanocyte loss in the eye. Pigment Cell & Melanoma Res. 26:769. Swaggerty, H.I. Chiang, H. Zhou M. H. Kogut. 2013. Concurrent analyses of host-pathogen transcriptomes in avian polymorphonuclear cells following exposure to Salmonella enterica serovar Typhimurium. American Association of Immunology Meeting, Honolulu, HI T.H. Kim, H. Zhou. 2013. Chicken IRF7 over-expressed and knock-downed HD11 cell line establishment applying piggyBac transposon vector system. IX Transgenic Animal Research Conference, Tahoe, CA. T.H. Kim, H. Zhou. 2013. Establish Overexpressed and Knock-down IRF7 Chicken HD11 Cell Line using PiggyBac Transposon System.102th Annual Poultry Science meeting, San Diego, CA Taylor, R. L., Jr., T. A. Burks, P. B. Siegel, and C. M. Ashwell. 2012. Temporal and treatment changes in embryonic bursal gene expression after testosterone exposure in high and low antibody lines. Poult. Sci. 91(Suppl. 1):29 U. Katneni, M.S. Parcells. 2013. Development of a Targeted Array to Assess Chicken Innate Signaling and Immune Patterning in Response to Marek’s Disease Vaccination. 85th NECAD, Sept. 18, 2013. Ulrich-Lynge S., Dalgaard T.S., Norup L.R., Olsen J.E., and Juul-Madsen H.R. The binding ability of purified chicken Mannose-Binding Lectin (MBL) to Salmonella enterica serotype B, C1, and D. 12th Avian Immunology Research Group Meeting 28 – 31. August 2012, Edinburgh, UK. Ulrich-Lynge, SL, Dalgaard, TS, Norup, LR, Olsen, JE & Juul-Madsen, HR 2013, 'Colonization Resistance and Immunological Differences in Response to An Experimental Salmonella Infection between Two Chicken Lines Selectively Bred for High or Low Concentration of Mannonse-Binding Lectin: SSI13-1018' Scandinavian Journal of Immunology, vol 77, nr. 4, s. 274-274. W. Peters, H.M. Dong, P. Kumar, V. Arumugaswami, P. Tavlarides-Hontz, M. S. Parcells. 2013. The Effect of Mutations in the Meq Oncoprotein of Marek's Disease Virus (MDV) on Lymphoma Composition. 85th NECAD, Sept. 18, 2013. Wang Y, J. Li, Q. Li, R. Li, X. Hu, N. Li, S. Hu, B. Lupiani, S. Reddy, S. Lamont, H. Zhou. 2013. Effects of Avian Influenza Virus infection on the Transcriptome and the DNA Methylome in Two Genetically Distinct Chicken Lines using Next Generation Sequencing. Plant & Animal Genome XXI, San Diego, CA. Wang, Y., Li, J., Li, Q., Hu, X., Li, N. Hu, S., Brahmakshatriy, V., Lupiani, B., Reddy, S., Lamont, S.J., and Zhou, H. 2013. Effects of avian influenza virus infection on the transcriptome and the DNA methylome in two genetically distinct chicken lines using next generation sequencing. Plant & Animal Genome XXI, January 2013, San Diego, CA Wang, Y., Lupiani, B., Reddy, S., Wang, H., Chen, R., Lamont, S.J., and Zhou, H. 2013. Lung transcriptome following avian influenza virus infection in two genetically distinct chicken inbred lines using RNA-seq. Epigenetics Conference, June 2013, Japan Wattrang E., Kjærup R. M., Norup L.R., Juul-Madsen H.R., and Dalgaard T.S. (2013). Degranulation of chicken cytotoxic T-cells during infectious bronchitis virus infection, 10th International Veterinary Immunology Symposium - IVIS, Milan, Italy Wattrang E., Norup L.R., Juul-Madsen H.R. Dalgaard T.S., Preliminary characterisation of CD107a and CD57 as potential activation markers of chicken cytotoxic T-cells. 12th Avian Immunology Research Group Meeting 28 – 31. August 2012, Edinburgh, UK. 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. Zavelo. A.E., Schmidt, C.J., Rothschild, M.F., Persia, M.E., Lamont, S.J., and Ashwell, C.M. 2013. Major histocompatibility complex diversity in local Ugandan birds. Proc. Poultry Sci. Ann. Mtg. San Diego, CA Zhou, H.J., Wang, Y., Lamont, S.J., and Ross, P. 2013. Re-annotation of chicken genome using RNA-seq data. Proc. Poultry Sci. Ann. Mtg. San Diego, CA

Impact Statements

Date of Annual Report: 04/14/2014

Report Information

Annual Meeting Dates: 10/03/2014 - 10/05/2014
Period the Report Covers: 10/01/2013 - 09/01/2014

Participants

Confirmed Attendance:;
Applegate, Todd;
Beckstead, Robert;
Berghod, Tom;
Berres, Mark;
Brennan, Paul;
Cotter, Paul;
Delany, Mary;
Du, Encun;
Fulton, Janet;
Heidari, Mohammad;
Kaiser , Michael;
Klasing, Kirk;
Kopulos, Renee;
Miller, Marcia;
Muir, William;
Okimoto, Ron;
Parmentier, Henk;
Reed, Willie;
Selvaraj, Ramesh;
Song, Jiuzhou;
Susan Eicher;
Taylor, Robert;
van Ginkel, Fritz;
Wakenell, Patricia;
Yates, Linda;
Zhang, Qian;

Absent:;
Ashwell, Chris;
Bowen-Faulkner, Olivia;
Byrne, Kristen;
Collison, Ellen;
Dalloul, Rami;
Drechsler, Yvonne;
Erf, Gisela;
Gallardo, Rodrigo;
Kaiser, Pete;
Keeler, Calvin;
Koci, Matthew;
Lamont, Susan;
Matukumalli, Lakshmi;
Odemuyiwa, Solomon;
Parcells, Mark;
Pevzner, Igal;
Poston, Rebecca;
Rath, Narayan;
Risdahl Juul-Madsen, Helle;
Rodriguez-Lecompte, Juan
Carlos;
Sharif, Shayan;
van der Poel, Jan;
Zhou, Huaijun

Brief Summary of Minutes

Chair: Dr. Mark Berres, University of Wisconsin-Madison

Secretary: Dr. Ramesh Selvaraj, The Ohio State University

10/04/2014. Saturday

Dr. Willie Reed, Dean, College of Veterinary Medicine, Purdue University, welcomed all the participants to Purdue University at 8 am.

Mr. Paul Brenan, Indiana poultry association, gave an introductory note at 8.15 am.

Dr. Kirk Klasing, UC Davis, gave a presentation at 8.30 am. The research focused on measuring the immune responses of three different lines to heat killed E.coli. Commercial birds produced less IL-1 in serum, had lower body temperature, though T cells produce more IL-1. Work is in progress to identify the mechanism through which commercial broilers decrease the inflammatory immune response against E. Coli. In collaboration with crystal biosciences, a heterozygote heavy chain Ig knock out birds were produced. These birds don’t produce B cells. The repertoire of other immune cells is similar between the wild and knock out birds.

Dr. Kaiser presented the report on behalf of Dr. Sue Lamont, Iowa state university at 9.00 am. The research priority was to develop ideal chicken lines that resist new castle disease virus and handle heat stress in Africa. The goal was to look at RNA expression data in two inbred lines and hyline brown to NDV infected birds. Trachea from Fayoumi line showed less damage and had higher antibody levels post-NDV infection. The second part of the research identified the SNPs associated with NDV resistance.

Dr. Mark Berres, University of Wisconsin-Madison presented the report at 9.30 am. The research characterized the genetic characterization of wild red jungle fowl. The objectives were to assess the extent and distribution of genetic variation in red jungle fowl in Cambodia. A variety of environmental habitats were identified in terms of density of trees, density of birds and other parameters. A total of 212 red jungle fowl were captured. There was a significant diversity among population as well as within population. The average spatial correlation effect was 5 km. A resistance allele to avian influenza virus was present only in red jungle fowl from certain lines.

Dr. Ramesh Selvaraj, The Ohio State University, presented the report at 10.30 am. The role of Regulatory T cells during Salmonella infection was discussed. During the infection with S. Entertidis, there was a significant increase in the number of regulatory T cells by day 4 post-infection that increased steadily throughout the course of the 14 days of infection. The amount of IL-10 mRNA content increased steadily in CD4+CD25+ cells from the S. Entertidis-infected animals over the course of infection

Dr. Ginkel, Auburn university, presented research about host immune response against IBV virus at 11.00 am. Birds were immunized against IBV. Age dependent increase in anti-IBV IgG was observed. Vaccination on day 1 delays significant delay in IgG response. Quality of antibody response was measured by using avidity studies. Anti-IBV IgG, tracheal lymphocyte score, mucosal thickness, and affinity index were lower in birds vaccinated at day 1 than the one vaccinated at 4 week of age.

Dr. Mary Delany, UC Davis, presented data on MDV-chicken genome interactions at 11.30 am. MDV integrates preferably in chromosome 9, 12 and 20. Infection with oncogenic strain targets T cells in spleen for integration into the chromosomes at 14 d of infection. Meq oncogene is absolutely essential for the virus to integrate in the host chromosome. MDV integrates early post infection.

Dr. Pat Wakenell, Purdue University, presented the impact of urban and backyard poultry on commercial operations at 12.05 am. The common diseases in backyard poultry are coccidia, MDV, Mycoplasma gallisepticum, Avian encephalitis, Pox, Parasites, Favus, histomonas and SE. Absence of MD vaccine, organic diets with no amprolium (anticoccidial), no mycoplasma-free-flock certification, mixed species in same locality, unvaccinated flocks, water source breeding mosquitoes, poorly cleaned coops, and poor management are some of the reasons for disease prevalence in these flocks.

Dr. Heidari, USDA presented MDV pathology in different organs at 1.30 pm. B cell follicle in cecal tonsils were destroyed by MDV at 5 d, though the follicle recovered by 21d. Unlike B cell follicle, thymus don’t recover from MDV infection. Macrophages migrate to MDV infected site and correlates to the amount of destruction post MDV infection. Destruction of germinal follicle centers were observed. Cytokine profile of MDV infected birds were studied. IL1, IL10, IL8, IL18, and IFNg mRNA levels were higher in susceptible line than in resistant lines.

Dr. J Song, University of Maryland, presented the role of long intergenic non coding RNAs in MDV infection at 2.00 pm. The basic properties of long intergenic RNAs were identified to have ~1Kb size, have 2.2 number of exons, have conserved sequences, have a mean 5.4 kb distance to neighboring protein genes. 425 unique lincRNAs were identified in resistant and 636 lincRNAs in susceptible lines. linc-satb1 may be involved in MD immune responses by regulating its upstream protein coding gene SATB1.

Dr. Bob Taylor, University of New Hampshire, presented the differences between line 6-3 and 7-2 in Rous Sarcoma virus response at 2.30 pm. Line 72 had higher RSV tumor profile index (TPI) and line J with lower TPI than other congenic lines. The rate of decline of peak antibody against SRBC was faster in line N.

Dr. Robert Beckstead, University of Georgia, presented research about treatments for blackhead disease in turkeys at 3.30 pm. Blackhead disease is caused by Histomaonas meleagridis and causes 30% to 100% mortality. Disease symptoms differ between difference species. An intermediate host is heterakis gallinarum worm. Zinc and copper sulfate has anti-parasitory effects on Histomonas in vitro. In vivo, Zinc and Copper decreased lateral transmission of Histomonas.

Dr. Marcia Miller, City of hope Beckman Research Institute, presented data on identifying the functions of genes in chromosome 16 of chickens at 4.00 pm. Olfactory receptors, scavenger receptor and CHIR-like sequences were assigned to chromosome 16. Many of the genes of OR14J are linked in heritability to MHC diversity. FISH analysis showed linkage of MHC-Y, NOR, SRCR and OR genes. Resequencing of MHC-Y region with Illumina technology validated the earlier data obtained from Sanger sequencing. MHC-Y class I gene function was defined to bind Vitamin metabolites.

Business meeting: 5.00 pm. 10/04/2014

Approval of minutes of 2013 meeting. Dr. Robert Beckstead moved a motion that the minutes be approved. Dr. Mary Delany seconded the motion. The motion passed.
The importance of impact statement was reiterated and it was decided to include all the impact statements from all participants in the final report.
The group welcomed Lakshmi Matukumali as NIFA representative.
The group would like to emphasize the importance of the presence of NIFA representative and or advisor to facilitate flow of ideas between the group members and NIFA. A motion was moved by Dr. Ginkel to authorize Dr. Bob Taylor to draft a letter to NIFA emphasizing the importance of NIFA advisors to attend the annual meeting. Dr. Mary Delany seconded the motion. The motion passed.
The importance of all group members attending annual meeting was discussed.
The venue of next annual meeting was discussed and agreed as follows. 2015: UC Davis. 2016: University of Wisconsin, Madison. 2017: University of Prince Edward Island
Concerns about university approval to international travel for the 2017 annual meeting were raised. A suggestion was put forth that a letter from NIFA administrators to university officials will help the participants to get support.

10/05/2014

Hank Parmentier, Wageningen University, presented research with Natural autoantibodies and transgenerational priming of immunity at 8.30 am. Layer birds, selected for 29 generations for high and low antibody titers, were analyzed for autoantibodies profile in healthy and diseased condition. At 5 week of age there were considerable overlap on the epitopes recognized by IgG and IgM autoantibodies in all three lines, but at 1 year the epitopes recognized diverged between lines. Igm and IgG antibodies showed distinct binding patters regardless of isotype.

Immunoglobulin binding patters are affected by age. Transgenerational epigenetic immunological phenomenon was identified in chickens.

Tom Beghod, Wageningen University, presented research about divergent selection for natural antibodies in poultry at 9.00 am. Heritability of anti-KLH Natural antibodiy titers for total Ig was 0.12. Eggshell color and egg breaking strength influence IgM. High genetic correlation between Ig isotypes (0.86-0.89) were identified.

Renee Kopulus and Linda Yates, Northern Illinois University, presented reserach on behalf of Dr. Briles at 9.30. Several chicken lines that are defined for MHC-B types were discussed. Collaboration on SNP genome mapping of A and C alloantigen systems were discussed. MHC-Y system identification was discussed. MHC haplotype differences in NIU line and Arkansas line were discussed. MHC-Y is highly polymorphic.

Dr. Janet Fulton, Hyline presented data about MHC recombinantion in chickens at 10.35 am. A new nomenclature for naming different haplotypes were proposed. Wild jungle fowl are highly heterogenous. SNP panel shows variability across entire MHC.

Dr. Paul Cotter, Cotter Lab, presented data about assessing stress using hematology at 11.15 am. Drawbacks of using H/L ration to measure stress were discussed. Stress can be defined as absence of tranquil hemogram, presence of normal blood picture with no fungus and absence of coccinocyes, cyanophils, plasmacytes and mott cells.

The group thanked Dr. Pat Wakenell and her team for the hosting the meeting.

The meeting was adjourned at 12.00 pm

Accomplishments

Publications

Impact Statements

Date of Annual Report: 05/01/2016

Report Information

Annual Meeting Dates: 10/09/2015 - 10/11/2015
Period the Report Covers: 10/09/2015 - 10/11/2015

Participants

Technical committee participants
Confirmed technical committee or guest (g) attendance:
Arsenault, Ryan (g)
Beckstead, Robert
Cheng, Hans (g)
Delany, Mary
Dreshler, Yvonne
Erf, Gisela
Fulton, Janet (g)
Gallardo, Rodrigo
Heidari, Mohammad
Kopulos, Renee (g; Briles lab)
Lamont, Susan
Miller, Marcia
Oakley, Brian (g)
Odemuyiwa, Wole
Owen, Jeb
Parcells, Mark
Selvaraj, Ramesh
Schat, Ton (g)
Taylor, Robert
van der Poel, Jan
Frits Ginkle
Wakenell, Patricia
Zhou, Huaijun

PhD Students-Staff-Post doctoral scholars:
Delany Lab: Marla McPherson, Ingrid Youngworth, Justin Smith
Erf Lab: Kristen Byrne
Cheng Lab: Alec Steep
Klasing Lab: Kevin Bolek
van der Poel & Parmentier Labs: Tom Berghof, Mandy Boa
Zhou Lab: Ying Wang,Perot Saelao, Khin Khine Zar Mon, Ganrea T Chanthavixay, Kelly Wilson,
Samantha Fousse
Gallardo Lab: Ruediger Hauck, Ana Paula Da Silvia

Absentees:
Berres, Mark
Collisson, Ellen
Dalloul, Rami
Koci, Matt
Klasing, Kirk
Matukumalli, Lakshmi (USDA-NIFA)
Reiger, Mark (Adm Advisor)
Parmentier, Henk
Risdahl Juul-Madsen, Helle
Rodriguez-Lecompte, Juan
Sharif Shayan
Song, Jiuzhou

Brief Summary of Minutes

2015 NE-1334 Annual Meeting

Genetic bases for resistance and immunity to avian diseases

October 9-10-11, 2015, University of California, Davis CA

Location: 2154 Meyer Hall (Weir Room)

Host: Mary Delany (medelany@ucdavis.edu)

Chair: Ramesh Selvaraj ( selvaraj.7@osu.edu )

Secretary: Robert Beckstead (beckstead.uga@gmail.com )

Registration fee: $50.00 Cash or checks payable to UC Regents (see Justin Smith during meeting)

SCHEDULE (10-5-15)

7:45 Opening Remarks - Mary Delany; Introductions - All

8:00 am Robert Beckstead, University of Georgia

8:30 am Ramesh Selvaraj, The Ohio State University

9:00 am Marla McPherson, UC Davis (Delany Lab)

9:30 am Perot Saelao & Kin Khine Zar Mon, UC Davis (Zhou Lab)

10:00 am BREAK

10:15 am Yvonne Dreshler, Western University of Health Sciences

10:45 am Gisela Erf, University of Arkansas

11:15 am Rodrigo Gallardo, UC Davis

11:45 am: LUNCH – MEXICAN BUFFET

12.30 pm: Mohammed Heidari, USDA

1.00 pm Sue Lamont, Iowa State University

1.30 pm Marcia Miller, City of Hope Beckman Research Institute

2.00 pm Wole Odemuyiwa, Tuskegee University

2:30 pm Mark Parcells, University of Delaware

3:00 pm BREAK

3:15 pm Jeb Owen, Washington State University (new member)

3:30 pm Ryan Arsenault, University of Delaware (guest)

3:45 pm Renee Kopulos, Northern Illinois University (guest)

4:00 pm Business Meeting

6:15 pm DINNER at Café Italia aka Dancing Tomato Café, 1121 Richards Blvd, Davis, CA 95616,

SUNDAY OCTOBER 11

8:15 am Bob Taylor, West Virginia University

8:45 am Tom Berghof, Wageningen University and Research Centre (Parmentier Lab)

9:15 am Mandy Bao, Wageningen University and Research Centre (Van der Poel & Parmentier Labs)

9:45 am Fritz Van ginkle, Auburn University

10:15 am Hans Cheng, USDA-ARS Avian Disease & Oncology Lab (guest); Alec Steep (Cheng Lab)

10:45 am Janet Fulton, Hyline International (guest)

11:15 am Pat Wakenell, Purdue University

11:45 am Open discussion as desired.

12:00 BAG LUNCHES to stay & eat or to pick up & go! END of Day 2

October 10, 2015

NE-1334 business meeting

4:00 Meeting called to order by Ramesh Selvaraj

Member present: Gisela Erf, Jeb Owen, Marcia Miller, Yvonne Dreshler, Sue Lamont, Bob Taylor, Mark Parcells, Mary Delany, Mohammad Heidari, Robert Beckstead, Jan van der Poel, Patricia Wakenell, Hauijun Zhou, Rodrigo Gallardo

4:10 Participants thanked Mary Delany for hosting the meeting

4:15 Motion was made by Patricia Wakenell to make Robert Beckstead the chair. Sue Lamont seconded the motion. The motion carried.

4:25 Motion was made by Hauijun Zhou to make Rodrigo Gallardo secretary. Patricia Wakenell seconded the motion. The motion carried.

4:30 Participants discussed the need for an impact statement to be included in the annual report and the requirement to turn in the report 1 week before the annual meeting.

4:40 There was significant discussion by the member regarding the absence of the NIFA representative Lakshmi Matukumalli and the need for his attendance at the annual meetings.

5:00 The 2014 NE-1334 minutes were corrected to state that Mark Reiger was absent from the meeting. Bob Taylor made a motion to accept the amended 2014 NE-1334 minutes. Robert Beckstead seconded the motion. The motion carried.

5:10 Announcement that next years meeting will be hosted by Mark Berres in Wisonsin.

5:15 Mark Parcells made a motion to accept Ryan Arsenault as a technical member of NE-1334. Mary Delany seconded the motion. The motion passed.

5:30 The meeting was adjourned.

Minutes recorded and prepared by

Robert Beckstead

Secretary NE-1334 2015

Accomplishments

Objective 1. Identify and characterize genes and their relationships to disease resistance in poultry with an emphasis on the major histocompatibility complex, as well as other genes encoding alloantigens, communication molecules and their receptors and other candidate systems.  Huaijun Zhou: Improving food security in Africa by enhancing resistance to Newcastle disease virus and heat stress in chickens: Effects of two stressors (biotic: NDV and abiotic: heat stress) were investigated. Birds of two genetically distinct and highly inbred lines (Fayoumi and Leghorn), and Hy-Line Brown were either exposed to NDV only (Iowa State) or NDV and heat stress (UCD). There were significant differences between Fayoumi and Leghorn birds in virus titers, antibody response to NDV infection and heat stress (Fayoumi is resistant and Leghorn is susceptible to both stressors). Salmonella enterica serovars Enteritidis infection alter the indigenous microbiota diversity in young layer chicks: Salmonella colonization in the gastrointestinal tract of the chickens has a direct effect on altering the natural development of the gastrointestinal microbiota. Significant inverse correlation between Enterobacteriaceae and Lachnospiraceae family in both non-infected and infected groups, suggest possible antagonistic interaction between members of these two taxa, which could potentially influences the overall microbial population in the gut. Our results also revealed that genetic difference between two genetic lines had minimal effect on the establishment of microbiota population. Overall, this study provided preliminary insights into the contributing role of Salmonella Enteritidis in influencing the overall makeup of chicken's gut microbiota. Robert Becksetead: Developed a PCR strategy that allows for the detection of H. gallinarum DNA from different types of samples: PCR results showed that all 3 primer sets were specific for H. gallinarum at an annealing temperature of 70 oC. Since all 3 primer sets were successful in detecting H. gallinarum DNA, a multiplex PCR diagnostic test may be developed to further increase the sensitivity of the primer sets. Developed a dry medium, which would allow storage at warm temperatures over a long period of time. Results showed that this dry medium may be used as an epidemiological tool to obtain H. meleagridis samples from the field. Established an accurate and reliable in vitro procedure to test alternative compounds against H. meleagridis cells. Inclusion of 93.5 ppm Nitarsone reduced mortality by over 60%. The inclusion of 3-nitrophenylboronic acid reduced mortality by almost 40%. Jiuzhou Song Transcriptome analysis revealed activation of major histocompatibility complex 1 and 2 pathways in chicken trachea immunized with infectious laryngotracheitis virus vaccine. The gene ontology analysis showed that genes included in the biological process cluster were related to antigen processing and presentation, positive regulation of immune system processes, T cell selection, and positive regulation of T cell activation. Chicken embryo origin vaccine activation of the major histocompatibility complex 1 and 2 pathways provided insight for evaluation and design of infectious laryngotracheitis vaccines. Methylome Analysis in Chickens Immunized with Infectious Laryngotracheitis Vaccine: Methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) method was employed in the detection of the 1,155 differentially methylated regions (DMRs) across the entire genome.  Mark Berres Distribution of genetic variation of RJF across the Annamite Range in south-central Vietnam was studied. Wild RJF populations are structured genetically at both coarse and fine-scales. Allelic variation is substantial and isolation-by-distance mechanisms appear not to operate at distances greater than 5 Km. These results establish conclusively that although RJF are widely distributed throughout SE and Central Asia, their populations are discontinuous and genetically distinctive. It remains to be determined if wild RJF are susceptible to endogenous threats such as risks to genetic introgression from native domestic chickens. We found no evidence of spatial dependence on two landscape features of landcover and topography. This suggests that the spatial genetic variation in the Red Junglefowl is more related to demography or specific movement characteristics (or both) rather than any dependence on landscape or sampling arrangements. 313 unique haplotypes in 398 chromosomes, none of which matched any known haplotype known from domestic chickens, were identified. The majority of genetic variation was partitioned at the within-individual level with only 0.83% apportioned at the between-population level. Evidence of recombination, including hotspots, and limited linkage disequilibrium among loci, were identified. Compared to domestic chickens, our results suggest extraordinarily high haplotype diversity remains in wild RJF and are consistent with a pattern of balancing selection. We conclude that wild RJF populations in Vietnam represent one of the richest resources of natural genomic variation that could directly help to improve agricultural diversity. Gisela Erf Assessed the melanocyte-specific autoimmune response to feather and embryo-derived melanocytes. Significant infiltration of CD4+ and CD8+ αβTCR+ cells were only observed in vitiliginous SL chickens injected with melanocytes (P<0.05). While ongoing, the results of this study strongly support a role for melanocyte-specific cell-mediated immunity in the pathology of vitiligo in SL chickens. Gene-expression analysis in collected tissues is underway to determine the functional activity of infiltrating leukocytes. Reduced expression of the RNase Dicer in primary melanocytes obtained from vitiligo-prone Smyth line chickens was identified. Dicer protein expression and localization were determined by immunofluorescence and by Western blot using cellular cytoplasmic and nuclear fractions. While the study is ongoing, results obtained thus far show that Dicer is localized in both the cytoplasm and the nucleus of chicken melanocytes corroborating previous studies in RPE cells. Its expression (mRNA and protein levels) was significantly downregulated (P<0.05) in SL compared to BL melanocytes. This is the first evidence that Dicer is dysregulated in SL melanocytes and that this dysregulation seems to occur at the transcription level. Uveitis and blindness in Smyth line chickens with autoimmune vitiligo: expression of cytokine- and melanogenesis-related-genes in eyes before and during loss of choroidal melanocytes were studied. Overall, autoimmune loss of melanocytes in choroids of SL chickens is associated with cytokine profiles similar to those observed in feather target tissue and with altered expression of melanogenesis-related genes.  Rodrigo Gallardo The response of six congenic lines plus a leghorn and a brown commercial line to a challenge with an IBV M41 strain looking for the resistance provided by the MHC was studied. Clinical signs such as respiratory sounds, tracheal lesions, and viral load were measured to determine disease resistance of the different MHC genetic lines. In addition, humoral immune responses were measured. All tested lines inbred and commercial were susceptible to the initial IBV M41 infection. Even though airsacculitis and clinical signs were detected in all the lines, mortality was only associated with groups B17, B19 and white leghorn. Reduced viral load seems (B18 and B21) to be associated with reduction of clinical signs but not with systemic antibody responses Variability Assessment of California Infectious Bronchitis Virus Variants studies identified seven different subpopulations. Sue Lamont Genomics and immunology of host response to avian pathogenic E. coli (APEC) was studied. Host transcriptional response of broilers to infection with avian pathogenic E. coli (APEC), with an overall objective to identify genes, signaling pathways and biological networks associated with infection and resistance to APEC in chickens was studied.  Genomics of host response to Newcastle Disease virus (NDV) was studied. Inbred Fayoumi (relatively resistant) and Leghorn (relatively susceptible) chicks were challenged with LaSota strain NDV. Samples were collected at 2 and 6 dpi to measure viral load (in tears, by qPCR) and at 10 dpi to measure circulating anti-NDV antibody. Birds were euthanized at multiple times (2, 6, 10 dpi) after challenge, and many tissues collected for RNA seq analyses. RNA was isolated from lung, trachea and Harderian glands, and 192 individual libraries generated for sequencing. All libraries have been sequenced and yielded high quality data. Detailed pathway analysis is on-going. J.J. van der Poel (ABG) and H.K. Parmentier (ADP) MHC analysis of parental and 1st generation chickens showed no indication of an MHC effect on Nab titers in lines selected for natural antibodies. .  Ellen Collisson, Maisie Dawes, & Yvonne Drechsler Impact of MHC on IBV associated clinical illness: We have demonstrated that B2 haplotype chickens display greater disease resistance to IBV with faster viral clearance from tissues than B19 haplotypes. We further showed that resistance can be partly attributed to enhanced innate immune responses of B2 chicks, particularly greater macrophage responses induced by IFNgamma or Poly I:C. RNA sequencing has shown that B2 macrophages show greater activation and tighter regulation of genes upon differentiation and activation. These findings suggest that the innate immune response and specifically macrophages have a significant impact on disease resistance and susceptibility of chicken haplotypes and ultimately will help us better understand the genetic basis of enhanced immunity. Mark Parcells In our study of the replication, innate gene expression and immune patterning elicited by rMd5ΔMeq, a recombinant Marek’s disease virus (MDV) having both copies of the meq oncogene deleted, we found MDV replication is sensed via TLR3 and MDA5. At 14 and 21 days post-infection, times typically corresponding to MDV lytic infection and latency, respectively, we have found an interesting difference in IL-12 subunit expression. At 14 days post-infection (dpi), we observed roughly equivalent expression of IL-12p19, p35 and p40 in the spleen total RNA from rMd5ΔMeq-infected chickens. This pattern would suggest roughly equivalent levels of IL-12p70 (IL-12p35 + IL-12p40) and IL-23 (IL-12p19 + IL-12p40). At 21 dpi, however, we observed upregulation of both IL-12p35 and IL-12p40, suggesting that IL-12p70 would be in greater abundance than IL-23. IL-12p70 is essential for TH1 immune patterning, the patterning associated with development of a CTL response. Marcia Miller Within the sequenced MHC-Y BACs there are many YF (MHC class I-like genes), many Ylec (putative c-type lectin-like NK cell receptor genes) and several YLβ (MHC class IIβ gene) sequences. Also present are numerous LINE/CR1 and retroviral LTR repeats. These comprise nearly half the cloned MHC-Y sequence. We are now completing an analysis of the inter-relationship between the MHC-Y genes and the CR1 and LTR sequences. To define the ligands bound by the MHC class I molecules encoded in the MHC- Y region, we are expressing YF class I proteins in E. coli as inclusion bodies. Currently mass spectrometry evidence suggests that YF1*7.1 (refolded tag-free heavy and light chains) binds a glycerophospholipid of microbial origin. We are moving ahead to confirm this observation and with a structural determination to reveal the interactions occurring between the ligand and YF1*7.1. Bob Taylor Antibody response and tumor growth differences among MHC identical populations with different background genes will reveal genes that can improve immune responses in commercial stocks. <ol> <li>Receptor expression, such as the ephrin B2 (EphB2) receptor, influences the bursa of Fabricius microenvironment for B-cell development</li> </ol>  Objective 2. Identify and characterize environmental, dietary and physiological factors that modulate immune system development, optimal immune function and immune system related disease resistance and welfare in poultry genetic stocks. Matt Koci: Typhimurium or S. Enteritidis serovars of Salmonella were able to colonize the cecum of both chicks and mice, and typically with greater colony forming units per gram (cfu/g) in the chick as compared with the mice. Salmonella; however, did not consistently colonize the liver and spleen of the chicks and when it did the cfu/g where 5-log lower than that detected in the cecum. Conversely, Salmonella was consistently recovered from the liver and spleens of the infected mice and at levels similar to that of the mouse cecum. Collectively this suggested that these serovars have a diminished capacity to cross the gut epithelium and invade the chicken host as compared to the mouse. Subsequent in vitro experiments using a series of S. Typhimurium mutants lacking and/or complemented with the various transcriptional regulators of the SPI-1 complex demonstrated that growth at 42°C, down-regulates expression of SPI-1 genes sipC, invF, hilA, and the SPI-1 activator rtsA as compared to expression at 37°C. Overexpression of the hilA activators fur, fliZ, or hilD were not able to overcome the inhibitory effects of growth at 42°C despite having similar levels of protein at both 37 and 42C. In contrast, overexpression of either hilC or rtsA was capable of inducing hilA and sipC at 42°C.  Collectively, these data indicate that poultry physiology results in environmental conditions, such as temperature, that have a profound impact on Salmonella modulating expression of key virulence markers. These findings help explain why Salmonella serovars associated with food borne disease do not result in clinical signs when infecting chickens.  Understanding the different gene expression profile of Salmonella in chickens as compared to mammals will allow for the development of better control and diagnostic tools. Jiuzhou Song Histone modifications in bursa induced by MDV infection at early cytolytic and latency phases were studied. Comprehensive analysis of chromatin signatures, therefore, revealed further clues about the epigenetic effects of MDV infection although further studies are necessary to elucidate the functional implications of the observed variations in histone modifications. Genome-wide mapping of DNase I hypersensitive sites and association analysis with gene expression in MSB1 cells: Results indicated that DNase I HS sites highly correlate with active genes expression in MSB1 cells, suggesting DHSs can be considered as markers to identify the cis-regulatory elements associated with chicken Marek's disease. Results demonstrated that lincRNAs may play an important role in MD resistance and provide a rich resource for hypothesis-driven functional studies.  Mark Berres: The global sPCA in all 212 Red Junglefowl samples resulted in high positive eigenvalues and uniformly low negative eigenvalues. This, together with the overall well-defined sPCA’s regressed gradient variances, illustrated monotonic clines of genetic similarities along the east and the west sides of the Annamite Mountain Range landscape. Specifically, we found evidence of strong local structure within individual sPCA models in the four major sampling. Overall, we found strong population genetic structure at coarse geographic scales and evidence of fine-scale genetic subdivision at distances as low as 5 km. Our results suggest that the current natural populations of Red Junglefowl in Vietnam are relatively small (locally) and contain substantial and genetic variation that differs considerably. Frits Ginkel Effects of early vaccination on memory response: We isolated head-associated lymphoid tissues (HALT) and spleen lymphocytes ~3.5 weeks after vaccination and stimulated them for 6 days in vitro with LPS and measured IBV-specific antibody levels in the culture supernatant. No significant differences in IBV-specific antibody levels for IgA in HALT culture supernatant or IgG in spleen cell culture supernatant were observed. However, when we measured IBV-specific B cells in spleen using FACS analyses significant higher levels of IBV-specific B cells were observed in the spleen of birds vaccinated at 3 weeks of age versus control birds. This increase was not observed in 1 day old vaccinated birds. The IBV-specific B cells frequency in the spleen of 1 day old birds did not differ from control bird cultures. Sue Lamont Interaction of response to inflammatory stimulus and heat stress in chickens was studied. Juan Carlos Rodríguez-Lecompte Proximal promoter region methylation patterns of TLR2b, TLR4, Igβ and MHCII β Chain was studied. We have found that the proximal promoter regions of our genes of interest have different numbers of CpG 2 dinucleotides: TLR2b has 1, TLR4 has 9, Igβ – 13 and MHCII β chain has 24. Furthermore, we have found that while some of these positions remained methylated or unmethylated throughout incubation with folic acid, some of them have changed their status, depending on concentration and time of incubation. Association between FA conc. and percent of proximal promoter methylation of TLR2b,TLR4, Igβ and MHCII β chain was studied. The effect of FA conc. at 4 and 8 hours incubation time on mRNA levels of TLR2b, TLR4, Igβ and MHCII β chain was studied. Effect of incubation time and FA concentration on TLR2b, TLR4, Igβ and MHCII β chain mRNA levels was studied. Effect of LPS on TLR2b, TLR4, Igβ and MHCII β chain mRNA levels was studied. Effect of different levels of vitamin D active form 1,25 (OH)2 D3 on chicken B-Cells associated with avian innate immune responses was studied. Taken together, the results above demonstrate that FA indeed has the capability to affect immune system related traits in the chicken. This immunomodulatory effect of FA under challenging and non-challenging conditions has significant impact. Using nutritional intervention in the form of FA supplementation to modulate the chicken’s immune capabilities is a promising and interesting notion, and it has the potential to affect the nutritional practices of the poultry industry. It is possible that the benefits from FA supplementation would affect not only one but several generations, a significant factor to consider in the chicken breeding industry. J.J. van der Poel and H.K. Parmentier Overall no effects of dietary beta-glucans were observed on performance and specific humoral immune responses. Only 40-50% of the broilers responded with an antibody response to the LPS/ HuSA challenge. When a distinction was made between immunological responders and nonresponders it was consistently found (in all 3 experiments) that beta glucans enhanced the cachectin response in the responder birds only. These results suggest that betaglucans will only influence the immune system in a part of the broiler population. Chicken like mammals have natural auto-antibodies which may be directed to neo-epitopes. IgG and IgM auto-antibodies were studied in 5 High line and 5 Low line families from the new KLH-Nab selection lines. Preliminary data indicated that recognition of auto-antigen fragments for IgG were age dependent but very individually restricted, whereas IgM profiles were less individually restricted. No or little obvious parental-neonatal alikeness was found. Binding to auto-antigens or related (mammalian) ‘auto-antigens’ was found in the NAb selection lines, which showed similar heritabilities and maternal effects as found for Nabs binding KLH (Mandy Bao, submitted). NAAb profiles were also studied for other species (bovine and pig). Deglycosylation of N-linked carbohydrates on KLH strongly reduced the binding of Natural antibodies as well as specific antibodies from KLH-immunized birds, indicating that the majority of circulating antibodies is directed towards carbohydrates. Since carbohydrates are also abundantly present on microorganisms, this may implicate that selection for higher NAb levels will enhance the first line of defense. Ramesh Selvaraj Two experiments were conducted to study Regulatory T cell (Treg [CD4+CD25+]) properties during the establishment of a persistent intestinal infection in broiler chickens. Four-day-old broiler chicks were orally gavaged with 5x106 CFU/mL Salmonella enteritidis or sterile PBS (control). Samples were collected at 4, 7, 10, and 14 d post infection. There was a significant (P < 0.05) increase in the number of CD4+CD25+ cells by day 4 post infection that increased steadily throughout the course of the 14 days of infection, whereas the number of CD4+CD25+ cells in the non-infected controls remained steady throughout the study. CD4+CD25+ cells from cecal tonsils of S. enteritidis-infected birds had a higher (P < 0.05) IL-10 mRNA content than CD4+CD25+ cells from the non-infected controls at all time points studied. The amount of IL-2 mRNA content in the cecal tonsil CD4+CD25- cells from the infected birds did not differ (P > 0.05) when compared to that of non-infected control birds. At a lower effector/responder cell ratio of 0.25:1, CD4+CD25+ cells from cecal tonsils of Salmonella-infected birds suppressed T cell proliferation at days 7 and 14 post S. enteritidis infection, while CD4+CD25+ cells from non-infected control groups did not suppress T cell proliferation. In the second experiment, day-old chickens were orally gavaged with PBS (control) or 1.25 X 108 CFU/bird of S. enteritidis. At 7 and 21 d post Salmonella infection, CD25+ cells collected from cecal tonsils of S. enteritidis-infected birds and restimulated in vitro with Salmonella antigen had higher (P < 0.05) IL-10 mRNA content compared to those in the control group. Spleen CD4+CD25+, CD4+ and CD8+ cell percentage did not differ (P > 0.05) between the Salmonella-infected and control birds. In conclusion, a persistent intestinal S. enteritidis infection increased the Treg percentage, suppressive properties, and IL-10 mRNA amounts in the cecal tonsils of broiler birds. Mark Parcells Over the course of this year, we have developed a modified pathogenesis model for MDV infection. Differential expression of interleukin 12 subunits (IL-12p19, IL-12p35 and IL-12p40) during the shift from Marek’s disease virus (MDV) lytic to latent infection, suggests that virulent, oncogenic strains (Md5) initially induce high levels of IL-23 (IL12p19/IL-12p40) followed by high levels of IL-12p40. The result of this is that IL-12p80, an inhibitor of TH1 patterning predominates, blocking the development of protective CTL responses. Alternatively, rMd5ΔMeq, a non-oncogenic MDV strain that elicits a highly protective vaccine response, elicits roughly equivalent levels of IL-12p70 (IL-12p35 + IL-12p40 heterodimer) and IL-23 at two weeks post-infection, but predominantly higher levels of IL-12p70 by three weeks post-infection. Objective 3. Develop, evaluate and characterize methodologies, reagents and genotypes to assess immune function and disease resistance to enhance production efficiency through genetic selection in poultry. Jiuzhou Song Advantage of the KEGG-PATH model through the functional analysis of the bovine mammary transcriptome during lactation was demonstrated. Gisela Erf: Monitoring tissue responses for the growing feather as an “in vivo test-tube” was conducted. The development of the “in vivo test-tube system” using the growing feathers as a dermal test-site provides an important tool to monitor and evaluate cellular immune system activities in complex tissues and the immunological mechanisms underlying disease susceptibility and resistance in poultry. With this test-system, humoral and cellular, innate and adaptive immune responses can be assessed and monitored in an individual. This ability is unique to the avian system and constitutes important opportunities to evaluate and develop effective prophylactic (e.g. vaccines) and immune modulating (e.g. adjuvants) treatments. Frits Ginkel We generated Ad5 vectors that induced antibody responses that were highly reactive to different IBV serotypes but these IBV-specific immune responses were not associated with great protection upon subsequent challenge with IBV. Therefor this approach needs to be improved to replace existing vaccination protocols Sue Lamont ISU genetic lines were used to assess antibody production after vaccination with a recombinant AI vaccine developed by HarrisVaccines. Ellen Collisson, Maisie Dawes, & Yvonne Drechsler Culture and stimulation of macrophages with poly I:C, IFN and LPS: Broad differences in B2 versus B19 macrophage function was identified in terms of innate and interacting adaptive immunity. While B2 macrophages upon isolation and culture upregulate a large number of genes followed by rapid downregulation of those genes. Evan more dramatic activation occurs following interferon or poly I:C stimulation.. B19 macrophages show a relatively delayed activation and subsequent disorganization of gene regulation. Furthermore, B19 macrophages are not tightly regulated after initial activation upon adhesion, and therefore subsequent stimulation is not effective. In addition, organizing the genes activated into Gene Ontology terms indicate that genes associated with macrophage activation and differentiation, as well as a large number of other biological mechanisms, are differentially regulated in the two haplotypes, also demonstrating differences in disease resistance. Dr. Collisson is also working with Professor Tim Gondwe at the Lilongwe University of Agriculture and Natural Sciences on providing villages in Malawi with hybrid birds with increased disease resistance. Wole Odemuyiwa Early response to double-stranded RNA in domestic chickens Using poly I:C transfected into HD-11 macrophage cell line as a model, we identified genes that are specifically upregulated or downregulated within 24 hours of exposure to dsRNA. We confirmed the pattern of response seen in HD-11 cells by using primary macrophage culture obtained from cells isolated from the bone marrow of chickens. We then investigated the effect of breeds on the activation of the MDA-5 pathway by injecting poly I:C into twelve different breeds of domestic chickens obtained from McMurray farms. We collected whole blood and harvested the spleen to evaluate the expression of the differentially upregulated genes previously identified in macrophage cultures. Our results showed significant breed differences in the expression of genes of innate immunity following activation of the MDA-5 pathway in the domestic chicken. We are now investigating differences in response among commercial lines.

Publications

Refereed Journals <ol> <li>V. A. Meliopoulos, S. A. Marvin, P. Freiden, L. A. Moser, P. Nighot, R. Ali, A. Blikslager, M. Reddivari, R. J. Heath, M. D. Koci, S. Schultz-Cherry. Oral administration of astrovirus capsid protein is sufficient to induce acute diarrhea. mBio. Sumbitted. 2015.</li> <li>A. L. Ballou, R. A. Ali, M. A. Mendoza, J. C. Ellis, H. M. Hassan, W. J. Croom, and M. D. Koci. Development of the chick microbiome: How early exposure influences future microbial diversity. Frontiers Veterinary Science. Submitted. 2015.</li> <li>B. Troxell, N. Petri, C. Daron, R. Pereira, M. Mendoza, H. M. Hassan, M. D. Koci. The body temperature of poultry contributes to the control of systemic invasion through reducing expression of SPI-1 genes in Salmonella enterica serovars Typhimurium and Enteritidis. Applied and Environmental Microbiology. In Press. 2015.</li> <li>Kim, T.H, H. Zhou. 2015. Functional Analysis of Chicken IRF7 in Response to dsRNA Analog Poly(I:C) by Integrating Overexpression and Knockdown: 10.1371/journal.pone.0133450</li> <li>Schmid M. et al. H. Zhou. 2015.Third Report on Chicken Genes and Chromosomes Cytogenet Genome Res 145:78-179 (DOI:10.1159/000430927)</li> <li>Leif, A. et al., The FAANG Consortium. 2015. Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project. Genome Biology 16:57. DOI: 10.1186/s13059-015-0622-4</li> <li>Shi S, Shen Y, Zhao Z, Hou Z, Yang Y, Zhou H, Zou J, Guo Y. 2014. Integrative analysis of transcriptomic and metabolomic profiling of ascites syndrome in broiler chickens induced by low temperature. Mol Biosyst. 10(11):2984-93. doi: 10.1039/c4mb00360h.</li> <li>&dagger;Luo, J., Carrillo, J.A., Menendez, K.R., Tablante, N.L., Zhao, K., and *Song, J. 2014 Transcriptome analysis reveals an activation of MHC-I and MHC-II pathways in chicken trachea immunized with infectious laryngotracheitis virus vaccine. Poultry Science. 10.3382/ps.2013-03624.</li> <li>Shang, S., Xie, Q., Duan, J.R., Ernst, C., Fulton, J.E., O’Sullivan, N., Song, J. Zhang, H. 2014. Host genetic resistance to Marek’s disease sustains HVT protective efficacy comparable to CVI988/Rispens in both experimental and commercial lines of chickens. Vaccine. 02/2014; DOI:10.1016/j.vaccine.2014.01.092.</li> <li>Du, J.l., Yuan, Z.F., Ma, Z.W., Song, J., Xie, X.L., Chen, Y.L. 2014. KEGG-PCA: Kyoto Encyclopedia of Genes and Genomes-based pathway correlation analysis using Principal Component Analysis method. Molecular BioSystem, 2014, DOI: 10.1039/C4MB00287C, Paper.</li> <li>&dagger;Mitra, A., Luo, J., He, Y., Gu, Y., Zhang, H. Zhao, K., Cui, K. and *Song, J. 2015 Histone modifications induced by MDV infection at early cytolytic and latency phases. BMC Genomics 13: 557. DOI 10.1186/s12864-015-1492-6.</li> <li>José A. C., He, Y., Luo, J., Menendez, K.R., Tablante, T. L., Zhao, K., Paulson , J.N., Li, B., and *Song, J. 2015 Methylome Analysis in Chickens Immunized with Infectious</li> <li>He Y., Ding, Y., Zhan, F., Zhang, H., Hu, G., Zhao, K., Han, B., Yang, N., Mao, L., *Song, J., 2015. The conservation and signatures of lincRNAs in Marek's disease of chicken. Scientific Reports 5, 15184; doi: 10.1038/srep15184</li> <li>Lian, L., Li, X., Zhao, C., Han, B., Qu, L., Song, J., Liu, C., and Yang, N. Chicken gga-miR-181a targets MYBL1 and shows an inhibitory effect on proliferation of Marek’s disease virus-transformed lymphoid cell line Poultry Science. (Accepted)</li> <li>He Y., José A. C, Luo, J., Ding, Y., Tian, F., *Song, J., 2014. Genome-wide mapping of DNase I hypersensitive sites and association analysis with gene expression in MSB1 cells. Front. Genet. | doi: 10.3389/fgene.2014.00308.</li> <li>Galvan,S.C., Carranc, A.G., Song J., and Félix RT., Epigenetics and animal virus infections. Front. Genet., 04 March 2015 | doi: 10.3389/fgene.2015.00048.</li> <li>Ma, M., Lin,R., Carrillo, J., Bhutani, M., Pathak, A., Ren, H., Li,Y., Song, J., Mao, L. ΔDNMT3B4-del Contributes to Aberrant DNA Methylation Patterns in Lung Tumorigenesis. EBioMedicine (2015), doi: 10.1016/j.ebiom.2015.09.002</li> <li>Nguyen-Phuc, Hoa, Fulton, Janet E., and M. E. Berres. 2015. Genetic variation of Major Histocompatibility Complex (MHC) in wild Red JungleFowl (Gallus gallus). Submitted to Poultry Science.</li> <li>Fulton JE, Lund AR, McCarron AM, Pinegar K, Korver D, Classen H, Aggrey S, Utterbach C,Anthony NB, and M. E. Berres. 2015. MHC Variability in Heritage chicken breeds. Accepted in Poultry Science.</li> <li>Nguyen-Phuc, Hoa and M. E. Berres. 2015. Spatial genetic structure of wild Red Junglefowl (Gallus gallus) in South Central Vietnam. Submitted to Molecular Ecology.</li> <li>Nguyen-Phuc, Hoa and M. E. Berres. 2015. Spatial dependence models and correlation of neutral genetic variation in wild Red Junglefowl (Gallus gallus). Submitted to Landscape Ecology.</li> <li>Byrne, K. A.*, D. M. Falcon*, and G. F. Erf. 2015. Novel approach to simultaneously monitor local and systemic in vivo effects of varying doses of intradermally injected LPS. J. Immunol. 193:146.11.</li> <li>Falcon, D. M.*, R. L. Dienglewicz, K. A. Byrne*, and G. F. Erf. 2015. Ex vivo killing of primary melanocytes by syngeneic mononuclear cells from active, autoimmune vitiligo lesions in Smyth chickens. J. Immunol. 193:115.12.</li> <li>Lyle, C. S.*, K. A. Byrne*, D. M. Falcon*, R. L. Dienglewicz*, HM. Jang, Z. Aguilar, and G. F. Erf. 2015. Immunostimulatory activity of indium phosphide quantum dots in vitro and in vivo. J. Immunol. 193:73.13 (in press).</li> <li>Erf, G. F., O. Alaamri*, H. Jang, K. A. Byrne*, D. M. Falcon*, Z. Aguilar, and R. L. Dienglewicz. 2015. Simultaneous monitoring of in vivo humoral and cellular immune responses in the avian model. J. Immunol. 193:73.8.</li> <li>Erf, G. F., HM Jang, K. A. Byrne*, O Alaamri*, C. S. Lyle*, D. M. Falcon*, and R.L. Dienglewicz. 2015. Differences in cellular and humoral primary and secondary immune responses to protein antigen administered with or without adjuvant. Poult. Sci. (E. Suppl. 1) 94: (in press).</li> <li>Byrne, K. A.* and G. F. Erf. 2015 Tissue and blood responses to peptidoglycan injection into chicken dermal tissue. Poult. Sci. (E. Suppl. 1) 94: (in press).</li> <li>Huett, W.*, Byrne, K. A. *, Sorrick, J.*, and G. F. Erf. 2015. Uveitis and blindness in Smyth line chickens with autoimmune vitiligo: expression of cytokine- and melanogenesis-related-genes in eyes before and during loss of choroidal melanocytes. Pigment Cell & Melanoma Res. 28 (in press).</li> <li>Falcon, D. M.*, R. L. Dienglewicz, and G. F. Erf. 2015. Monitoring of leukocyte infiltration responses to melanocytes injected into growing feathers of Smyth line chickens with autoimmune vitiligo. Pigment Cell & Melanoma Res. 28 (in press).</li> <li>Falcon, D. M.*, G. F. Erf and S. Dridi. 2015. Reduced expression of the RNase Dicer in primary melanocytes obtained from vitiligo-prone Smyth line chickens. Pigment Cell & Melanoma Res. 28 (in press).</li> <li>Gallardo R.A., Carrasco-Medanic R., Zhou H., Lyu S., Wang Y., Woolcock P.R., Hoerr F.J. Effects of challenge with very virulent infectious bursal disease virus reassortants in commercial chickens. Avian Dis, 58(4): 579-86.</li> <li>Gallardo R.A., Aleuy O.A., M. Pitesky, G. Senties-Cue, A. Abdelnabi, P.R. Woolcock, M. R. Hauck, H. Toro. Variability Assessment of California Infectious Bronchitis Virus Variants. Avian Dis. Submitted for publication.</li> <li>van Ginkel, F.W., J. Padgett, G. Martinez-Romero, M.S. Miller, K. Joiner, S.L. Gulley. 2015. Age-dependent immune responses and immune protection after avian coronavirus vaccination. Vaccine 33: 2655-2661.</li> <li>Sun, H., Liu, P., Nolan, L.K., and Lamont, S.J. 2015. Avian pathogenic Escherichia coli (APEC) infection alters bone marrow transcriptome in chickens. BMC Genomics 16:690 DOI 10.1186/s12864-015-1850-4.</li> <li>Walugembe, M., Hsieh, J.C.F., Koszewski, N.J., Lamont, S.J., Rothschild, M.F., and Persia, M.E. 2015. Effects of dietary fiber on cecal short fatty acid and cecal microbiota of broiler and laying hen chicks. Poultry Sci. 94:2351–2359.</li> <li>Kim, D. K., Lillehoj, H.S., Jang, S.I., Lee, S.H., Hong, Y.H., and Lamont, S.J. 2015. Genetically disparate Fayoumi chicken lines show different response to avian necrotic enteritis. J. Poultry Sci. doi.org/10.2141/jpsa.0140203.</li> <li>Schmid, M., Smith, J., Burt, D.W., Aken, B.L., Antin, P.B. et al. 2015. Third Report on Chicken Genes and Chromosomes 2015. Cytogenet Genome Res 145: 78-179.</li> <li>Yitbarek A, Echeverry H, Munyaka P, and Rodriguez-Lecompte JC, 2015. Innate immune responses in chicken pullets fed probiotic and symbiotic. Poult. Sci. 94:1802-1811.</li> <li>Waititu SM, Yitbarek A, Matini E, Echeverry H, Kiarie E, Rodriguez-Lecompte JC,. Nyachoti CM.            2014. Effect of supplementing direct-fed microbials on broiler performance, nutrient digestibilities, and immune responses. Poult. Sci. 93:625-635</li> <li>Jing M, Munyaka P, Tactacan G, Rodriguez-Lecompte, JC, O K, and House JD. 2014. Performance,            serum biochemical responses and gene expression of intestinal folate transporters of young and older laying hens in response to dietary folic acid supplementation and challenge with Escherichia coli lipopolysaccharide. Poult. Sci. 93:122-131</li> <li>A. van Knegsel, H. M. Hammon, U. Bernabucci, G. Bertoni, R.M. Bruckmaier, R. M. A. Goselink, J. J.Gross, B. Kuhla, C. C. Metges, H. K. Parmentier, E. Trevisi, A. Tröscher, A. M. van Vuuren. Metabolicadaptation during early lactation: key to cow health, longevity and a sustainable dairy production chain.: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resourches. CAB Review 9: doi:10.1079/PAVSNNR20149002, 2014.</li> <li>F. Molist, E. van Eerden, H. K. Parmentier, J. Vuorenmaa. Effects of inclusion of hydrolyzed yeast on the immune response and performance of piglets after weaning. Animal Feed Science and Technology 195:136-141, 2014.</li> <li>H. K. Parmentier, E. Harms, A. Lammers, M. G. B. Nieuwland. Age and genetic selection affect autoimmune profiles of chickens. Dev. Comp. Immunol. 47: 205-214, 2014.</li> <li>J. O. Khobondo, M. G. B. Nieuwland, L. E. Webb, E. A. M. Bokkers, H. K. Parmentier. Natural (auto)antibodies in calves are affected by age and diet. Veterinary Quarterly 35: 64-75, 2015.</li> <li>D. B. de Koning, E. P. W. C. Damen, M. G. B. Nieuwland, E. M. van Grevenhof, W. Hazeleger, B.Kemp, H. K. Parmentier. Association of natural (auto-) antibodies at an early age with osteochondrosis at slaughter in growing gilts. Livestock Science 176: 152-160, 2015.</li> <li>T. V. L. Berghof, S. A. S. van der Klein, J. A. J. Arts, H. K. Parmentier, J. J. van der Poel, H. Bovenhuis. Genetic and non-genetic inheritance of natural antibodies binding keyhole limpet hemocyanin in a purebred chicken line. Plos One, e0131088, 2015.</li> <li>A. Koppenol, E. Delezie, H. K. Parmentier, J. Buyse, N. Everaert. Limited evidence for transgenerational effects of maternal dietary supplementation with omega-3 fatty acids on immunity in broiler.Veterinary Journal 203: 244-249, 2015.</li> <li>N. Mayasari, G. de Vries Reilingh, M. G. B. Nieuwland, G. J. Remmelink, H. K. Parmentier, B. Kemp,A. T. M. van Knegsel. Effect of maternal dry period length on colostrum immunoglobulin content, natural and specific antibodies titers and development of calves. J. Dairy Science 98: 3969-3979, 2015.</li> <li>S. A. S. van der Klein, T. V. L. Berghof, J. A. J. Arts, H. K. Parmentier, J. J. van der Poel, H. Bovenhuis. Genetic relations between natural antibodies binding keyhole limpet hemocyanin and production traits in a purebred layer chicken line. Poultry Science 94: 875-882, 2015.</li> <li>E. Wondmeneh, E. H. van der Waaij, H. K. Parmentier, B.J. Ducro, J. A. M. van Arendonk. High natural antibody titers of indigenous chickens are related with increased hazard in confinement. Poultry Science 94: 1493-1498, 2015.</li> <li>B. de Klerk, B. Ducro, H. Heuven, I. den Uijl, J. van Arendonk, H. K. Parmentier, J. J. van der Poel. Phenotypic and genetic relationships of natural antibodies binding keyhole limpet hemocyanin in bovine plasma and milk. J. Dairy Science 98: 2746-2752, 2015.</li> <li>M. Bao, H. Bovenhuis, M. G. B. Nieuwland, H. K. Parmentier, J. J. van der Poel. Genetic parameters of IgM and IgG antibodies binding autoantigens in healthy chickens. In press, Poultry Sci. 2015.</li> <li>K Simon, De Vries Reilingh G and Lammers A. 2014. Development of ileal cytokine and immunoglobulin expression levels in response to early feeding in broilers and layers. Poultry Science. In press.</li> <li>Banat G. R., Tkalcic S., Dzielawa J. A., Jackwood M. W., Saggese M. D., Yates L., Kopulos R.,Briles W. E., Collisson E. W. Association of the chicken MHC B haplotypes with resistance to avian coronavirus. Dev. Comp. Immunol. 2013;39:430-437.</li> <li>Dawes M.E., Griggs L.M., Collisson E.W., Briles W.E., Drechsler Y. Dramatic differences in the response of macrophages from B2 and B19 MHC-defined haplotypes to interferon gamma and polyinosinic:polycytidylic acid stimulation. Poultry Science 2014 Vol 93: 830-838</li> <li>Miller MM and Taylor, Jr.RL. Brief Review of the Chicken Major Histocompatibility Complex – the Genes, their Distribution on Chromosome 16 and their Contributions to Disease Resistance. Submitted.</li> <li>Fulton JE, McCarron AE, Lund AR, Pinegar K, Wolc A, Chazara O, Bed’Hom B, Berres ME, Miller MM. A high density SNP panel reveals extensive diversity, frequent recombination and multiple recombination hotspots within the chicken major histocompatibility complex B region between BG2 to CD1A1. To be submitted in October 2015.</li> <li>Miller, MM, Goto, RM, McPherson, M, Delgado MH, Dalton J, Warden C, Wu X, Hosomichi K, Delany ME, and Shiina T. FISH analysis and sequencing of the red jungle fowl MHC-Y region reveals multiple compartmentalized clusters of MHC class I-like, MHC class IIβ, c-type-lectin-like genes interspersed with repetitive elements. To be submitted in December 2015</li> <li>Miller, MM, Goto, RM, Gugui, G., Bjorkman P. Nature of ligands bound byYF1*7.1. To be submitted in August 2016.</li> <li>Taylor, R. L., Jr., J. L. Anderson, and S. C. Smith, 2014. Commentary on: Atherosclerosis-susceptible and atherosclerosis-resistant pigeon aortic cells express different genes in vivo. <a href="http://www.athero.org/commentaries/comm1188.asp">http://www.athero.org/commentaries/comm1188.asp</a></li> <li>Taylor, R. L., Jr. 2015. Letter to the Editor – An incomplete story told by a single number. Poult. Sci. 94:1995-1996 doi:10.3382/ps/pev221</li> <li>Taylor, R. L., Jr., Z. Medarova, and W. E. Briles. Immune effects of chicken non-Mhc alloantigens. Poult. Sci. 95: in press doi:10.3382/ps/pev331 (review)</li> <li>Taylor, R. L., Jr. The future of poultry science research: Challenges as opportunities. AMENA, Asociación Mexicana de Especialistas en Nutrición Animal, Puerta Vallarta, Mexico in press</li> <li>CAST (Long., J. H. Blackburn, A. Martin, F. Silversides, R. L. Taylor, Jr., and C. Youngs). The need for agricultural innovation to sustainably feed the world by 2050: Protecting food animal gene pools for future generations. in press</li> <li>Miller, M. M., and R. L. Taylor, Jr. Brief review of the chicken major histocompatibility complex – the genes, their distribution on chromosome 16 and their contribution to disease resistance. (review)</li> <li>Weathers, B., S. L. Branton, R. Jacob, E. D. Peebles, R. L. Taylor, Jr., and G. T. Pharr. Expression of the ephrin receptor B2 in the embryonic chicken bursa of Fabricius.</li> </ol>   Book Chapters <ol start="2016"> <li>D. Koci and S. Schultz-Cherry. “Astrovirus”. Food Microbiology Series: Laboratory Models for Foodborne Infections. Ed. D. Liu. Springer Science. New York. 2016.</li> </ol> Presentations <ol> <li>Koci, M. Intestinal immunology and the role of enteric viruses in intestinal health. Zoetis Intestinal Health Seminar. North Carolina State University (NCSU), June 2015.</li> <li>Koci, M No Theses Without the Feces. RTP180. Research Triangle Park, NC. June 2015.</li> <li>Koci, M Resistance, Immunity and Vaccines: Lessons from our Feathered Friends. Triangle Immunology and Virology Interest Group. Research Triangle Park, NC. February 2015.</li> <li>Koci, M Innovation in Action: Novel Research in Agriculture and Life Sciences. Stewards of the Future: the Future of Foods. Raleigh, NC. November 2014.</li> <li>Koci, M The Future With or Without Anitbiotics. 41st Poultry Nutrition Conference. Raleigh NC.</li> <li>Saelao, P., Y. Wang, D,A, Bunn, R. Gallardo, S.J. Lamont. Zhou, H. 2015. Transcriptome Response of Two Distinct Highly Inbred Lines to Combined Stressors of Newcastle Disease Virus and Heat. International Symposium on Animal Functional Genomics, Piacenza, Italy.</li> <li>Herrmann, M. S. H. Zhou, R. Gallardo, D. Bunn, S. Lamont. 2015. Differential response of resistant and susceptible chicken lines to a lentogenic Newcastle Disease Virus strain. Global Alliance for Research on Avian Diseases (GARAD) Conference, UK.</li> <li>Rowland, K., H. Zhou, R. Gallardo, D. Bunn, S. Lamont. 2015. Identifying genes and genetic markers associated with NDV resistance in chickens. Poultry Breeders Roundtable meeting, St Louis, MO.</li> <li>Zhou, H., D,A, Bunn, R. Gallardo, S. J. Lamont, J. C.M. Dekkers, A. Muhairwa, P. Msoffe, B. Kayang, A. Naazie, G. Aning, C. J. Schmidt. 2015. Improving Food Security in Africa By Enhancing Resistance to Newcastle Disease and Heat Stress in Chickens: Genomics to Improve Poultry. Plant & Animal Genome XXIII, San Diego, CA.</li> <li>Y. Wang, P. Saelao, D,A, Bunn, R. Gallardo, S.J. Lamont. Zhou, H. 2015. Transcriptome Analysis of Genetic Resistance to Heat Stress in Two Genetically Distinct Chicken Inbred Lines. Plant & Animal Genome XXIII, San Diego, CA.</li> <li>Saelao, P. H. Zhou. 2015. Tissue-Specific Transcriptome Regulation through Histone Modifications in Chickens. Plant & Animal Genome XXIII, San Diego, CA. November 2014.</li> <li>Giuffra , E., S. Foissac ,O. Madsen ,M. A.M. Groenen , R. Crooijmans , P. J. Ross , I. Korf ,H. Zhou. 2015. The Functional Annotation of Animal Genomes (FAANG) Initiative. Plant & Animal Genome XXIII, San Diego, CA.</li> <li>Zhou H., Wang Y, N. Huefner, Lupiani B, Reddy SM, Lamont SJ. 2015. Systems Biology Analysis of Mechanism of Host Response to Avian Influenza Virus Infection in Two Genetically Distinct Chicken Inbred Lines. Plant & Animal Genome XXIII, San Diego, CA.</li> <li>Kim, T.H, Y. Wang, Z. Zhao, S. J. Lamont, H. Zhou. 2015. RNA-Seq Based Genome-Wide Analysis of Genomic Imprinting in Chicken Lungs. Plant & Animal Genome XXIII, San Diego, CA.</li> <li>Saelao, P., Y. Wang, A. Nazmi, D,A, Bunn, R. Gallardo, S.J. Lamont. Zhou, H. 2015. Transcriptional Analysis of Resistance to Newcastle Disease Virus Infection in Two Genetically Distinct Inbred Chicken Line. Plant & Animal Genome XXIII, San Diego, CA.</li> <li>Gallardo R.A., M. Pitesky, B. Crossley. 2014. Understanding Variability of California Infectious Bronchitis Viral Strains. American Association of Avian Pathologists Annual Meeting. Denver, CO.</li> <li>Gallardo R.A., H. Zhou, Y. Wang, K. Smith. 2015. Major Histocompatibility Complex and Genetic Resistance Towards Infectious Bronchitis Virus. American Association of Avian Pathologists Annual Meeting. Boston, MA.</li> <li>C. Breedlove, A.M. Ghetas, S. Gulley, F.W. van Ginkel, K. Joiner, V.L. van Santen, H. Toro. Effects of Supplemental Fermentation Product of S. cerevisiae in Chicken Diets on Resistance against Infectious Bronchitis Virus. Southern Conference on Avian Diseases, Atlanta, GA, January 2015.</li> <li>Miller, S.L. Gulley, and F.W. van Ginkel. Effects of vaccination on IBV-specific antibody production and avidity in chickens. Phi Zeta Research Emphasis Day. November 5, 2014, Auburn AL.</li> <li>Elad O, Rodríguez-Lecompte JC, Sharif S, McKenna P. 2014. Effect of Folic Acid on the Expression of TLR2b, TLR4 and Igβ Genes in Chicken B cells, with or Without LPS Challenge. Avian Immunology Research Group (AIRG) meeting 2014, University of Guelph, ON, Canada July 16-19 2014 (Poster)</li> <li>Rodríguez-Lecompte JC, Elad O, Yitbarek A, Sharif S, McKenna P. 2014. Effect of different levels of vitamin D active form 1,25 (OH)2 D3 on chicken B-Cells associated with avian innate immune responses. Avian Immunology Research Group (AIRG) meeting 2014, University of Guelph, ON, Canada July 16-19 2014 (Poster)</li> <li>Alizadeh M, Rodriguez-Lecompte JC, Slominski BA. 2014. The effect of different formulation of yeast-derived products on growth performance, innate and humoral immunity in broiler chickens. The International Scientific Conference on Probiotics and Prebiotics – IPC2014, 24–26th June 2014, Budapest, Hungary (Oral presentation).</li> <li>Rodríguez-Lecompte JC, Echeverry H, Yitbarek A, Parada M. 2014. Innate immune response of chicken macrophages to Escherichia coli derived-LPS and Propionibacterium acnes and granulosum challenge. 63rd Western Poultry Conference & XXXIX ANECA Joint Annual Meeting, Puerto Vallarta, Jalisco, Mexico April 2-5, 2014 (Oral presentation)</li> <li>Drechsler Y: International Anatomy & Pathology meeting, Zagreb, Croatia, 2014. The role of CD8 T lymphocyte in virally associated protective immunity.</li> <li>Drechsler Y: GARAD. London, UK 2015: RNA Sequencing Elucidates Large-Scale Temporal Dysregulation of Gene Expression in B19 versus B2 Haplotype Activated Macrophages WVPA, Capetwon, South Africa 2015.</li> <li>Drechsler Y and Collisson EW: RNA sequencing shows significantly different gene expression of macrophages in B2 compared to B19 haplotype chickens</li> <li>Collisson EW, Griggs LM, Drechsler Y: Macrophages are Key Players in B Haplotype Associated Enhanced Adaptive Immunity to Avian Influenza Virus.</li> <li>Platform Report at NC-1170 - Advanced Technologies for the Genetic Improvement of Poultry and the NSRP-8, National Animal Genome Research Program held at XXII Plant and Animal Genome Meeting, San Diego, CA, January 10, 2015</li> </ol>   Abstracts <ol> <li>V. A. Meliopoulos, S. A. Marvin, B. Sharp, P. Freiden, L. A. Moser, P. Nighot, R. Ali, A. Blikslager, M. Reddivari, R. J. Heath, M. D. Koci, and S. Schultz-Cherry. The Astrovirus Capsid Protein: A Novel Viral Enterotoxin That Increases Intestinal Barrier Permeability And Sodium Malabsorption In A Small Animal Model. July 2015. 34th Annual Meeting of the American Society for Virology. London ON, Canada.</li> <li>B. Troxell, R. Ali, M. Mendoza, H. M. Hassan, and M. D. Koci. Growth at the Body Temperature of Poultry Inhibits the Function of the HilD Protein and Reduces Expression of SPI-1 Genes within Salmonella enterica serovar Typhimurium. May 2015. 115th General Meeting of the American Society for Microbiology. New Orleans, LA.</li> <li>A. L. Ballou and M. D. Koci. Impact of Gut Microbiome Modulation on Gut Inflammation. March 2015. Experimental Biology 2015. Boston, MA.</li> <li>A. L. Ballou, R. A. Ali, J. Croom, M. D. Koci. Where do probiotics live and work? January 2015. 2015 International Poultry Scientific Forum. Atlanta, GA.</li> <li>Zhang, H., Xie, Q.M., Chang, S., Ernst, C.W. Black-Pykosz, A., He, Y., *Song J. 2014. Differential expression profiling of miRNAs between a Marek’s disease resistant and a susceptible line of chickens by deep sequencing. The 10th International Symposium on Marek’s Disease and Avian Herpes viruses. East Lansing, Michigan.</li> <li>He, Y., Luo, J., Ding, Y., Zhang, H., Cheng, H., and *Song, J. 2014. LINCRNA Identification of Marek’s Disease in CD4+ T cells. The 10th International Symposium on Marek’s Disease and Avian Herpes viruses. East Lansing, Michigan.</li> <li>He, Y., Luo, J., Ding, Y., Zhang, H., Tian, F., and *Song, J. 2014 Epigenetics pattern and Host-virus interaction. International Society Animal Genetics 2014 XiAn, China.</li> <li>Ding, Y., He, YH., Carrillo, J., Zhang, HM., and *Song, J. Transcriptomic signatures of Marek’s disease in immune organs. Poultry Science Association Annual Meeting. Louisville, Kentucky, United States. July 27-30, 2015</li> <li>Han, B , He, YH. Ding, Y. Zhang, L. Yang, N. and *Song, J. 2015 Identification of LincRNAs and their modeling of knockdown systems associated with chicken Marek’s disease. Poultry Science Association Annual Meeting. Louisville, Kentucky, United States. July 27-30, 2015</li> <li>He, YH, Zhang, HM. and *Song, J. 2015 Differential transcriptome analysis of CD4+ T cells of chickens induced by Marek’s disease virus challenge. International Plant & Animal Genome XXIII. San Diego, CA, USA. January 10-14, 2015</li> <li>He,YH, Zhang, HM. Taylor, RL. and *Song, J. 2015 DNA methylation patterns associated with the resistance of Marek's disease. Poultry Science Association Annual Meeting. Louisville, Kentucky, United States. July 27-30, 2015</li> <li>Herrmann, M.S., Gallardo, R., Bunn, D.A., Zhou, H., and Lamont, S.J. 2015. Differential response of resistant and susceptible chicken lines to a Newcastle Disease Virus vaccine strain. Proc. International Symposium on Vaccines Against Antigenically Variable Viruses (VAAVV), November 5-8, 2015; Ames, IA.</li> <li>Herrmann, M.S., Gallardo, R., Bunn, D.A., Zhou, H., and Lamont, S.J. 2015. Differential response of resistant and susceptible chicken lines to a lentogenic Newcastle Disease Virus strain. Proc. Global Alliance for Research on Avian Diseases, June 28-July 1, 2015; London, England.</li> <li>Herrmann, M.S., Gallardo, R., Bunn, D.A., Zhou, H., and Lamont, S.J. 2015. Differential response of resistant and susceptible chicken lines to a lentogenic Newcastle Disease Virus strain. Proc. National Breeders Roundtable, May 7-8, 2015; St. Louis, MO.</li> <li>Herrmann, M.S., Gallardo, R., Bunn, D.A., Zhou, H., and Lamont, S.J. 2015. Differential response of resistant and susceptible chicken lines to a lentogenic Newcastle Disease Virus strain. Proc.2nd Graduate and Professional Research Conference, April 2, 2015; Ames, IA.</li> <li>Rowland, K., Zhou, H., Gallardo, R., Bunn, D., Lamont, S. 2015. Identifying genes and genetic markers for NDV resistance in commercial layer chickens. Proc. 9th European Symposium on Poultry Genetics, June 16-18, Tuusula, Finland.</li> <li>Rowland, K., Zhou, H., Gallardo, R., Bunn, D., Lamont, S. 2015. Identifying genes and genetic markers associated with NDV resistance in chickens. Proc. Poultry Breeders Roundtable, May 7-8, St. Louis, MO.</li> <li>Rowland, K., Zhou, H., Gallardo, R., Bunn, D., Lamont, S. 2015. Identifying host genes and genetic markers for antibody production to Newcastle Disease Virus (NDV) vaccine strain in commercial layer chickens. Proc. Vaccines Against Antigenically Variable Viruses, November 5-8, Ames, IA.</li> <li>Casebere, K., Kaiser, M., and Lamont, S. 2015. Bacterial component induced inflammatory response in roosters from diverse genetic lines. Animal Science Leaflet R2998, Iowa State University, Ames IA USA</li> <li>Herrmann, M.S., Gallardo, R., Bunn, D.A., Zhou, H., and Lamont, S.J. 2015. Differential response of resistant and susceptible chicken lines to a Newcastle Disease Virus vaccine strain. Proc. International Symposium on Vaccines Against Antigenically Variable Viruses (VAAVV), November 5-8, 2015; Ames, IA.</li> <li>Herrmann, M.S., Gallardo, R., Bunn, D.A., Zhou, H., and Lamont, S.J. 2015. Differential response of resistant and susceptible chicken lines to a lentogenic Newcastle Disease Virus strain. Proc. Global Alliance for Research on Avian Diseases, June 28-July 1, 2015; London, England.</li> <li>Herrmann, M.S., Gallardo, R., Bunn, D.A., Zhou, H., and Lamont, S.J. 2015. Differential response of resistant and susceptible chicken lines to a lentogenic Newcastle Disease Virus strain. Proc. National Breeders Roundtable, May 7-8, 2015; St. Louis, MO.</li> <li>Herrmann, M.S., Gallardo, R., Bunn, D.A., Zhou, H., and Lamont, S.J. 2015. Differential response of resistant and susceptible chicken lines to a lentogenic Newcastle Disease Virus strain. Proc.2nd Graduate and Professional Research Conference, April 2, 2015; Ames, IA.</li> <li>Hsieh J.C., Jernigan R.L., Lamont S.J. 2015. The Structure of Newcastle Disease Virus Fusion Protein Bound to Chicken Protein Disulfide Isomerase A3 Suggests a Molecular Target for New Therapies. Proc. National Breeders Roundtable; 2015 May 7-8; St. Louis</li> <li>MO. Hsieh J.C., Walugembe M., Koszewski N.J., Lamont S.J., Persia M.E., Rothschild M.F. 2015. Whole Genome Shotgun Sequencing Metagenomics Analysis for the “Common” Scientist – Poultry. International Plant & Animal Genome Conference XXIII; 2015 Jan 10-14; San Diego, CA.\</li> <li>Rowland, K., Zhou, H., Gallardo, R., Bunn, D., Lamont, S. 2015. Identifying genes and genetic markers for NDV resistance in commercial layer chickens. Proc. 9th European Symposium on Poultry Genetics, June 16-18, Tuusula, Finland.</li> <li>Rowland, K., Zhou, H., Gallardo, R., Bunn, D., Lamont, S. 2015. Identifying genes and genetic markers associated with NDV resistance in chickens. Proc. Poultry Breeders Roundtable, May 7-8, St. Louis, MO.</li> <li>Rowland, K., Zhou, H., Gallardo, R., Bunn, D., Lamont, S. 2015. Identifying host genes and genetic markers for antibody production to Newcastle Disease Virus (NDV) vaccine strain in commercial layer chickens. Proc. Vaccines Against Antigenically Variable Viruses, November 5-8, Ames, IA.</li> <li>Sauer, Z., Kaiser, M., and Lamont, S.J. 2015. RNA expression levels of 2 immunologically related genes upon lipopolysaccharide stimulation amongst 3 distinct genetic lines of chicken. Animal Science Leaflet R2999, Iowa State University, Ames IA USA</li> <li>Sun, H., Liu, P., Nolan, L.K., and Lamont, S.J. 2015. Combination analysis of three primary lymphoid tissues in response to extraintestinal pathogenic Escherichia coli (ExPEC) infection. Next generation sequencing USA congress and single cell analysis USA congress: 2015 Oct 27-28; Boston, USA.</li> <li>Walugembe M., Hsieh J.C., Koszewski N.J., Lamont S.J., Persia M.E., Rothschild M.F. 2015. Metagenomics Analysis of Broiler and Layer Chicks Cecal Content from High Fiber Diets - Poultry. International Plant & Animal Genome Conference XXIII; 2015 Jan 10-14; San Diego, CA.</li> <li>Elad O, Rodriguez-Lecompte JC, Sharif S and McKenna P. 2015. Epigenetic characterization of the effect of folic acid on chicken B cell receptors methylation patterns and mRNA expression. Poult. Sci.9(E-suppl.1)4:29</li> <li>Rodriguez-Lecompte JC, Elad O, Reyes J, Yitbarek A, Sharif S and McKenna P. 2015. Vitamin D active form 1,25-(OH)2D3 supplementation on toll-like receptors, BCR, MHC II, cytokines and chemokine profile in chicken B cells. Poult. Sci. 94 (E-suppl.1):64</li> <li>He, Y., H. Zhang, R. L. Taylor, Jr., and J. Song. 2015. DNA methylation patterns associated with the resistance of Marek's disease. Poult. Sci. 94(E-Suppl. 1):50</li> <li>Taylor, R. L., Jr., S. J. Nolin, Z. S. Lowman, A. E. Zavelo, and C. M. Ashwell. 2015. Antibody kinetics differ among Mhc-identical recombinant congenic strains. Poult. Sci. 94(E-Suppl. 1):64</li> </ol> Thesis/Dissertation Completed: <ol> <li>Kallie Sullivan. Honors (POSC) Innate and adaptive anti-tumor immunity in naïve and tumor-bearing Arkansas Rous sarcoma Regressor and Progressor chickens” (Sp 2015). University of Arkansas. G.F. Erf, mentor.</li> <li>Innate patterning of the immune response to marek's disease Virus (mdv) during pathogenesis and vaccination, Upendra k.Katneni, august 2015, phd</li> <li>Transcriptional analysis of the unfolded protein Response (upr) and lymphoma microenvironment during Marek’s disease virus (mdv) infection, sabarinath neerukonda, August 2015, MS</li> </ol>

Impact Statements

Date of Annual Report: 02/16/2017

Report Information

Annual Meeting Dates: 10/08/2016 - 10/09/2016
Period the Report Covers: 10/08/2016 - 10/09/2016

Participants

Participants Attachment:

Participants 2016.pdf

Brief Summary of Minutes

Minutes Attachment:

Brief Summary of Minutes.pdf

Accomplishments

Mark Berres (U. Madison Wisconsin) Goal: Characterize genetic variation of Vietnamese jungle fowl, specifically immunologically active genes, distribution of allelic variation, risk of genetic endangerment, new reference genome for red jungle fowl. Work done in South East Asia MHC Genetic diversity -84 SNP chip panel is used from 199 individuals, high MHC nucleotide diversity was detected and high levels of heterozygocity -This suggests diversifying selection of MHC -A substantial amount of recombination was found, certain areas much higher than the normal rate. This suggests that recombination is an important factor contributing to diversity -Compared to domestic lines the amount of diversity is huge.  Genome wide neutral variation (not MHC related) -Lots of private alleles -Global scale and local scale differentiation was detected -Lowland shows less intraspecific variation, highlands show more intraspecific variation -Twelve or thirteen populations were detected -Population discontinuity was demonstrated due to physical barriers such as a river -Landscape genetic model was created. Is used to observe and evaluate the effect of topography and landscape on the diversity of these chickens. No effect of topography was found. -Movement monitoring will be needed to explain their genetic differences       Conclusions <ul> <li>MHC high diversity, no population structure, huge array of novel haplotypes</li> <li>Neutral sites, high diversity, high global and local structure <ul> <li>5km home size range</li> <li>not influenced by landscape, or land cover</li> <li>No evidence of domestic introgression</li> </ul> </li> </ul> Future  <ul> <li>Re-sequencing RJF with PacBio SMRT</li> <li>Apply genotyping by sequencing</li> <li>Investigate regulation patterns (methylation)</li> </ul> The methylation patterns of RJF are extremely different than the methylation seen in commercial leghorn lines.    Susan Lamont (Iowa State University) Poultry immunogenetics 3 focus areas: -Adapting chicken production to climate change through breeding -Improving food security in Africa by enhancing resistance to NDV and heat stress in chickens -Host resistance to avian pathogenic E. Coli. Harderian gland transcriptome response to NDV Oculo-nasal site of exposure with NDV (lentogenic), mostly T cells, produces antibodies; also T cells increase with immune stimulation. This is the first time the HG transcriptome is investigated. -Fayoumi’s (village type bird rep) and leghorns (relatively susceptible) were used in live bird studies in order to come up with a resistance – susceptible model. -In terms of viral load Fayumi’s are able to clear the virus faster than leghorns at 2 and 6dpi. -Fayoumi’s are producing much more antibodies than leghorns confirming the resistance – susceptibility model.  Harderian Gland Transcriptome:       -High level of difference between lines as early as 2dpi, becoming lower as we reach 10dpi. -Regardless of the challenge there are lots of differences in the gene expression of these birds. -Hyper expressed genes are related with IF2 signaling in response to viruses, cytokines, B cell receptor signaling, etc. -Leghorns have a big difference at 6dpi, this change can be unsuccessful efforts of leghorns to deal with the infection. Since the challenge was done with a lentogenic strain a confirmation of the model were done submitting birds to SEPRL for challenges with Velogenic strains (Burkina Faso).  Conclusions: -Important immune related pathways were detected -Large difference detected between lines specially at 2dpi -Additional studies are needed to understand 6dpi leghorn responses -HD11 cells reacted to heat stress by activation of protective mechanisms and inhibition of apoptosis -HSPs were not activated by LPS alone -Heat stress reinforced expression of LPS-activated chemokines: CCL4, CCL5, IL-8, pro-inflammatory IL-1.    Mohammad Heidari (USDA)  MDV and Skin interaction -RNA seq was used to understand better the skin infection by MDV -10, 20 and 30dpi virus was detected Upregulation was detected mostly at 20dpi -Differential expression of different genes is under characterization -The gene expression of the virus in the skin was also investigated, in order to compare them the different days post infection were compared.  Mechanisms of vaccine induced protection -It has been demonstrated protection after vaccination as early as 1 dpi -Interestingly there is an involvement of the innate immune system most likely NK cells, studies by Lee have shown that at 1 day post vaccination there is some protection, full protection is acquired after 5dpv. -5 Days post infection there is production of IFNg, a, CD107a, suggesting NK involvement. -10DPI you also see IFNg, even though this will reflect more the adaptive immune response -At CT there were some changes in the expression of IFNg  Deletion of adaptive immune system No time to present B and T cells are the target cells, if you deplete them and vaccinate you can weight the innate immune response.   Ramesh Selvaraj (Ohio State University)  Salmonella infection on splenic macrophages T Regs were involved in the suppression of Salmonella growth Macrophages role: IL-10. Upregulated, TLR-2 down regulated after salmonella infection (in vitro) IL-10. similar to what was see in vitro, IFNg down regulated, NO production decreased in salmonella infected chickens (In vivo) Conclusion: Salmonella survives in macrophages Nanoparticle based vaccine for Salmonella The goal is to target the M cells, they are designing a methyl vinyl ether and maleic anhydride based nanoparticles for mucosal delivery. The idea is to include the Salmonella in the nanoparticle, no outside Cecal tonsil cells were imaged after labeling the nanoparticles with DAPI and red fluorescent protein. 24 hours after feeding the nanoparticles the image shows that the nanoparticles are sticking to the CT.   Matt Koci (North Carolina State University)  How does the chicken Microbiome develops and how do probiotics affect it? What happens over time? -Age has the strongest impact on composition of the microbiome -The diversity gets more complex with age (microbiome) -Complexity increases rapidly post hatch -Early events have long term effects, early colonizers set the biome, their residue impacts what happens later on. Is very difficult to change this setting later in life. -Individual species do not affect much the function in the chicken gut. The gut inly cares about what it needs. -Enterobacterias dominate early, firmicutes in maturity -Early exposure to microbial treatments changes microbiome development  How do different regions of the GI compare? -Location has strongest impact in composition, major influence of population ecology and composition -Cyanobacteria and firmicutes dominate -Ileal segmented filamented bacterias (SFB’S) decrease, Ruminococcaceae increases -Probiotics are able to reduce the existence of clostridiaceaes -Diet and probiotic status impact composition of the ceca and ileum biome   Bob Taylor (West Virginia University) -Golden Sebright and Lakenvelder have been studied in terms of their MHC with a high-density panel. -Lakenvelders there is only one MHC haplotype in the Sebrights 3 MHC haplotypes segregating -BSNP-Q02: New similar to others -BSNP-K02 -BSNP-A09A  Lakenvelders: BSNP-C06 similar to one described in broilers  Conclusions -High density chicken MHC SNP panel revealed multiple, non serologically defined haplotypes -Novel and defined haplotype differences may indicate recombination  Chicken RBC alloantigens  The idea is to detect the location of the different alloantigens  There is no information about the susceptibility and resistance of the two described lines   Gisela Erf (University of Arkansas)  Spontaneous autoimmune disease  -Smithline autoimmune vitiligo used as a model for human vitiligo -Hashimoto thyroiditis in the obese strain chickens   -UCD200 autoimmune scleroderma   They are important models for non-communicable multifactorial diseases -Genetic susceptibility -Environmental factors -Immunopathology   Growing feathers as dermal test site to monitor in-vivo cellular tissue responses Need to be growing feathers because it has living tissue, you can find dermis and epidermis. Feather infections are done (18ds old regenerating feathers). Barbs are cutted above the epidermis and they are inoculated, feathers are collected different time after infection and the pulp is extracted, these are digested and strained in nylon mesh fir cell suspensions, they can be fixed and histo analyzed.   LPS, PGN were injected and profiles in blood and tissues was investigated: -Blood heterophyl increased -Macrophages and heterophyls in feathers. Lymphocytes came in in great number B and T cells too very diverse response. -Cytokine expression in tissues LPS increased IL-1B -Same profile was detected when chickens were inoculated with LPS and PGN at the same time -Cutaneous GF in vitro test reveals differences in the responses of different lines of chickens -She also has studied adjuvant effects on cellular and humoral adaptive immune responses to mouse IgG using this technique showing how powerful is this technique. Summary: -Is a window to look into cellular/tissue responses -Is minimally invasive -You can examine responses also in blood   Marleen Visker (Wageningen University) Natural antibodies (Nab) -High levels of maternal antibodies provides a longer and productive life to hens -Antibodies in individuals generated without the exposure to an antigen -Generation 5 generated low and high Nab subsets -There has been an increase in the total Ig production over lines and generations  Breeding for general disease resistance -High Nab birds were able to reduce the mortality compared with med and low lines after infection with E. Coli. -Next experiments will compare different challenge titers -A field test was planned Checking fir maturity, egg production etc. they need to make sure the high levels of Nabs do not affect the productive parameters   Summary -Nabs are heritable and selection is possible -Low or no relation between Nabs and productive traits -Chromosome 4 seems to play a huge role in the production of Nab   B cells can be activated by different cells, they have several receptors. Nabs can be produced by B cells activated in a non-conventional route and provide an effect. They are part of the innate immune response.                Marcia Miller (City of Hope Beckman Research Institute) MHC-Y On chromosome 16 near MHC-B, CD1 is on the same chromosome, MHC B is highly polymorphic <ol> <li>Genome assembly</li> </ol> four contigs. In addition, two more trying to fill the gaps   <ol start="2"> <li>Polymorphism of the YF class I-like genes</li> </ol> It has been detected that YF is similar to MR1 in humans, their role in humans is to basically respond to bacterias <ol start="3"> <li>PCR based method for MHC-type binding</li> </ol>  Summary -Doubled MHC-Y sequence -Strong data revealed nature of YF ligands -Insights on YF molecular signaling -Developed a simpler MHC-Y typing method   Mark Parcells (University of Delaware) Mechanism of MD vaccine response Meq gene products Polycomb Bmi-1, upregulated in several cancer’s, binds to DNA elements and represses transcription. Polycomb repressive complexes are recruited through tissue development, used by herpesvirus to establish latency   Bmi-1 localizes primarily to nucleoplasm   Christina Swaggerty (USDA) Selection of broilers for innate immune response -Early studies -Initial selection -Current selection   Selecting for innate immune response is considered as a pre-harvest intervention for reducing food borne pathogens The selection strategy was to obtain a high and a low line Challenge models; high line birds had lower pathology scores than low lines (Eimeria tenella, SE, NE) New project: They will be screening and selecting using dams and sires.    Jeb Owens (Washington State University)  Agriculture, Ecology, Immunology and Disease   Reality resides in co-infection <ol> <li>Northern fowl mite</li> </ol> The chicken MHC confers protection to chickens against this parasite B15 resistant B21 susceptible, this is due to the inflammation A study involved the association of the resting metabolic rate, feed conversion and mite intensity (Murillo, 2016) 17% decline of the feed conversion In infested birds The cost has been calculated in 10C per bird per week… Resting metabolic rate is not significantly different   Changing production environment, increasing complexity and decreasing biosecurity= INCREASED RISK.   Robert Beckstead (North Carolina State University) Blackhead Disease Culture system is still the best way of screening compounds that inhibit H. Meleagridis Sometimes they do not correlate with in vivo experiments Future: Screen resistance to Blackhead             Screen different industry genetic lines             Genomic comparison between chickens and turkeys             Targeted probiotic RNAi     Henk Parmentier (Wageningen University)  Nabs are heritable Natural autoantibodies, there is a certain level of normal autoantibodies 1/3 of your B cells are autoimmune Nat autoab are potential markers for health, diseases and disorders By ELISA they checked the presence of diff antibodies       The location of the Auto Ab is in chromosome 4 Conclusion: Common genetic components underlying the b cell recognizing different antigens CLL recognized by self IgM and IgG antibodies, they show different binding patterns based on lines and individuals     The IgM auto-antibody profiles at hatch seems species related (no correspondence with mother) The IgG auto-antibody profiles at hatch are related to the mother     M.G.R. Matthijs (Utrecht University)  Hypothesis: Differences in susceptibility of broilers to colibacillosis  They found differences on susceptibility to colibacillosis in differebt broilers lines    Jason Payne (NC State) CRISPR/CAS used for gene editing and reincorporating DNA to the animal    Paul Cotter Present (Framingham University) Bashophia and basophiliosis Normal expected presence of basophils is around 2 to 3%

Publications

Impact Statements

Date of Annual Report: 02/20/2018

Report Information

Annual Meeting Dates: 09/30/2017 - 10/01/2017
Period the Report Covers: 10/01/2016 - 09/30/2017

Participants

PARTICIPANTS:
Technical Committee Members present at the meeting:
Dr. Gisela Erf (U. Arkansas),
Dr. Rodrigo Gallardo (UC-Davis),
Dr. Mark Parcells (U. Delaware),
Dr. Ramesh Selvaraj (University of Georgia),
Dr. Sue Lamont (Iowa State University),
Dr. Rami Dalloul (Virginia Tech. U.),
Dr. Henk Parmentier (Wageningen University),
Dr. Robert Taylor (West Virginia University),
Dr. Juan Carlos Rodriguez (UPEI)

Technical Committee Members absent from the meeting:
Dr. Vicky van Santen (Auburn University),
Dr. Marcia Miller (Beckman Institute, City of Hope Hospital),
Dr. Kirk Klasing (UC-Davis),
Dr. Mary Delany (UC-Davis),
Dr. Huaijin Zhou (UC-Davis),
Dr. Ellen Collisson (Western U. of Sciences),
Dr. Yvonne Drechsler (Western U. of Sciences),
Dr. Ryan Arsenault (U. Delaware),
Dr. Shayan Sharif (U. Guelph),
Dr. Patricia Wakenell (Purdue U.),
Dr. Jiuzhou Song (U. Md.),
Dr. Mohammad Heidari (USDA-ADOL),
Dr. Robert Beckstead (NC-State U.),
Dr. Matt Koci (NC-State U.),
Dr. Christi Swaggerty (USDA-ARS, College Station),
Dr. Jan van der Poel (Wageningen U.),
Dr. Mark Beres (U. Wisconsin)

Collaborators/Guests present at the meeting:
Dr. Chris Ashwell (NC-State U.),
Dr. Paul Cotter (Framingham University),
Dr. Katharine Magor (University of Alberta),
Dr. Lisa Bielke (Ohio State University)

Brief Summary of Minutes

NE-1334 2017: Genetic Bases for Resistance and Immunity to Avian Diseases
Meeting Location: Atlantic Veterinary College, Charlottetown, Prince Edward Island, 550 University Avenue, C1A 4P3

Meeting Host: Dr. Juan Carlos Rodriguez (jrodriguez@upei.ca)
Meeting Chair: Dr. Rodrigo Gallardo (ragallardo@ucdavis.edu)
Meeting Secretary: Dr. Mark Parcells (parcells@udel.edu)

Saturday, September 30^th, 2017

7:30 Registration (Fee: $50) and Continental Breakfast

8:00 Welcome – Juan Carlos Rodriguez

8:15 Opening Remarks – Rodrigo Gallardo

Station reports: 25-minute presentation including 5-10 minutes for discussion

8:30 Vicky van Santen (Auburn University) – did not attend

8:55 Gisela Erf (University of Arkansas)

9:20 Rodrigo Gallardo (University of California, Davis)

9:45 Huaijun Zhou (University of California, Davis) – did not attend

10:10 – 10:30 Break

10:30 Mark Parcells (University of Delaware)

10:55 Ramesh Selvaraj (University of Georgia)

11:20 Sue Lamont (Iowa State University)

11:45 Robert Beckstead (North Carolina State University) – did not attend

12:15 – 1:15 Lunch (on your own)

1:15 Christopher Ashwell (North Carolina State University)

1:40 Henk Parmentier (Wageningen University)

2:05 Katharine Magor (University of Alberta)

2:30 Robert Taylor (West Virginia University)

2:55 – 3:15 Break

3:15 Janet Fulton (Hy Line international)

3:40 Paul Cotter (Framingham University)

4:05 Lisa Bielke (Ohio State University)

4:30 pm: Business meeting

Items

The Minutes of the Business Meeting from the October 8, 2016 meeting were read by Chair Rodrigo Gallardo.

There was one correction to the minutes regarding the location for future meetings:

Meeting in UWV in 2018, (host by Robert Taylor, perhaps in Pittsburgh)

Meeting in 2019 UGA, (hosted by Ramesh)

Meeting at Virginia Tech to possibly meet with AIRG or in affiliation with this meeting. 2019 meeting to be discussed in New business.

Sue Lamont made a motion to approve the minutes. The motion was seconded by Ramesh Selvaraj, and passed unanimously.

Old Business:

This project will need to be submitted for re-authorization in 2018 (5 years since last renewal).

Rick Rhoads, requested the submission of an administrative summary and

revision of the project in terms of revised objectives.

It was decided that there would be two people per project goal and that this should be submitted by December 15, 2017.

The target for a working draft of the revised project proposal would be completed by the end of October.

The would be a poll of the existing membership to determine those willing to continue to participate in the revised project.

Mark Parcells, as secretary for 2017 will submit the annual report to NIMSS.

Sought funding support from NIFA (Margo Holland and Peter Johnson) – through regular channels. Perhaps best for 2020 year (in combination with AIRG).

New Business:

The new Program Administrator for this project is now Dr. Robert Taylor (who authorizes uploading the annual report into NIMSS)

Following the prior succession plan for our project group,

The Chair for the 2018 Meeting will be Mark Parcells

Lisa Bielke agreed to serve as Secretary for 2018

This selection of chair and secretary were moved by Rodrigo Gallardo, seconded by Ramesh Selvaraj and passed unanimously

Juan Carlos Rodriguez was thanked for hosting a very productive and interesting meeting and being a gracious host.

Robert Taylor provided options for the 2018 meeting:
- Morgantown, WV is somewhat difficult to reach given one small airport with single flights in and out per day.
- On a non-football weekend, hotel rooms are in the $120/night range)
- Taylor offered the alternative of hosting in Pittsburgh, PA (~1.5 hrs from UWV), which has a larger airport
- Rooms at the Courtyard Marriott in downtown Pittsburgh (October 12 – 14, 2018) would run ~$159/night.
- The members felt that this was the preferred meeting location and more details would be following.

Stakeholder input needed for new century of NIFA

Ramesh Selvaraj offered to host the 2019 meeting at the University of Georgia. This offer was put to a motion by Mark Parcells and seconded by Sue Lamont. The motion passed unanimously.

It was again noted that no NIFA representatives were present at our meeting.

There being no additional business, the meeting adjourned at 5:27 PM, on motion.

Accomplishments

ACCOMPLISHMENTS: The annual reports presented are focused on the accomplishments addressing the three objectives of the NE-1334 Project: OBJECTIVE 1: Characterize the function of genes and their relationships to disease resistance in poultry with an emphasis on the MHC as well as other genes encoding alloantigens, communication molecules and their receptors.  OBJECTIVE 2: Identify and characterize environmental, dietary and physiologic factors that modulate immune system development, optimal immune function and disease resistance in poultry genetic stocks. OBJECTIVE 3: Develop and evaluate methodologies and reagents to assess immune function and disease resistance to enhance production efficiency through genetic selection in poultry.  These are described based on the investigators addressing each project: <h2>Objective 1 </h2> Investigators addressing this objective are: <ol> <li>Marcia Miller-CA, BRI</li> <li>Rodrigo Gallardo-CA, UCD</li> <li>Huaijin Zhou – CA, UCD</li> <li>Yvonne Drechsler and Ellen Collison – CA, WU</li> <li>Mark Parcells - DE</li> <li>Sue Lamont – IA</li> <li>Matt Koci – NC</li> <li>Rami Dalloul – VA</li> <li>Robert Taylor – WV</li> <li>Mark Beres – WI</li> <li>Henk Parmentier - WUNL</li> </ol>  1. Contributions of Dr. Marcia Miller (CA, BRI): Advances in the completion of genomic sequence for the MHC-Y region (Miller, Goto, Warden, Wu, Kang and Delany).  During the past year we have assembled completed RJF BAC clone sequences into three contigs for the MHC-Y.  The USDA NRSP-8 funds provided in 2016 were of great importance in letting us use Single Molecule Real-Time (SMRT) sequencing to determine the sequences of these clones that are filled with repetitive sequences that made several previous attempts to assembly MHC-Y sequence data. Annotation of the sequences and analysis of the genes within are nearly complete.  2. Contributions of Dr. Rodrigo Gallardo (CA, UCD): 2.1. Our focus in this objective is the understanding immune resistance to infectious bronchitis virus (IBV) using chicken lines of different MHC-I haplotypes. Our goal was to determine resistance and susceptibility of MHC B haplotype in congenic and inbred chicken lines in order to establish a resistant–susceptible model. Eight congenic lines (253/B18, 254/B15, 330/B21, 312/B24, 331/B2, 335/B19, 336/B21, and 342/BO), two inbred lines (003/B17 and 077/B19), and three commercial lines (white leghorn, brown layers, and broilers) were used in two experiments. We identified 331/B2 as the most resistant and 335/B19 as the most susceptible congenic chicken lines. These two lines will be used in subsequent experiments to understand the mechanisms by which the immune system in chickens generates resistance to infectious bronchitis virus.In a second experiment we hypothesized that chicken lines B2 and B19 were relatively resistant and susceptible respectively to challenges with different IBV types (M41 and Ark). It was not possible to determine resistance levels using innate immune parameters. Humoral responses (IgG and IgA), especially in tears, were good predictors of resistance to both IBV challenges.   2.2. Molecular Biology and Experimental Characterization of an Infectious Bronchitis Virus with Increased Enteric Tropism. We have previously reported the detection and isolation of Cal ent an IBV-like coronavirus causing lesions in the intestines of red broiler chickens showing typical signs of runting stunting syndrome (RSS). When the virus was isolated, it was detected in embryo intestines, but not in the allantoic fluid. In a preliminary animal study using SPF birds, the virus showed and enhanced tropism for intestinal epithelium and caused mild intestinal symptoms. The S1 gene had a 94% homology to IBV Cal 99. The aim of this study was to compare tissue tropism and shedding of Cal ent to respiratory strain M41 in commercial broilers as well as sequencing the whole genome of Cal ent for comparison with other IBV types. In a broiler-based study, we observed that Cal ent did not increase enteric signs or pathology in this experiment but showed higher viral load in the small intestines and cloacal swabs compared to M41. Cal ent caused mild respiratory disease and less viral load in the upper respiratory tract. We did not see major differences between inoculation routes. 2.3. Understanding the latest Coryza outbreaks. More than 40 commercial poultry cases  of infectious coryza have been diagnosed from January to August 2017. In order to understand the occurrence and increased severity of these cases we molecularly characterized 3 strains of Avibacterium paragallinarum using Next Generation Sequencing (NGS) to determine if the latest outbreaks were caused by variants types of the bacteria. We focused the molecular characterization analysis on the HMPT 210 gene the hemagglutinin protein of the Avibacterium in order to align with the commonly used serological characterization assays (Kume and Page) using the hemagglutination properties of the bacteria. This characterization suggests that the isolates belong to the group C of Avibacterium paragallinarum. Homologies of 100% were encountered when the isolated sequences were compared with H18 and Modesto Avibacterium paragallinarum reference isolates. These two strains are part of the inactivated vaccines used in the field. The results of the applied research showed that the Avibacterium was not able to persist in infected isolators (seeder birds and infected bedding) for more than 12 hours after the seeder birds were euthanized. Mild respiratory signs and swollen heads were detected in exposed birds. Gallibacterium anatis was isolated inconsistently from those cases.                2.4. Molecular Characterization as a Surveillance Strategy for Clinically Relevant Reoviruses. Since 2015, hundreds of clinically relevant Reoviruses; associated with a history of leg problems, poor performance and lack of uniformity; have been isolated from broiler chickens at the California Animal Health and Food Safety (CAHFS) Laboratory. Two sets of isolates: the first with twenty-eight Reoviruses collected between September 2015 to October 2016, and the second with fifty collected between October 2016 to February 2017 were chosen for further characterization. Reovirus isolates were confirmed by a diagnostic RT-PCR amplifying a conserved region of Sigma 4 gene. After confirmation a different RT-PCR amplified a segment of Sigma C being the substrate for sequencing and phylogeny studies. Most of the isolates from both characterized sets grouped in cluster 1 (vaccine cluster). However, homologies of these Reoviruses to S1133 are below 78% for both sets. The rest of the isolates grouped in clusters 2, 3 and 4 and their homologies to S1133 were below 58.9, 57.5 and 55.7% respectively. In regards to the full genome sequence from the eight viruses only 7 were Reoviruses, and one proved to be a Fowl Adenovirus, demonstrating the need of better tests to confirm Reovirus isolation. In terms of variability the S1 gene was one of the variable genes. In addition, L3 and M2 (still under analysis*) also show high variability. 3. Contributions of Dr. Huaijin Zhou (CA, UCD): 3.1. Improving food security in Africa by enhancing resistance to Newcastle disease virus and heat stress in chickens. This project is part of a 5-year, multi-investigator award. Birds of two genetically distinct and highly inbred lines (Fayoumi and Leghorn), and Hy-Line Brown were either exposed to NDV only (Iowa State) or NDV and heat stress (UCD). Measures of body temperature, blood gas parameters, NDV titers from tears, and antibody response in serum were taken on the live birds, and tissues were collected for transcriptome analysis. Three ecotypes each in Ghana and Tanzania will be exposed to NDV. DNA isolated from Hy-Line Brown were genotyped using chicken 600K SNP for GWAS. At UCD, the RNA-seq data of 144 individual cDNA libraries (focusing on infection: 3 tissues (lung, trachea, and harderian gland), 2 genetic lines of chickens, NDV challenge and control, 3 times points at 2, 6 and 10 dpi) and 96 individual cDNA libraries (focusing on heat stress: 3 tissues (liver, breast muscle, and hypothalamus) at 4 hours and 9 days post-heat treatment, 2 genetic lines of chickens, heat stress and control) were generated from the combined NDV challenge with heat stress study of highly inbred chicken lines.   Genes that responded to NDV infection and differed between resistant and susceptible genetic lines were identified (for NDV infection, 500-800 genes at 2 dpi, 100-400 genes at 6 dpi, and 50-200 genes at 10 dpi; and for heat stress, 50-200 genes at both time points).  Enriched gene groups and pathways were also identified and validated (NDV resistance: such as cytokine-cytokine receptor interaction, regulation of T cell activation, cell adhesion molecules, MHC class I protein complex antigen processing and presentation, regulation of type I interferons, T-helper 1 immune response etc.; heat tolerance: oxidative response, oxidative de-ethylation, and ossification etc.). To assess the response to heat stress, thirteen blood physiological parameters were measured using the iSTAT system. Completed genome-wide association analysis for challenge experiments on Hy-Line Brown: In general, most of the economically important traits have low to medium heritabilities (0.1-0.4), except body weight with high heritability (0.5-0.7). Birds were genotyped using the 600K SNP panel to conduct a genome-wide association study (GWAS). For African ecotype NDV challenges, replicate trials involving a total of 2,653 chicks (UOG) and 1789 chicks (SUA) were completed in the challenge facilities. Following natural NDV exposure, data on survival times, body weight, antibody response, and pathological lesion scores were collected. Data analyses are underway. 3.2. Salmonella enterica serovars Enteritidis infection in young layer chicks. The main objective of the research project is to elucidate molecular and cellular mechanisms of Salmonella enterica subsp. enteric serovar Enteritidis (SE) persistent infection in chickens by studying the interaction of the following trio: host, pathogen, and microbiome using next generation sequencing (NGS). The objective of the current study was to profile SE associated microbiome during the developmental stage of young chicks. Chicks were challenged with SE at two weeks of age and cecum microbiome was analyzed with 16S rRNA sequencing post-infection at 3, 7, 14 and 21 days. Our results suggest that as SE colonization continues to persist over time with infection, its functional activity could play a role in creating a beneficial environment for other incoming species. Overall, the presence of a developing microbial community in 2 weeks old chicks was effective in inhibiting pathogen-induced microbiota alteration. 3.3. Development of colonization resistance in chicks. The main objective to this project was to dissect the mechanism of SE colonization in newly hatched chicks by investigating aerobic respiration as possible mechanisms behind the bloom of facultative anaerobe in gut during SE infection. Our results indicated that the host inflammatory response triggered by Salmonella virulence factors contribute to microbial dysbiosis and increase oxygenation of the gut epithelial that drives the bloom of the SE growth in gut of the newly hatched chicks.To further assess the competition for available of oxygen in the gut between the facultative anaerobic members of the microbial community, we did in vivo bioluminescence imaging utilizing the transformed avian E.coli harboring the bacterial luciferase and fatty acid reductase genes that will emit visible light. Our results suggested that early colonization by E. coli allows it to establish a colonization site in the gut that is closest in proximity to the oxygen rich niche. Subsequent colonization by SE later on however has to settle on colonizing the site that was not already occupied by the first colonizer, thus resulting in SE having limited access to the oxygen for aerobic growth and diminishing its ability to compete effectively against E.coli for oxygen utilization. 4. Contributions of Drs. Yvonne Drechsler and Ellen Collison (CA, WU): 4.1. Impact of MHC on macrophage responses.  We characterized the molecular basis for dramatically different nitric oxide production and immune function between the B2 and the B19 haplotype chicken macrophages. We employed RNA-seq analysis of macrophages from each haplotype during differentiation and after stimulation. We found that a large number of genes exhibit divergent expression between B2 and B19 haplotype cells both prior and after stimulation. These differences in gene expression appear to be regulated by complex epigenetic mechanisms that need further investigation. 4.2. Macrophages, not T-cells are driving the differential immune response in B2 vs B19 haplotypes. In the current study, in vitro T lymphocyte activation measured by IFNg release was significantly higher in B2 versus B19 haplotypes. AIV infection of macrophages was required to activate T lymphocytes and prior in vivo exposure of chickens to NP, AIV plasmid enhanced responses to infected macrophages. Our data suggest that the demonstrated T lymphocyte activation is in part due to antigen presentation by the macrophages, as well as cytokine release by the infected macrophages, with B2 haplotypes showing stronger activation. These responses were present both in CD4 and CD8 T lymphocytes. In contrast, T lymphocytes stimulated by ConA showed greater IFNg release from B19 haplotype cells, further indicating the greater responses in B2 haplotypes to infection is due to macrophages, but not T cells. In summary, resistance of B2 haplotype chickens appears to be directly linked to a more vigorous innate immune response and the role macrophages play in activating adaptive immunity. 5. Contributions of Dr. Mark Parcells (DE): Project 5.1. Identification of innate immune patterning of acquired immune responses to MD vaccination.  This project was focused on the differences between rMd5, a very virulent MDV and rMd5∆Meq, a derivative lacking both copies of the oncogene. rMd5∆Meq replicates in vivo for ~2 weeks and then drops to near detectable levels, yet provides vaccine protection superior to an attenuated MDV strain, CVI988 (Rispens) which establishes a more long-term infection. Our analysis focused on measuring mRNA expression of genes involved in: innate sensing, second messengers, pro-inflammatory cytokine, interferons and interferon-inducible, cell-specific markers, immune modulatory cytokine, and lineage-determining transcription factors via qRT-PCR. Our results showed similar levels of innate sensing, second messenger and pro-inflammatory cytokine gene expression. however in each case, rMd5-induced expression was significantly above rMd5∆Meq for pro-inflammatory and interferon- inducible genes. However, SOCS1 and 3 were also more highly-upregulated, suggesting that despite increased pro-inflammatory and interferon expression, their signaling and effects may be blocked. Our data suggested that rMd5∆Meq induces higher levels of GM-CSF with concomitant increased expression of CD11c as well as increased numbers of CD11c mAb-positive cells at 14 and 21 dpi. Accompanying and following this were increased levels of CD8 expression and CD8+ cells in the spleen. A key finding was the observation of differential expression of IL-12 subunits, at 14 and 21 dpi. In rMd5-infected chickens at 14 dpi, the genes for IL-12p19 and IL-12p40 were induced to a greater extent that IL-12p35, suggesting that IL-23 (p19 + p40 heterodimer) would be in greater abundance than IL-12p70 (p35 + p40). As IL-23 is associated with proliferation of naïve T-cells and in tumor progression, our data suggest that differential subunit expression by Meq may mediate expansion of latently-infected T-cells. By 21 dpi, we observed upregulation of only IL-12p40 and downregulation of both p19 and p35. IL-12p80 (homodimer of IL-12p40 that suppresses the development of cytotoxic T-cells). 5.2. MDV induction of the unfolded protein response (UPR). The UPR is activated via three somewhat distinct pathways (ATF6, PERK and IRE-1a ) that have some distinct downstream target genes. We examined these pathways at 4, 7, 14, 21 and 28 days post-infection using (3) pools of spleen tissue per time point, per virus with each set of genes being compared to mock-infected, age-matched chickens. Our findings are that during MDV vaccination, the unfolded protein response is upregulated via ATF4 and IRE-1a  pathways at early times post-infection (4 and 7 dpi), with IRE-1a being highly active (in terms of splicing of XBP-1) at 14 and 21 dpi. As the replication of rMd5∆Meq is detectable primarily at 4 and 7 dpi, with little to no replication being detected at 14 - 28 dpi, our data suggest that this activation of UPR via IRE-1a is likely due to the repopulation of the spleen after the early lytic infection. In terms of rMd5 pathogenic infection, we similarly observed induction of the UPR via ATF4 and IRE-1 -induced pathways, however splicing of XBP-1 at 14 and 21 dpi was less than that induced by rMd5∆Meq. As this is the peak time of lytic infection in the spleen (14 dpi) and the onset of latency (21 dpi), our data suggest that rMd5 may actively suppress UPR activation. As the sole difference between the vaccine and pathogenic viruses is the absence or presence of the meq gene, our data suggest that Meq gene products actively repress the UPR response. In MDV-induced spleen tumors, versus non-tumorous spleen tissue, there were significant increases in chaperone BiP (GRP78), GRP94, as well as spliced XBP-1, indicative of both ATF6 and IRE-1a  activation. 5.3. MDV and regulation of metabolism. Recently, a clear connection has been established between immune patterning and cellular metabolism. During an inflammatory response, early induction of reactive oxygen species and nitric oxide shift cells towards anaerobic metabolism indicative of the Warburg effect. Alternatively, a less inflammatory, M2/TH2 response is associated with oxidative phosphorylation. We have examined target gene expression indicative of Warburg  and OX/Phos metabolism in RNA samples from mock, rMd5∆Meq- and rMd5-infected chickens. Our data suggest that rMd5∆Meq (vaccine MDV), induces primarily OX/PHOS metabolism with the exception of 7 dpi, during the peak of lytic replication and induction highest induction of inflammation. Conversely, rMd5 induced early OX/PHOS metabolic programming (4 dpi) that shifted to Warburg during peak lytic infection, OX/PHOS during latency establishment and in transformed cells. 5.4. Role of Exosomes in MDV-mediated Immune Suppression and Vaccine Responses. We are in the process of examining the transcriptomic and proteomic profiles of exosomes purified from the serum of tumor-bearing MDV-infected, as well as vaccinated and protected chickens. Exosomes are small vesicles actively secreted by all cells. These vesicles are approximately the size of viruses, being 50 - 100 nm in diameter, and are secreted from cells. Working with Carl Schmidt, Robing Morgan, Erin Bernberg, Ryan Arsenault and Fiona McCarthy, we are examining the transcriptomes, proteomes, and functional relevance of exosomes in the affecting MDV tumor progression, immune suppression and conversely, in mediating systemic immune protection during MDV challenge. 6. Contributions of Dr. Sue Lamont (IA): 6.1  Genomics and immunology of host response to avian pathogenic E. coli (APEC) Our research on infection with avian pathogenic E. coli (APEC) has an overall objective to identify genes, signaling pathways and biological networks associated with infection and resistance to APEC in chickens.In 2017, we initiated the genome-wide association study (GWAS) of data from an APEC challenge of approximately 400 chicks of the broiler X Fayoumi advanced intercross line (AIL) bacterial counts to identify genomic regions controlling host response to bacterial colonization with APEC. In 2017, RNA-seq was done various tissues collected from an APEC-challenge study of reciprocal F1 crosses of two sets of lines: Broiler X Fayoumi; Leghorn X Fayoumi. The RNA sequencing is to determine gene expression profile and allele-specific expression of various tissues in response to APEC challenge. Refined analyses are on-going. 6.2 Genomics of host response to Newcastle Disease virus (NDV) The objective of the research conducted at ISU is to determine the response in ISU research lines and a commercial line to Newcastle Disease Virus (NDV) challenge. Inbred Fayoumi (relatively resistant) and Leghorn (relatively susceptible) chicks were challenged with LaSota strain NDV. Samples were collected at 2 and 6 dpi to measure viral load in tears, and at 10 dpi to measure circulating anti-NDV antibody. Designated birds were euthanized at time points (2, 6, 10 dpi) after challenge, and tissues collected for RNA seq analyses. In the past year, detailed pathway analysis was completed and identified many pathways of interest, and highlighted that the NDV response of the two lines is distinctly different, as well as being different among the tissues. In another study at ISU, commercial layer chicks (Hy-Line Brown) were challenged with NDV. Birds were genotyped using the 600K SNP panel to conduct a genome-wide association study (GWAS) and, with an NE1334 collaborator, also selected candidate genes. Analysis was completed and a manuscript was submitted on splenic gene expression in the NDV-challenged and non-challenged birds as characterized by RNA-seq.  7. Contributions of Dr. Matt Koci (NC): For over 30 generations two lines of white leghorn chickens have been undergoing continuous divergent selection for high (HAS) or low (LAS) antibody titer to sheep red blood cells (SRBCs) at 5 days post-injection.  This has been a well utilized model for immunology and genetic trials, and many differences between the lines have been observed in terms of performance and response to diseases. Using RNA-seq analysis, significant differences in gene expression were observed between lines with over four times as many genes up regulated in HAS as compared to LAS. Upregulated HAS genes are involved with immune response, particularly interferon signaling and antigen processing.  Genes up regulated in LAS largely involve fatty acid transport and cell membrane integrity. We also implemented a pooled genome re-sequencing approach to investigate the consequences of 39 generations of bidirectional selection in these same HAS and LAS lines. We observed wide genome involvement in response to this selection regime. Many genomic regions were highly differentiated resulting from this experimental selection regime, an involvement of up to 20% of the chicken genome (208.8 Mb). While genetic drift has certainly contributed to this, we implemented gene ontology, association analysis and population simulations to increase our confidence in candidate selective sweeps. Three strong candidate genes, MHC, SEMA5A, and TGFBR2 were identified as major functional candidates. The extensive genomic changes observed highlight the polygenic genetic architecture of antibody response in these chicken populations, which were derived from a common founder population, demonstrating the extent of standing immunogenetic variation available at the onset of selection.  8. Contributions of Dr. Rami Dalloul (VA):       8.1. MIF receptors. Efforts continue to characterize the pluripotent cytokine MIF (macrophage migration inhibitory factor) and its receptors in both host (chicken, turkey) and pathogen (Eimeria parasites).  Receptor-specific transformants were constructed and the interactions tested using an array of assays with avian cell lines and primary chicken monocytes.  Receptor binding tests included pull-down assay, co-immunoprecipitation, immunofluorescence, and flow cytometry.  In addition, CXCR4 internalization assay and co-localization of CXCR4 and CD74 were performed.  Preliminary data indicate that both avian and parasite MIFs interact with the receptors CD74, CXCR4, as well as their complex. 9. Contributions of Dr. Robert Taylor (WV): 9.1  West Virginia University (WVU) research antisera [Taylor, Kopulos].  Selected alloantisera produced by Dr. W. E. Briles at Northern Illinois University (NIU) were transferred to West Virginia University (WVU).  Other antisera remain at NIU for the present.  Any NE-1334 investigator may acquire these antisera for their work for the shipping charges. 9.2  West Virginia University (WVU) genetic stocks [Taylor, Kopulos, Delany, Ashwell].  University of California-Davis inbred lines UCD 001 (Jungle fowl, reference) and UCD 003 (white Leghorn) came courtesy of Mary Delany.  Chris Ashwell at N. C. State kindly sent lines congenic for MHC recombinants.  Line 003.R2, a better responder against Marek's disease (MD) and Rous sarcoma virus (RSV), lacks a 225 bp insert in the 3' UTR of BG1 that exists in Line 003.R4. Chicks produced from NIU parents segregate for multiple alloantigen alleles.  A synthetic line, Whiting Blue, having the blue egg gene (O-), was acquired from a commercial source.  Two stocks were typed with a chicken MHC high-density 90 SNP panel encompassing 210,000 bp (Fulton et al., 2016).  Lakenvelders are homozygous for a novel MHC haplotype BSNP-C06, formerly RLT-LAK01.  Golden Sebrights segregate for three haplotypes: novel BSNP-Q02, BSNP-K02 found in Barred Plymouth Rock and broiler lines (BRL), and BSNP-A09A, identical to BSNP-A09 (serotype BQ) from Red Jungle Fowl and BRL. 9.3  SNP mapping of alloantigens (Ashwell, Kopulos, Fulton, Taylor).  Alloantigen systems A, C, D, I, and L are being studied.  For each system, pedigreed progeny segregating for two alloantigen alleles were produced from single sires mated with multiple dams/sire (Table 2).  DNA from birds of known alloantigen genotypes in the NIU archive will be used as well.  A 600 K SNP panel will be used to examine snp associations with particular alloantigen.  That data, combined with sequencing will facilitate identification of the specific alloantigen gene and its location. 9.3.1. L system.  Matings of L1L2 sires and dams produced progeny segregating for three alloantigen L system genotypes (L1L1, L1L2, L2L2).  Mating 729 had 84 total progeny. Mating 2, unrelated to the first, yielded 82 total progeny.  A 60K SNP analysis located alloantigen L on chromosome 4.  9.3.2. A and C systems.  Matings of A3A4, C2C5 sires and dams produced progeny segregating for three A system (A3A3, A3A4, A3A4) and three C system (C2C2, C2C5, C5C5) genotypes.  Matings A860 and A861 had 111 and 85 progeny, respectively.  9.3.3. D and I systems.  Matings of D1D2, I2I8 sires and dams produced progeny segregating for three D system (D1D1, D1D2, D2D2) and three I system (I2I2, I2I8, I8I8) genotypes. Matings 916, 917, and 918 had 22, 18 and 30 progeny, respectively.  9.3.4. Adaptation of a PCR to detect blue egg (Kopulos, Taylor).  Blue egg (O-) results from insertion of a retrovirus in SLCO1B3 which encodes the membrane transporter OATP1B3.  Blue egg is linked to pea comb (P-) by 4.28 cM (Bitgood et al., 1983).  This gene can serve as a marker in certain stocks. Genotypes for blue egg chickens were determined through test crosses.  A three primer PCR to identify the blue egg genotypes, developed by Wang et al., (2013), was adapted to the WVU lab.  Thermocycler conditions were 94°C for 5 min, 36 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 20 s, and a final extension at 72°C for 5 min. Products were separated by 2% agarose gel electrophoresis.  10. Contributions of Dr. Mark Beres (WI):       10.1. Wild Junglefowl Genome Sequencing. Domestication and long-term artificial selection by humans has caused significant evolutionary change in species of agricultural importance. Evaluations of genetic diversity are of central importance to the identification and conservation of genetic resources in agriculture species. Yet, very little is known about the genetic diversity and structure of wild progenitors and if still extant, face increasing threats of extinction from habitat loss and reduction of genetic diversity, including genetic alteration from introgression with domesticated stocks. Using three different next-generation sequencing platforms, we are generating genomic sequences of two wild Red Junglefowl (male and female) obtained from Yok Don Province, Vietnam. We have completed 72X coverage for 2x125 Illumina reads, 62X coverage for PacificBiosciences reads (average read length= 29Kb), and are preparing for complete runs on Oxford Nanopore MinION (average read length to date= 135Kb) to acquire 100X coverage for each chromosome (except for W chromosome, which will be approximately half of the full coverage across all three platforms). Scaffolds of Pacific Biosciences reads finished with Illumina reads have been assembled into chromosomes and mirror the number present in the current galgal5 release. However, many additional smaller scaffolds exist, which may reflect the presence of uncharacterized microchromosomes. Compared to the Shiina MHC-B sequence, MHC from Red Junglefowl is larger and exhibits many structural rearrangements and substantial numbers of SNPs. 11. Contributions of Dr. Henk Parmentier (WUNL): 11.1. SNP associations with natural antibody (NAb) isotypes IgM and IgG binding KLH and auto-antigens in laying hens. Work is in progress using a dedicated SNP set consisting of 384 SNP chosen on the results of a previous SNP analyses. Starting from the Wageningen SRBC-selection lines of the 29th generation from the control line we applied a new pilot selection on either high or low KLH NAb titers for 2 generations. Animals from this 2nd generation high or low KLH NAb originating from the non-selected Control line from the SRBC selection experiment were genotyped along with parental animals of the founding generation. In addition, animals from the SRBC High and Low selection lines at the 31st generation were also genotyped. In total, 960 animals were genotyped on our BEAD Xpress machine (Illumina). Genotypes were checked but the analysis will be done coming period (Man Bao). A Genome Wide Association study (GWAS) was performed using a 3k and 11k SNP set (imputed to 60k) for the baseline population of the current KLH NAb selection lines (originating from a White Leghorn pure-bred elite line). A very strong association is found on GGA4 with IgM titers binding KLH, total IgM levels in blood and titers of IgM binding auto-antigens, respectively. Little significant associations were found for IgTotal, IgA, and IgG. Furthermore complete genomic sequences available from 70 key ancestors of the founding population were used to identify TLR1A variants with a causal mutation in TLR1A. Follow-up actions are being considered. The current G6 H line birds consisted mostly of CC and CG variants, suggesting full dominance, whereas the Low line birds were primarily GG variants. We intend to breed the next NAb selection generations (G7 and onwards for homozygous H line CC variants and L line GG variants. <h2>Objective 2</h2>  Investigators addressing this objective are: <ol> <li>Gisela Erf – AR</li> <li>Mark Parcells – DE</li> <li>Sue Lamont – IA</li> <li>Matt Koci – NC</li> <li>Rami Dalloul – VA</li> <li>Henk Parmentier - WUNL</li> </ol> 1. Contributions of Gisela Erf (AR) 1.2.1 Autoimmune vitiligo Smyth line chickens. The mutant Smyth Line (SL) chickens develop spontaneous, post-hatch, autoimmune vitiligo-like loss of pigmentation in the feather and eye. In addition, SL chickens may also develop autoimmune thyroiditis, an alopecia-like feathering defect, and blindness. Loss of pigment in feathers of SL chickens appears to involve several factors, including an inherent melanocyte defect, immune system components and environmental factors (e.g., herpesvirus of turkey). Together, these factors will result in post-hatch autoimmune loss of melanocytes (vitiligo). The complete animal model consists of MHC-matched lines of chicken that are homozygous for the B101-MHC haplotype. B101 sublines include the Light Brown Leghorn line (LBL control, vitiligo resistant; no incidence of vitiligo), the Brown line (BL parental control, vitiligo-susceptible; but <2% incidence of vitiligo in the population) and the Smyth line (SL, vitiligo-susceptible; between 80% and 95% incidence of vitiligo in the population) 3.2.1a. Immunosuppressive activities in the target tissue of autoimmune vitiligo-susceptible, but non-expressing Brown line chickens. Daniel Falcon is continuing his work on establishing the immunological mechanisms underlying the initiation of autoimmune vitiligo in Smyth chickens (SL). Specifically, he monitored and assessed: 1) infiltration of leukocytes into growing feathers (target tissue) before and throughout vitiligo development in SL chickens using BL chickens as controls, 2) alterations in the T cell receptor repertoire throughout vitiligo development in SL chickens using BL chickens as controls and 3) activities of the leukocyte infiltrate using gene expression analysis.Gene expression analysis suggested active recruitment (CCL19, CCR7) of lymphocytes prior to onset and a sustained Th1-like gene signature (IFN-γ, FASLG, GZMA) throughout disease progression. Spectratype analysis of CDR3 regions of T-cell receptor cDNA suggested skewing of the T-cell repertoire prior to visual onset indicative of a clonal T-cell response. Unexpectedly, while no BL chickens showed any signs of depigmentation, in some individuals a transient recruitment and infiltration of CD4+ and CD8+ T-cells was observed, with CD4+ cells being the dominant population. In contrast to SL however, infiltration was accompanied by elevated expression of immunosuppressive genes (CTLA-4 and IL-10) without increases of IFN-γ, FASLG or GZMA. These results reveal, for the first time, what appear to be immunoregulatory activities in vitiligo-susceptible BL chickens. Taken together these data suggest triggering of melanocyte-specific immune system responses in growing feathers of both SL and BL chickens with the latter responding in an immuno- suppressive manner and the former progressing to a sustained cell-mediated immune response. 3.2.1b. Innate immune system responses in the UCD-200 autoimmune scleroderma/systemic sclerosis line of chickens. The UCD200 line of chickens spontaneously develop scleroderma/systemic sclerosis which is a complex autoimmune connective tissue disease. The objective of this study was to examine the tissue/cellular responses to injection of lipopolysaccharide (LPS), Mycobacterium butyricum bacterin (Mb), functionalized graphene-based nanomaterial (F-GBN), or vehicle (endotoxin-free PBS) into the pulp of growing feathers (GFs). In GFs from both lines, vehicle injection resulted in a small increase in heterophil levels (% pulp cells) at 6 h and 1 d (8-10% of pulp cells at 6 h). GF-injection with LPS also had similar effects in UCD-200 and LBL chickens, whereby heterophil levels peaked at 6 h (18-22 %) and remained elevated (5-10%) at 1-2 d, before returning to baseline levels by 3 d (1%). Macrophage infiltration followed a similar time-course, reaching peak levels of 4-6 %, whereas lymphocyte infiltration was not significant (P > 0.05). However, GF-injection of F-GBN and Mb resulted in greatly higher leukocyte-, specifically lymphocyte-, infiltration in UCD-200 compared to LBL (P < 0.05). For both lines, F-GBN resulted in lymphocyte infiltration by 6 h that reached peak levels on 1-3 d, and remained elevated on 5 and 7 d. Lymphocyte levels in UCD-200 GFs at these time-points were 20%, 55-62%, 45% and 25% of pulp cells, respectively, whereas those in LBL GFs were 11% at 0.25 d and 15-20% on 1, 2, 3 & 7 d. In UCD-200 GFs, 50% of infiltrating lymphocytes were IgM+ B cells, 25% CD4+ T cells, and 25% CD8+ T cells), whereas in LBL chickens IgM+ B cells made up 20% and T cells 80%. Compared to LBL chickens, UCD-200 chickens also had substantially higher lymphocyte infiltration levels in Mb-injected GFs.  2. Contributions of Mark Parcells (DE). Projects in the laboratory addressing Objective 2 are focused on fundamental mechanisms of Marek's disease virus (MDV) pathogenesis and the evolution of virulence of MDV field strains.  2.2.1 Role of Splice Variant-derived Meq Proteins in MDV-induced Lymphoma Formation and Progression. As MDV establishes latency in CD4+ T-cells, the genome expresses splice-variants of the main oncogene, meq (Meq/vIL8, etc.). With collaborators Fiona McCarthy, Shane Burgess and Ken Pendarvis at the University of Arizona, we identified polycomb protein Bmi-1 that associates with Meq/vIL8 and Meq/vIL8∆exon 3 and transport this protein into the nucleolus. Bmi-1 is part of Polycomb Repressive Complex 1 (PRC-1), a complex associated with the silencing of genetic loci during embryogenesis and development. Using small molecule inhibitor, PTC-209, which blocks Bmi-1 expression transcriptionally, we found that the proliferation of MDV-induced cell lines UD35 and UA53, but not MSB-1 cells, was blocked, and that the inhibitory concentration was similar to that used to inhibit human AML and ALL cell lines. We expanded our analysis to examine proteins of PRC-2, which works in tandem with PRC-1 to silence genomic loci. PRC-2 is comprised of the histone 3, lysine 27 trimethyltransferase (H3K27me3) enhancer of zeste 2 (EZH2), embryonic ectodermal development (EED), retinoblastoma binding protein 4 or 7(RBBP4/7), zinc finger protein SUZ12, and Jumangi AT-rich interaction domain protein 2 (Jarid2). Of these, a subset is enriched in the proteome of CD30Hi MDV-induced lymphoma cells (EZH2, EED, RBBP7 and Jarid2). In terms of its relevance to MDV-mediated transformation, we found that the inhibitor of EZH2 (GSK-126) induced cell cycle arrest in MDV-transformed T-cell lines and that this effect was additive with the inhibitor for Bmi-1 (PTC-209). Our current hypothesis is that as MDV establishes latency, the Meq to vIL8 region generates splice variants that have increased affinity for chromatin remodeling complexes (CtBP-1, PRC-1 and PRC-2) and that these are involved in silencing the MDV genome, but are also involved in the transformation of T-cells. 2.2.2 Role of Meq Mutations in Affecting MDV Evolution of Virulence. For this project, collaborators Benedikt Kaufer and Shiro Murata provided us with recombinant viruses in which the meq genes from vaccine strains CVI988 and a vv+MDV strain (N strain, with mutations common to strains 648A and 686) were inserted into the common backbone virus, RB-1B. In a study using these viruses to infect unvaccinated SPF chickens at one day of age, we found no Meq effects on virus replication in spleen cells and PBMC, in mortality or in tumor incidence. In fact, the pRB-1B-CVI988 Meq virus (having the Meq gene from the vaccine CVI988/Rispens) was the most pathogenic in terms of mortality and tumor incidence. In follow-up work to this study, we treated CEF and spleen cells (SPC) infected by these viruses with innate agonists (LPS, Poly I:C, or cGAMP) for 2 hrs prior to evaluating infection via plating on CEF. The number of plaques formed and their relative areas were calculated on triplicate samples, with 50 - 100 plaques per virus. We found that in CEF-infected with the viruses, showed marked inhibition of plaque number and plaque are and that this corresponded to the meq gene encoded in the same background virus. The most stark effects were observed for spleen cells treated with LPS, Poly I:C, and cGAMP compared to medium only. For the pRB-1B CVI988 Meq virus, there was a consistent 30% decrease in plaque area with all three treatments. For the pRB-1B parent virus, there was a consistent 10% decrease in plaque area, and with the pRB-1B N strain Meq virus, there was only a 4% decrease in plaque area. 3. Contributions of Sue Lamont (IA). 3.2.1 Interaction of response to inflammatory stimulus and heat stress in chickens Within a now-terminated USDA-AFRI Climate Change project (led by C. Schmidt, UDEL), we investigated the interaction of two stressors: heat stress and exposure to an inflammation-inducing PAMP (LPS). Birds of two distinct and highly inbred lines (broiler, Fayoumi) that were either exposed to daily cyclic heat episodes or kept at control temperatures were injected with either LPS or saline. Bioinformatic analyses of RNA-seq generated from tissues from birds of each of the four treatment groups was conducted to identify genes and pathways associated with response to the stressors. A paper was submitted and published on the spleen response. Analysis of the bursa and thymus transcriptome of the same birds was completed and the manuscripts are being drafted.  4. Contributions of Matt Koci (NC). 4.2.1 Selection of Salmonella-resistant Chickens. Salmonella causes an estimated one million food-borne illnesses in the US annually, and poultry and poultry products are believed to be one of the major sources of Salmonellosis.  This makes preventing Salmonella colonization of poultry a major priority in the hopes that this will reduce the amount of contaminated poultry products consumed by humans. Our research group has focused on developing ways to augment the chicken’s gut microbiota to aid in preventing colonization.  We have explored how the use of prebiotics (GOS (1 % w/w)) and attenuated Salmonella (attST) as ways to affect the development and structure of the chicken intestinal microbiome, and how these changes affect Salmonella colonization and host immune function. Initial studies examined changes in the microbiome from intestinal samples from 300 animals, collected once a week for 8 weeks. These studies demonstrated that both GOS and the attST strain modified the gut microbiome but the changes were in very different taxonomic groups. The attST treatment resulted in increases of Alistipes and undefined Lactobacillus, while GOS treatment led to increases in Christensenallacea and L. reuteri. Interestingly, the microbiome changes induced by both treatments resulted in a faster clearance after Salmonella challenge at 4 weeks of age.  These studies demonstrate that manipulation of the gut microbiota can enhance the bird’s ability to resist Salmonella colonization. Subsequent studies focused on how the host mucosal immune response may have changed due to the treatments and/or the change in microbiome. Day-old pullet chicks were fed control diets or diets supplemented with GOS (1 % w/w) and then challenged with a cocktail of Salmonella Typhimurium and S. Enteritidis at 3 days of age. As before, GOS treatment group altered the development and structure of the microbiome, and appeared to enhance the bird’s resistance to Salmonella colonization. Interestingly, there was no evidence of any anti-Salmonella specific immune response. There were treatment and challenge specific changes in the expression of various innate immune modulators in the cecal tonsil, while these changes were transient the overall trend suggested a reduction in immune activation following GOS treatment. Collectively these data demonstrated that treatment with the prebiotic GOS can modify both cecal tonsil gene expression and the cecal microbiome, suggesting that this type of treatment may be useful as a tool for altering the carriage of Salmonella in poultry. 5. Contributions of Rami Dalloul (VA) 5.2.1. In ovo probiotics application modulates post hatch gene expression We investigated the effects of in ovo delivery of water-soluble probiotics on hatchability, early post-hatch performance, expression of immunity markers, and response to an enteric challenge in broiler chicks.  Multiple doses of a defined probiotic were administered into the amnion of 18-day-old broiler embryos as they were transferred from the incubator to the hatcher.  Parameters were evaluated and samples collected on multiple days early post hatch and end of each trial.  No negative effects on hatchability were observed in any of the treatments compared to non-injected controls.  Medium doses of the probiotic enhanced early performance as well as reduced coccidia lesion scores in the challenged birds.  Generally, in ovo supplementation of probiotics downregulated innate immune markers up to 10 days post hatch, with differential effects among tissues especially between ileum and cecal tonsils. 6. Contributions of Henk Parmentier - (WUNL) Our studies focus on the following topics: <ol> <li>Effects of husbandry (hygienic conditions, organic feed, housing, e.g. battery cage versus Free range-like) on immune responsiveness of chickens and chicken breeds. - noreport for this period</li> <li>Immuno-modulation of the immune response, especially via the innate immune system, with special emphasis on Natural antibodies, probiotics and PAMP's. - no report for this period</li> <li>Natural antibodies and natural auto-antibodies in chickens (and other species: bovine and pig).</li> <li>Divergent selection of layers to KLH</li> <li>Immunity and behavior - no report for this period</li> <li>Immuno-development - no report for this period</li> <li>Transgenerational priming of innate immunity</li> </ol>  6.2.3.Immunity and natural (auto-) antibodies (H.K. Parmentier, M. Bao, H. Bovenhuis, J vander Poel) Chickens, like mammals have 'natural auto-antibodies' which may be directed to neo-epitopes. These NAAb show heritabilities alike the heritability of natural antibodies binding KLH. IgG and IgM auto-antibody profiles were studied in 5 High line and 5 Low line families from the old SRBC lines and from the new KLH-NAb selection lines by Western blotting. Recognition of auto-antigen fragments for IgG were age dependent but very individually restricted suggesting a stochastic origin, whereas IgM profiles were less individually restricted. No or little obvious parental-neonatal alikeness (including between full sibs) was found. Binding to auto-antigens or related (mammalian) 'auto-antigens' was found in the NAb selection lines using ELISA, which showed similar heritabilities and maternal effects as found for NAbs binding KLH (Mandy Bao). NAb and NAAb profiles were also studied for other species: bovine and pig (Parmentier). An important target for NAAb are phosphate-protein conjugates originating from dying or apoptotic cells. The presence of such antibodies in the selection lines is topic of future studies.  6.2.4. Divergent selection of layers to KLH Nabs: effect on disease resistance (T. Berghof, M. Matthijs, M. Visker, H.K. Parmentier, H. Bovenhuis, and J. vander Poel) An infection experiment with intratracheally administered E. coli of the G4 NAb selection lines revealed a remarkably high difference between the highly resistant High NAb line on the one hand, and the Low NAb line (and the parental elite line on the other hand), suggesting that breeding for higher general resistance on the basis of natural antibody levels may be feasible. Repeating this experiment in the G6, also using a lower dose and including parameters of morbidity and immunity revealed the same results. As yet, the mechanisms behind resistance or susceptibility for mortality and morbidity remained unknown. We intend to write a new project on the resistance of layer birds to E. coli infections. No significant differences were found between the H and L NAb lines with respect to peripheral blood leucocyte cells apart from thrombocytes and numbers of B cells, the latter being significantly higher in the H line.  6.2.5 Transgenerational epigenesis (M. Verwoolde, A. Lammers and H.K. Parmentier) In vitro stimulation assays with PAMP's such as β-glucans and LPS using HD11 cells and primary phagocytes from various tissues including bone marrow revealed 'innate training' as indicated by NO production and cytokine PCR after second challenges with PAMP's in vitro. This will be studied also in vivo using dietary treatments and finally over generations.  <h2>Objective 3 </h2> <h3>Investigators addressing this objective are:</h3> <ol> <li>Gisela Erf-AR</li> <li>Marcia Miller-CA, BRI</li> <li>Rodrigo Gallardo-CA, UCD</li> <li>Yvonne Drechsler and Ellen Collison – CA, WU</li> <li>Mark Parcells - DE</li> <li>Sue Lamont – IA</li> <li>Rami Dalloul – VA</li> </ol> 1. Contributions of Dr. Gisela Erf (AR): 1.3.1. Rescue, establishment and characterization of UCD-200 and Obese-strain chicken populations at the University of Arkansas. Chicken research lines selected for spontaneous and predictable development of autoimmune disease, have made significant contributions to our understanding of the components and mechanisms involved in complex, non-communicable diseases. Two such lines include the Obese strain (OS) originally developed at Cornell University and the UCD-200 originating from the University of California Davis. The OS is one of the most-valued models for studying spontaneously occurring Hashimoto's thyroiditis.  The UCD-200 chicken line is the only model for spontaneously occurring systemic sclerosis/scleroderma that presents the combination of symptoms observed in humans (Wick et al., 2006).  Following their establishment as biomedical research models at Medical schools in Austria (Innsbruck) and Sweden (Uppsala), US maintenance of the UCD-200 and OS lines was discontinued and the genetic stocks destroyed. However, in 2014, urgent requests were sent from both Innsbruck and Uppsala to adopt and rescue these valuable animal models.  A plan was implemented to relocate the respective lines to the University of Arkansas by importing pedigreed hatching eggs. From the imported eggs only one chick from the UCD-206 line imported from Innsbruck survived.  This females was included in the breeding population.  The UCD-206 is a subline of the UCD-200 expressing the B15 MHC-genotype instead of the B2 and B17 in the UCD-200 line. Most of the UCD-200 chicks hatched were from the Sweden population previously established from the Innsbruck populations. Recent MHC-typing conducted by Dr. Janet Fulton, Hy-Line International using a chicken MHC SNP panel revealed the presence of the B2 and B15 MHC haplotypes, no B17, but unexpected expression of the B13 haplotype in the AR-UCD-population.  B13 likely came from the OS population, which was found to be homozygous B13.  All of the OS fertile eggs were imported from Sweden [the original OS population was terminated in Innsbruck after sending fertile eggs to Sweden]. It is not clear, were the mix-up occurred, but all B13 carrying UCD chickens will be excluded from future breeding. Additionally, major fertility problems were encountered with the OS males, even with thyroid hormone supplementation. Improvements have been made and more effective pedigree breeding is underway. Joseph Hiltz, Dr. Anthony's graduate student is conducting various phenotypic measurements on both the OS and UCD lines and is pursuing a plan to generate and document a pedigree that will facilitate the rescue and reestablishment of the respective genetic lines, including a UCD-206 line with B15 MHC haplotype. 1.3.2. Tools to monitor and assess in vivo cellular/tissue responses: the growing feather as an "in vivo test-tube". We continue our research on developing the growing feather as an "in-vivo test-tube" and window into cutaneous in vivo immune activities to intradermally injected test-materials (Erf and Ramachandran, 2016). Special focus was on refining the method and demonstrate its uses. 1.3.2a. Simultaneous assessment of antibody responses and cutaneous cellular responses to protein antigen in chickens following a primary and secondary i.m. immunization. As reported in part last year, we examined local innate (primary exposure), primary effector, and secondary effector responses by injecting antigen (mouse IgG) into GFs of non-immunized and antigen-immunized chickens. To gain insight into effector responses, GFs were injected with Ag during the height of the primary response (10 d after primary intramuscular immunization) or 5 days after the secondary immunization (during the height of the memory response). In addition to local leukocyte infiltration profiles cytokine expression was also assessed in antigen-injected GFs of all treatment groups (no-immunization, primary immunization, and secondary immunization).  Based on the relative quantity, type, and time-course of leukocytes infiltration as well as cytokine expression, this method revealed immune system activities expected for an innate, primary effector and memory effector responses established in mammals. Moreover, sampling of the peripheral blood in the same chickens allowed for simultaneous establishment of antibody responses to the Ag (based on ELISA) that followed classic primary and memory antibody response profiles expected for a T-dependent antigen (in terms of time-course, quantity and isotype switching). This novel approach provided a first insight into systemic humoral as well as local antigen-specific immune responses in the same individuals over a 7 d time-course. This methodology will find important application in vaccine development.  1.3.2b. Simultaneous assessment of cutaneous responses to different test-materials injected into different GF in the same individual. We continue our efforts to determine whether the GF "in vivo test-tube system" can be used to evaluate responses to multiple test-materials in the same chicken. (i.e. i.d. injection of GFs with different test-materials in the same bird, similar to multi-cutaneous testing of responses to allergens in humans). Time-course studies examining leukocyte infiltration profiles following injection of LPS, PGN, PBS or no-injection in individual GFs of the same chickens, revealed the unique leukocyte response profiles expected based on evaluation of a single test-material in an individual.  Similarly, injection of PBS and F-GBN or LPS and Mb in the same chicken resulted in the same response profiles as those observed following injection of only one of the test-materials in a chicken. Importantly, our studies have shown that the GF as a cutaneous test-site reveals differences in immune system responses of different chicken populations. 2. Contributions of Dr. Marcia Miller (CA, BRI): 2.3.1. Development and evaluation of a method for MHC-Y genotyping (Miller, Goto, Zhang, Psifidi, and Fulton).  The sequence determinations have allowed us to further improve MHC-Y typing based on patterns produced by PCR reaction products.  These products reflect differences in sequence and sequence-length of a region immediately upstream of the MHC class I-like YF genes in the region.  We are now genotyping birds in trials for colonization by bacteria. 3. Contributions of Dr. Rodrigo Gallardo (CA, UCD): 3.3.1. The Effect of Diatomaceous Earth in Live Attenuated Infectious Bronchitis Vaccine, Immune Responses and Protection Against Challenge (PI: R. A. Gallardo) Live virus vaccines are commonly used in poultry production, particularly in broilers. Massive application and generation of a protective local mucosal and humoral immunity with no adverse effects is the main goal for this strategy. Live virus vaccines can be improved by adding adjuvants to boost mucosal innate and adaptive responses. In a previous study we showed that diatomaceous earth (DE) can be used as adjuvant in inactivated vaccines. The aim of this study was to test DE as adjuvant in an ArkDPI live infectious bronchitis virus (IBV) vaccine after ocular or spray application.  Titrating the virus alone or after addition of DE showed that DE had no detrimental effect on the vaccine virus. However, adding DE to the vaccine did not induce higher IgG titers in the serum and IgA titers in tears. It also did not affect the frequency of CD4+ T cells, CD8+ T cells and monocytes/macrophages in the blood and the spleen determined by flow cytometry. In addition, protection generated against IBV homologous challenges, measured by viral load in tears, respiratory signs and histopathology in tracheas, did not vary when DE was present in the vaccine formulation. Finally, we confirmed through our observations that Ark vaccines administered by hatchery spray cabinet elicit weaker immune responses and protection against an IBV homologous challenge compared to the same vaccine delivered via ocular route. 4. Contributions of Yvonne Drechsler and Ellen Collison (CA, WU) Yvonne Drechsler has started a collaboration during her sabbatical at the University of Washington to expand her research into epigenetic regulation of differential immune responses in chicken B haplotypes. She has been optimizing ChiP seq and ATAC seq on chicken cells and tissues. Contributions of Mark Parcells (DE) 5.3.1. Innate sensing and mechanism of action of Victrio® Victrio® (https://www.bayerlivestock.com/show.aspx/dna-immunostimulant-victrio, formerly BAY98059, JVRS-100) is a DNA-liposome immune modulator that elicits general protection against bacterial pathogenesis. It has been validated in an avian pathogenic E. coli (APEC) challenge model and can be included with MD vaccination. Our laboratory has performed the licensing tests for this product and have these past two years worked on identifying the mechanism of action (sensor, signaling and outcomes) of this immune stimulant.  With Dr. Ryan Arsenault and Rolf Joerger, we have examined the kinomic signature of a macrophage cell line (HTC), PBMC and primary CEF to Victrio®, and the spectrum of efficacy of antimicrobial peptides elicited within 24 hrs. In short, our data showed the Victrio® was signaling primarily as a dsDNA in the cytoplasm of treated cells, in a manner very similar to kit-prepared plasmid DNA/commercial liposome. Victrio® primarily induced a type I interferon response via STING (stimulator of interferon genes), either through DDX41 or direct activation of cGAS.  5.3.2 Cloning and Expression of Chicken Innate Sensors, Signaling Molecules and Meq-interacting Proteins. We have cloned the chicken innate sensors, second messengers, etc., into various vectors, with epitope-tags in most cases. These chicken protein expression vectors are available to NE-1334 members, upon request. These are useful for protein-protein interaction, co-localization, functional and mobility studies. Innate Sensors: MDA-5, LGP2, DDX41, STING,  Interferon Regulatory Factors: IRF1, IRF4, IRF5, IRF7,  IFN-stimulated genes: ISG15, IRG1, Adapters and Transcription Factors: MyD88, STAT1, STAT3, c-Jun, NFIL-3, C-EBPbeta, PU.1, Maf-B, Chromatin Remodeling Proteins: CtBP-1, Bmi-1, EZH2 and EED Contributions of Sue Lamont (IA) 6.3.1 Genetic population development, maintenance, and characterization. Iowa State University maintains 13 unique chicken genetic lines, which are of two basic genetic structures: (a) highly inbred lines or (b) advanced intercross lines (AIL). Highly inbred lines (70-100 generations of sib-mating) of defined MHC type are maintained, with the inbreeding of the earliest line (Line 8) starting in 1925. Lines are primarily of egg-type origin, but also include the non-commercial Fayoumi and Spanish lines, and one non-inbred broiler line of genetics from about 25 years ago, as well as two advanced intercross lines (now at generation F28) that were initiated by crossing an outbred broiler male with females of two distinct, highly inbred lines (Leghorn and Fayoumi). Birds of the MHC-defined inbred lines are serologically typed each generation with line-specific anti-erythrocyte antisera to verify line purity; all birds are typed as chicks and then potential breeders are typed a second time before mating takes place. These lines are used in research at Iowa State, and shared as chicks, fertile eggs, tissues or DNA with collaborating researchers, including within NE-1334.  Contributions of Rami Dalloul (VA) 7.3.1 ELISA system for house finch IL-1β. We previously reported on the cloning, expression, and characterization of house finch IL-1β in collaboration with Dana Hawley (Biology, VT) and James Adelman (Natural Resource Ecology & Management, Iowa State). This project continued and culminated this year in the development and validation of a direct ELISA system for HfIL-1β using a commercial anti-ChIL-1β pAb.  Two different coating methods were used: carbonate and dehydration. In both methods, antigens (recombinant HfIL-1b or house finch plasma) were serially diluted in carbonate-bicarbonate coating buffer and either incubated at 4 °C overnight or at 60 °C on a heating block for 2 hr. As a result, rHfIL-1β could not be detected by an anti-ChIL-1β pAb when the antigen was coated with carbonate- bicarbonate buffer at 4°C overnight. However, rHfIL-1β was detected by the anti-ChIL-1β pAb when the antigen was coated using a dehydration method by heat (60°C). Using the developed direct ELISA for HfIL-1β with commercial anti-ChIL-1β pAb, we were able to measure plasma IL-1β levels from house finches. 7.3.2 Host defense peptides in turkey poults. In avian species, there are three classes of host defense peptides (HDPs): avian beta defensins (AvBDs), cathelicidins (Cath) and liver-expressed antimicrobial peptide 2 (LEAP-2). The objective was to compare expression of HDPs in male turkey poults at day of

Publications

113 total publications from NE-1334 Project participants 2016-2017  *=17 Cooperative publications among 2 or more project participants *Da Silva A. P., Hauck R., H. Zhou, and R. A. Gallardo. 2017. Understanding immune resistance to infectious bronchitis using major histocompatibility complex chicken lines. Avian Dis. 61(3): 358-365. *Deist M. S., R. A. Gallardo, D. A. Bunn, T. R. Kelly, J. C. M. Dekkers, H. Zhou, and S. J. Lamont. 2017. Novel mechanisms revealed in the trachea transcriptome of resistant and susceptible chicken lines following infection with Newcastle disease virus. Clin. Vacc. Immunol. (In Press) http://cvi.asm.org/content/24/5/e00027-17.short *Deist M.S., R. A. Gallardo, D. A. Bunn, J. C. M. Dekkers, H. Zhou, and S. J. Lamont. 2017. Resistant and susceptible chicken lines show distinctive responses to Newcastle disease virus infection in the lung transcriptome. BMC Genomics. (In press) *Deist, H., R. Gallardo, D. Bunn, T. Kelly, J. Dekkers, H. Zhou, and S. Lamont. 2017. Novel mechanisms revealed in the trachea transcriptome of resistant and susceptible chicken lines following infection with Newcastle disease virus. Clin Vaccine Immunol 24(5). pii: e00027-17. doi: 10.1128/CVI.00027-17. *Deist, M. S., R. A. Gallardo, D. A. Bunn, T. R. Kelly, J. C. M. Dekkers, H. Zhou, and S. J. Lamont. 2017. Resistant and susceptible chicken lines show distinctive responses to Newcastle disease virus infection in the lung transcriptome. BMC Genomics. in press *Deist, M. S., R. A. Gallardo, D. A. Bunn, T. R. Kelly, J. C. M. Dekkers, H. Zhou, and S. J. Lamont. 2017. Novel mechanisms revealed in the trachea transcriptome of resistant and susceptible chicken lines following infection with Newcastle disease virus. Clin. Vaccine Immunol. 24:e00027-17. doi:10.1128/CVI.00027-17. *Dunn, J.R., S. M. Reddy, M. Niikura, V. Nair, J.E. Fulton and H.H. Cheng. 2017.  Evaluation and identification of Marek’s disease virus BAC clones as standardized reagents for research.  Avian Dis. 61:107-114. *Fulton, J.E., A.M. McCarron, A.R. Lund, K.N. Pinegar, A. Wolc, O. Chazara, B. Bed’Hom, M. E. Berres and M.M. Miller, 2016.  A high-density SNP panel reveals extensive diversity, frequent recombination and multiple recombination hotspots within the chicken major histocompatibility complex B region between BG2 and CD1A1.  Genetics Sel. Evol. 48:1 *Fulton, J.E., M. E. Berres, J. Kantanen and M. Honkatukia. 2017.  MHC-B variability within the Finnish Landrace chicken conservation program.  Poult. Sci. in press *Irizarry, K. J. L., E. Downs, R. Bryden, J. Clark, L. Griggs, R. Kopulos, C. M. Boettger, T. J. Carr, C. L. Keeler, E. Collisson, and Y. Drechsler. 2017. RNA sequencing demonstrates large-scale temporal dysregulation of gene expression in stimulated macrophages derived from MHC-defined chicken haplotypes. Plos One 12. *Irizarry, K. J., A. Chan, D. Kettle, S. Kezian, D. Ma, L. Palacios, Q. Q. Li, C. L. Keeler, and Y. Drechsler. 2017. Bioinformatics analysis of chicken miRNAs associated with monocyte to macrophage differentiation and subsequent IFNγ stimulated activation. MicroRNA 6:53–70. *Kogut, M. H., C. L. Swaggerty, J. A. Byrd, R. Selvaraj, and R. J. Arsenault. 2016. Chicken-Specific Kinome Array Reveals that Salmonella enterica Serovar Enteritidis Modulates Host Immune Signaling Pathways in the Cecum to Establish a Persistent Infection. Int. J. Mol. Sci. 17:1207. *Lee, S. H., X. Dong, H. S. Lillehoj, S. J. Lamont, X. Suo, D. K. Kim, K.-W. Lee, and Y. H. Hong. 2016. Comparing the immune responses of two genetically B-complex disparate Fayoumi chicken lines to Eimeria tenella. Brit. Poult. Sci. 57:165–171. doi:10.1080/00071668.2016.1141172 *Miller, M. M., and R. L. Taylor, Jr. 2016.  Brief review of the chicken major histocompatibility complex – the genes, their distribution on chromosome 16 and their contribution to disease resistance. Poult. Sci. 95:375-392 doi:10.3382/ps/pev379 (review) *Nguyen-Phuc, H., J.E. Fulton, and M.E. Berres, 2016.  Genetic variation of Major Histocompatibility Complex (MHC) in wild Red JungleFowl (Gallus gallus).  Poultry Science 95:400-411. *Wang, Y., P. Saelao, K. Chanthavixay, R. A. Gallardo, D. Bunn, S. Lamont, J. Dekkers, T. Kelly, and H. Zhou. 2017. Physiological responses to heat stress in two genetically distinct chicken inbred lines. Poult. Sci. in press *Zhang, J., M. Kaiser, M. Deist, R. A. Gallardo, D. Bunn, T. Kelly, J. Dekkers, H. Zhou, and S. J. Lamont. 2017. Transcriptome analysis in spleen reveals differential regulation of response to Newcastle disease virus in two chicken lines. Sci. Rep. in press   96 publications from individual project participants Ahrens, B. J., L. Li, A. K. Ciminera, J. 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Hsd3b2 associated in modulating steroid hormone synthesis pathway regulates the differentiation of chicken embryonic stem cells into spermatogonial stem cells. J Cell Biochem 119:1111-1121. doi 10.1002/jcb.26279 Zhao, C., X. Li, B. Han, Z. You, L. Qu, C. Liu, J. Song, L. Lian, and N. Yang. 2017. Gga-miR-219b targeting BCL11B suppresses proliferation, migration and invasion of Marek's disease tumor cell MSB1. Sci Rep 7:4247. doi 10.1038/s41598-017-04434-w Zhou, H., P. J. Ross, C. Kern, P. Saelao, Y. Wang, J. L. Chitwood, I. Korf, M. Delany, and H. Cheng. 2016. Genome-wide functional annotation of regulatory elements in chickens. Pp:48-52. The Proceedings of XXV World’s Poultry Congress, Beijing, China. Zhu,Y.,  W. Wang, T. Yuan, L. Fu, L. Zhou, G. Lin, S. Zhao, H. Zhou, G. Wu, and J. Wang. 2017. MicroRNA-29a mediates the impairment of intestinal epithelial integrity induced by intrauterine growth restriction in pig. Am J Physiol Gastrointest Liver Physiol. 312(5):G434-G442. doi: 10.1152/ajpgi.00020.2017. Epub 2017 Mar 9. Zuo, Q., K. Jin, Y. Zhang, J. Song, and B. Li. 2017. Dynamic expression and regulatory mechanism of TGF-beta signaling in chicken embryonic stem cells differentiating into spermatogonial stem cells. Biosci Rep 37. doi 10.1042/BSR20170179 Zuo, Q., Y. Wang, S. Cheng, C. Lian, B. Tang, F. Wang, Z. Lu, Y. Ji, R. Zhao, W. Zhang, K. Jin, J. Song, Y. Zhang, and B. Li. 2016. Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology. G3 (Bethesda) 6:1787-1792. doi 10.1534/g3.116.028803   5 total book chapters from NE-1334 Project participants 2016-2017 Erf, G. F., and I. C. Le Poole. 2017. Animal Models for Vitiligo; in: Vitiligo, 2nd edition. M. Picardo and A. Taieb, editors; Springer, SPi Global in press He, Y. and J. Song. 2017 Bioinformatics analysis of Epigenetics. Bioinformatics in Aquaculture: Principles and Methods, First Edition. © 2017 John Wiley & Sons Ltd. Koci MD and S. Schultz-Cherry. 2017. Astrovirus. Pages 26-38 in Food Microbiology Series: Laboratory Models for Foodborne Infections. D. Liu, ed. CRC Press, Boca Raton. 2017. ISBN: 978-1-4987-2168-4. Webb, K. C., S. W. Henning, G. F. Erf, and I. C. Le Poole. 2017. Autoimmune Pathology of Vitiligo; in: Vitiligo, 2nd edition. M. Picardo and A. Taieb, editors; Springer, SPi Global in press Wolc, A, and J.E. Fulton, 2016.  Molecular breeding techniques to improve egg quality.  In Achieving Sustainable Production of Eggs. Chapter 19.   Ed. J. Roberts.

Impact Statements

Impact Statements of Objective 3: Develop and evaluate methodologies and reagents to assess immune function and disease resistance to enhance production efficiency through genetic selection in poultry. Investigators addressing this objective are: 1. Gisela Erf-AR 2. Marcia Miller-CA, BRI 3. Rodrigo Gallardo-CA, UCD 4. Yvonne Drechsler - CA, WU Ellen Collison - CA, WU 5. Mark Parcells - DE 6. Sue Lamont - IA 7. Rami Dalloul - VA 1. Dr. Gisela Erf (AR): Impacts: 1.1 The development of the "in vivo test-tube system" using the growing feathers as a dermal test-site provides an important tool to monitor and evaluate local cellular immune system activities in a complex tissue and explore the immunological mechanisms underlying disease susceptibility and resistance in poultry. 1.2 The GF in vivo test-system together with sampling the peripheral blood or other body fluids provides a minimally invasive, two-window approach for comprehensive monitoring and assessment of local and systemic immune system activities. 2. Dr. Marcia Miller (CA, BRI): Impact: 2.1 These determinations are greatly advancing understanding how MHC-Y class I molecules likely function. Hypotheses have been developed for how MHC-Y may affect host pathogen interactions and are now under investigation. 3. Dr. Rodrigo Gallardo (CA, UCD): Impact: 3.1 Testing new adjuvants to use in poultry vaccines may provide a novel method to boost immune responses. 4. Yvonne Drechsler and Ellen Collison (CA, WU) Impact: (none listed) 5. Mark Parcells (DE) Impacts: 5.1 Use of the reagents will aid in the elucidation of their functions in immunity, as well as targets in MDV pathogenesis. 6. Sue Lamont (IA) Impacts: (none listed) 7. Rami Dalloul (VA) Impacts: 7.1 House finch IL-1β ELISA: A unique clade of the bacterium Mycoplasma gallisepticum (MG) has resulted in annual epidemics of conjunctivitis in North American house finches since the 1990s. Currently, few immunological tools have been validated for this songbird species. IL-1β is a prototypic multifunctional cytokine and can affect almost every cell type during Mycoplasma infection. This project developed and validate a direct ELISA assay for house finch IL-1β using a cross-reactive chicken antibody. This work provides a critical tool to continue studying the ecological disease model and host-pathogen interactions of house finches and MG, with implications on transmission to poultry species. 7.2 Host defense peptides: HDPs are a large group of small positively charged peptides that play an important role in innate immunity. Their role is more critical at early ages when other components of the immune system have not fully developed. There are three classes of avian HDPs: avian beta defensins (AvBDs), cathelicidins (Cath) and liver-expressed antimicrobial peptide 2 (LEAP-2). Understanding the differential expression of HDPs, in health and disease conditions, could reveal the innate immune status of male and female poults, and may subsequently allow improvement of their health through appropriate early mitigation strategies.