Brief Summary of Minutes of Annual Meeting

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:

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:

Objective 1 

Investigators addressing this objective are:

1. Marcia Miller-CA, BRI
2. Rodrigo Gallardo-CA, UCD
3. Huaijin Zhou – CA, UCD
4. Yvonne Drechsler and Ellen Collison – CA, WU
5. Mark Parcells - DE
6. Sue Lamont – IA
7. Matt Koci – NC
8. Rami Dalloul – VA
9. Robert Taylor – WV
10. Mark Beres – WI
11. Henk Parmentier - WUNL

 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.

Objective 2

 Investigators addressing this objective are:

1. Gisela Erf – AR
2. Mark Parcells – DE
3. Sue Lamont – IA
4. Matt Koci – NC
5. Rami Dalloul – VA
6. Henk Parmentier - WUNL

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:

1. 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
2. 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
3. Natural antibodies and natural auto-antibodies in chickens (and other species: bovine and pig).
4. Divergent selection of layers to KLH
5. Immunity and behavior - no report for this period
6. Immuno-development - no report for this period
7. Transgenerational priming of innate immunity

 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. 

Objective 3 

Investigators addressing this objective are:

1. Gisela Erf-AR
2. Marcia Miller-CA, BRI
3. Rodrigo Gallardo-CA, UCD
4. Yvonne Drechsler and Ellen Collison – CA, WU
5. Mark Parcells - DE
6. Sue Lamont – IA
7. Rami Dalloul – VA

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

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. Chea, E. Poku, J. R. Bading, M. R. Weist, M. M. Miller, D. M. Colcher, and J. E. Shively. 2017. Diagnostic PET Imaging of Mammary Microcalcifications Using 64Cu-DOTA-Alendronate in a Rat Model of Breast Cancer. J. Nuclear Med. 58:1373-1379.

Allali, I., J. W. Arnold, J. Roach, M. B. Cadenas, N. Butz, H. M. Hassan, M. Koci, A. Ballou, M. Mendoza, R. Ali, and M. A. Azcarate-Peril. 2017. A comparison of sequencing platforms and bioinformatics pipelines for compositional analysis of the gut microbiome. BMC Microbiol 17:194. doi 10.1186/s12866-017-1101-8.

Arsenault, R. J., J. T. Lee, R. Latham, B. Carter, and M. H. Kogut. 2017.  Changes in immune and metabolic gut response in broilers fed β-mannanase in β-mannan-containing diets. Poult. Sci. Sep 14. doi: 10.3382/ps/pex246.

Arsenault, R. J., K. J. Genovese, H. He, H. Wu, A. S. Neish, and M. H. Kogut. 2016. Wild-type and mutant AvrA−Salmonella induce broadly similar immune pathways in the chicken ceca with key differences in signaling intermediates and inflammation. Poult. Sci. 95:354–363.

Ballou, A. L., R. A. Ali, M. A. Mendoza, J. C. Ellis, H. M. Hassan, W. J. Croom, and M. D. Koci. 2016. Development of the Chick Microbiome: How Early Exposure Influences Future Microbial Diversity. Front Vet Sci 3:2. doi 10.3389/fvets.2016.00002

Barrios, M. A., A. Kenyon, and R. B. Beckstead. 2017. Development of a dry medium for isolation of Histomonas meleagridis in the field. Avian Dis. 61:242-244

Bickhart, D. M., L. Xu, J. L. Hutchison, J. B. Cole, D. J. Null, S. G. Schroeder, J. Song, J. F. Garcia, T. S. Sonstegard, C. P. Van Tassell, R. D. Schnabel, J. F. Taylor, H. A. Lewin, and G. E. Liu. 2016. Diversity and population-genetic properties of copy number variations and multicopy genes in cattle. DNA Res 23:253-262. doi 10.1093/dnares/dsw013

Carrillo, J. A., Y. He, Y. Li, J. Liu, R. A. Erdman, T. S. Sonstegard, and J. Song. 2016. Integrated metabolomic and transcriptome analyses reveal finishing forage affects metabolic pathways related to beef quality and animal welfare. Sci Rep 6:25948. doi 10.1038/srep25948

Chen, H., Q. Zuo, Y. Wang, J. Song, H. Yang, Y. Zhang, and B. Li. 2017. Inducing goat pluripotent stem cells with four transcription factor mRNAs that activate endogenous promoters. BMC Biotechnol 17:11. doi 10.1186/s12896-017-0336-7

Chen, H., Q. Zuo, Y. Wang, M. F. Ahmed, K. Jin, J. Song, Y. Zhang, and B. Li. 2017. Regulation of Hedgehog Signaling in Chicken Embryonic Stem Cells Differentiation Into Male Germ Cells (Gallus). J Cell Biochem 118:1379-1386. doi 10.1002/jcb.25796

Chen, Y., J. Stookey, R. Arsenault, E. Scruten, P. Griebel, and S. Napper. 2016. Investigation of the physiological, behavioral, and biochemical responses of cattle to restraint stress. J. Anim. Sci. 94:3240–3254.

Chou, W.-K., C.-H. Chen, C. N. Vuong, D. Abi-Ghanem, S. D. Waghela, W. Mwangi, L. R. Bielke, B. M. Hargis, and L. R. Berghman. 2016. Significant mucosal sIgA production after a single oral or parenteral administration using in vivo CD40 targeting in the chicken. Res. Vet. Sci. 108:112–115.

Clarke, L. L, R. B. Beckstead, J. R. Hayes, and D. R. Rissi. 2017. Pathologic and molecular characterization of histomoniasis in peafowl (Pavo cristatus). J. Vet. Diagn. Invest. 29:237-241

Collisson, E., L. Griggs, and Y. Drechsler. 2017. Macrophages from disease resistant B2 haplotype chickens activate T lymphocytes more effectively than macrophages from disease susceptible B19 birds. Developmental & Comparative Immunology 67:249–256.

Cooper, J., Y. Ding, J. Song, and K. Zhao. 2017. Genome-wide mapping of DNase I hypersensitive sites in rare cell populations using single-cell DNase sequencing. Nat Protoc 12:2342-2354. doi 10.1038/nprot.2017.099

Derksen T. J., Lampron R., Hauck R., M. Pitesky, and R. A. Gallardo. 2017. Biosecurity assessment and seroprevalence of respiratory diseases in backyard poultry flocks located close and far from commercial premises. Avian Dis. (In press).

Drobik-Czwaro, W., A. Wolc, J.E. Fulton, J. Arango, T. Jankowski, N.P. O’Sullivan and J.C.M. Dekkers, 2017.  Identifying the genetic basis for resistance to avian influenza in commercial egg layer chickens.  Animal.  doi: 10.1017/S1751731117002889

Erf, G. F., and I. R. Ramachandran. 2016. The growing feather as a dermal test-site: comparison of leukocyte profiles during the response to Mycobacterium butyricum in growing feathers, wattles, and wing webs. Poult. Sci.: 95:2011-2022.

Erf, G. F., D. M. Falcon, K. A. Sullivan, and S.E. Bourdo. 2017. T lymphocytes dominate local leukocyte infiltration in response to intradermal injection of functionalized graphene-based nanomaterial. J. Applied Toxicol. 37:1317-1324.

Erf, G. F., H. R. Kong, K. A. Byrne, D. M. Falcon, and Z. Aguilar. 2017. Novel approach to simultaneously assess and monitor an individual’s humoral and cellular immune responses in the chicken model. PLOS ONE (submitted; PONE-D-17-42058).

Figueroa A., R. Hauck, J. Saldias-Rodriguez, and R. A. Gallardo. 2017. Combination of quaternary ammonia and glutaraldehyde as a disinfectant against enveloped and non-enveloped viruses. J. Appl. Poult. Res. 26(4): 491-497.

Fulton, J.E., A.R. Lund, A.M. McCarron, K.N. Pinegar, D.R. Korver, H.L. Classen, S. Aggrey, C. Utterbach, N.B. Anthony and M.E. Berres, 2016.  MHC variability in heritage breeds of chickens.  Poultry Science 95:393-399.

Grenier, B., I. Dohnal, R. Shanmugasundaram, S. D. Eicher, R. K. Selvaraj, G. Schatzmayr, and T. J. Applegate. 2016. Susceptibility of Broiler Chickens to Coccidiosis When Fed Subclinical Doses of Deoxynivalenol and Fumonisins-Special Emphasis on the Immunological Response and the Mycotoxin Interaction. Toxins (Basel) 8.

Han, B., L. Lian, X. Li, C. Zhao, L. Qu, C. Liu, J. Song, and N. Yang. 2016a. Chicken gga-miR-103-3p Targets CCNE1 and TFDP2 and Inhibits MDCC-MSB1 Cell Migration. G3 (Bethesda) 6:1277-1285. doi 10.1534/g3.116.028498

Han, B., L. Lian, X. Li, C. Zhao, L. Qu, C. Liu, J. Song, and N. Yang. 2016b. Chicken gga-miR-130a targets HOXA3 and MDFIC and inhibits Marek's disease lymphoma cell proliferation and migration. Mol Biol Rep 43:667-676. doi 10.1007/s11033-016-4002-2

Han, B., Y. He, L. Zhang, Y. Ding, L. Lian, C. Zhao, J. Song, and N. Yang. 2017. Long intergenic non-coding RNA GALMD3 in chicken Marek's disease. Sci Rep 7:10294. doi 10.1038/s41598-017-10900-2

Hauck R., B. Crossley, D. Rejmanek, H. Zhou, and R. A. Gallardo. 2017. Persistence of high and low pathogenic avian influenza viruses in footbaths and poultry manure. Avian Dis. 61:64-69.

Hauck R., C. G. Sentíes-Cué, Y. Wang, C. Kern, H. L. Shivaprasad, H. Zhou, and R. A. Gallardo. 2017. Evolution of avian encephalomyelitis virus during embryo adaptation. Vet. Microbiol. 204:1-7.

Hauck, R., C. G. Sentíes-Cué, Y. Wang, C. Kern, H. L. Shivaprasad, H. Zhou, and R. A. Gallardo. 2017. Evolution of avian encephalomyelitis virus during embryo-adaptation. Vet Microbiol 204:1-7. doi: 10.1016/j.vetmic.2017.04.005. Epub 2017 Apr 9.

Hauck, R., D. Crossley, D. Rejmanek, H. Zhou, and R. A. Gallardo. 2017. Persistence of high and low pathogenic avian influenza viruses in footbaths and poultry manure. Avian Dis 61:64-69.

He, Y. H., S. Y. Pu, F. H. Xiao, X. Q. Chen, D. J. Yan, Y. W. Liu, R. Lin, X. P. Liao, Q. Yu, L. Q. Yang, X. L. Yang, M. X. Ge, Y. Li, J. J. Jiang, W. W. Cai, and Q. P. Kong. 2016. Improved lipids, diastolic pressure and kidney function are potential contributors to familial longevity: a study on 60 Chinese centenarian families. Sci Rep 6:21962. doi 10.1038/srep21962

He, Y., M. Song, Y. Zhang, X. Li, J. Song, Y. Zhang, and Y. Yu. 2016. Whole-genome regulation analysis of histone H3 lysin 27 trimethylation in subclinical mastitis cows infected by Staphylococcus aureus. BMC Genomics 17:565. doi 10.1186/s12864-016-2947-0

He, Y., Y. Ding, F. Zhan, H. Zhang, B. Han, G. Hu, K. Zhao, N. Yang, Y. Yu, L. Mao, and J. Song. 2016. Corrigendum: The conservation and signatures of lincRNAs in Marek's disease of chicken. Sci Rep 6:19422. doi 10.1038/srep19422

Heidaritabar, M., A. Wolc, J. Arango, J. Zeng, P.Settar, J.E. Fulton, N.P.O’Sullivan, J.W.M, Bastiaansen, R.L. Fernando, D.J. Garrick, JCM. Dekkers, 2016. Impact of fitting dominance and additive effects on accuracy of genomic prediction of breeding values in layers. J. Anim. Breed. Genet. 5:334-346.

Hughes, R. A., R. A. Ali, M. A. Mendoza, H. M. Hassan, and M. D. Koci. 2017. Impact of dietary galacto-oligosaccharide (GOS) on chicken's gut microbiota, mucosal gene expression, and Salmonella colonization. Front Vet Sci 4:192. doi 10.3389/fvets.2017.00192

Jia, X., Q. Nie, X. Zhang, L. K. Nolan, and S. J. Lamont. 2017. Novel miRNA involved in host response to avian pathogenic Escherichia coli identified by deep sequencing and integration analysis. Infect. Immun. 85:e00688-16. doi.org/10.1128/IAI.00688-16

Kogut, M. H., and R. J. Arsenault. 2017. Immunometabolic Phenotype Alterations Associated with the Induction of Disease Tolerance and Persistent Asymptomatic Infection of Salmonella in the Chicken Intestine. Front. Immunol. 8

Kogut, M. H., K. J. Genovese, H. He, and R. J. Arsenault. 2016a. AMPK and mTOR: sensors and regulators of immunometabolic changes during Salmonella infection in the chicken. Poult. Sci. 95:345–353.

Kropp, J., J. A. Carrillo, H. Namous, A. Daniels, S. M. Salih, J. Song, and H. Khatib. 2017. Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos. BMC Genomics 18:280. doi 10.1186/s12864-017-3673-y

Lai, F. N., H. L. Zhai, M. Cheng, J. Y. Ma, S. F. Cheng, W. Ge, G. L. Zhang, J. J. Wang, R. Q. Zhang, X. Wang, L. J. Min, J. Z. Song, and W. Shen. 2016. Whole-genome scanning for the litter size trait associated genes and SNPs under selection in dairy goat (Capra hircus). Sci Rep 6:38096. doi 10.1038/srep38096

Lan, X., Y. Wang, K. Tian, F. Ye, H.-D. Yin, X.-L. Zhao, H.-Y. Xu, Y. Huang, H. Liu, J. Hsieh, S. Lamont, and Q. Zhu. 2017. Integrated host and viral transcriptome analyses reveal pathology and inflammatory response mechanisms to ALV-J injection in SPF chickens. Sci. Reports 7:46156. doi: 10.1038/srep46156

Lee, M. O., H.-J. Jang, D. Rengaraj, S.-Y. Yang, J. Y. Han, S. J. Lamont, and J. E. Womack. 2016. Tissue expression and antibacterial activity of host defense peptides in chicken. BMC Vet Res 12:231. doi: 10.1186/s12917-016-0866-6

Lee, M. O., L. Andersson, S. J. Lamont, J. Chen, and J. E. Womack. 2016. Duplication of defensin7 gene in Fayoumi chickens generated by gene conversion and homologous recombination. PNAS 113:13815–13820. doi: 10.1073/pnas.1616948113

Li, D., Y. Ji, F. Wang, Y. Wang, M. Wang, C. Zhang, W. Zhang, Z. Lu, C. Sun, M. F. Ahmed, N. He, K. Jin, S. Cheng, Y. Wang, Y. He, J. Song, Y. Zhang, and B. Li. 2017. Regulation of crucial lncRNAs in differentiation of chicken embryonic stem cells to spermatogonia stem cells. Anim Genet 48:191-204. doi 10.1111/age.12510

Luoma, A., A. Markazi, R. Shanmugasundaram, G. R. Murugesan, M. Mohnl, and R. Selvaraj. 2017. Effect of synbiotic supplementation on layer production and cecal Salmonella load during a Salmonella challenge. Poult Sci. doi: 10.3382/ps/pex251.

Markazi, A. D., V. Perez, M. Sifri, R. Shanmugasundaram, and R. K. Selvaraj. 2017. Effect of whole yeast cell product supplementation (CitriStim(R)) on immune responses and cecal microflora species in pullet and layer chickens during an experimental coccidial challenge. Poult. Sci. 96:2049-2056.

Mason, A.S., J.E. Fulton, P.M Hocking and D.W. Burt, 2016.  A new look at the LTR retrotansposon content of the chicken genome.  BMC Genomics 17:688. doi: 10.1186/s12864-016-3043-1

McPherson, M.C., and M.E. Delany. 2016. Virus and host genomic, molecular and cellular interactions during Marek’s disease pathogenesis and oncogenesis. Poultry Science 95:412-429. http://ps.oxfordjournals.org/content/early/2016/01/14/ps.pev369.full.pdf?papetoc

McPherson, M.C., H.H Cheng, and M.E. Delany. 2016. Marek’s disease herpesvirus vaccines integrate into chicken host chromosomes yet lack a virus-host phenotype associated with oncogenic transformation.  Vaccine 34:5554-5561 http://dx.doi.org/10.1016/j.vaccine.2016.09.051 (Highlighted article November 2016: http://www.journals.elsevier.com/vaccine/highlighted-articles/highlighted-article-november-2016

Meliopoulos, V. A., S. A. Marvin, P. Freiden, L. A. Moser, P. Nighot, R. Ali, A. Blikslager, M. Reddivari, R. J. Heath, M. D. Koci, and S. Schultz-Cherry. 2016. Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo. MBio 7. doi 10.1128/mBio.01494-16

Nazmi A., R. Hauck, A. Davis, M. Hildebrand, L.B. Corbeil, and R. A. Gallardo. 2017.  Diatoms and diatomaceous earth as novel poultry vaccine adjuvants.  Poult. Sci. 92:288-294

Neerukonda, S.N., U. K. Katneni, S. Golovan, and M. S. Parcells. 2016. Evaluation and validation of reference gene stability during Marek’s disease virus (MDV) infection. J. Virol. Methods 236:111-116.

Neerukonda, S. N., N. A. Egan, and M. S. Parcells. 2017. Exosomal communication during infection, inflammation, and virus-associated pathology. J. Cancer and Thera. Sci.,1(1)https://inscienz.com/journals/cancer/JCTS-1-104.php

Nazmi A., R. Hauck, L. B. Corbeil, and R. A. Gallardo. 2017. The effect of diatomaceous earth in live attenuated infectious bronchitis vaccine, immune responses and protection against challenge. Poult. Sci. 96:8 2623-2629.

Padhi, A., and M. S. Parcells. 2016. Positive selection drives rapid evolution of the meq oncogene of Marek’s disease virus. PLoS ONE Sep 23;11(9):e0162180. doi: 10.1371/journal.pone.0162180.

Perez, V., R. Shanmugasundaram, M. Sifri, T. M. Parr, and R. K. Selvaraj. 2017. Effects of hydroxychloride and sulfate form of zinc and manganese supplementation on superoxide dismutase activity and immune responses post lipopolysaccharide challenge in poultry fed marginally lower doses of zinc and manganese. Poult. Sci.. doi: 10.3382/ps/pex244

Placek, K., G. Hu, K. Cui, D. Zhang, Y. Ding, J. E. Lee, Y. Jang, C. Wang, J. E. Konkel, J. Song, C. Liu, K. Ge, W. Chen, and K. Zhao. 2017. MLL4 prepares the enhancer landscape for Foxp3 induction via chromatin looping. Nat Immunol 18:1035-1045. doi 10.1038/ni.3812

Rezvani, M., M. Mendoza, M. D. Koci, C. Daron, J. Levy, and H. M. Hassan. 2016a. Draft Genome Sequence of Lactobacillus crispatus C25 Isolated from Chicken Cecum. Genome Announc 4. doi 10.1128/genomeA.01223-16

Rezvani, M., M. Mendoza, M. D. Koci, C. Daron, J. Levy, and H. M. Hassan. 2016b. Draft Genome Sequences of Lactobacillus animalis Strain P38 and Lactobacillus reuteri Strain P43 Isolated from Chicken Cecum. Genome Announc 4. doi 10.1128/genomeA.01229-16

Schaal, T.P., J. Arango, A. Wolc, J.V. Brady, J.E. Fulton, I. Rubinoff, I.J. Ehr, M.E. Persia and N.P. O’Sullivan, 2016.  Commercial Hy-Line W-36 pullet and laying hen venous blood gas and chemistry profiles utilizing the portable i-STAT 1 analyzer.  Poultry Science 95:466-471.

Schock, E.N., C.-F. Chang, I. Youngworth, M. Davey, M.E.  Delany, and S.A. Brugmann. 2016. Utilizing the chicken as an animal model for human craniofacial ciliopathies. Developmental Biology 45(2):326-337. http://dx.doi.org/10.1016/j.ydbio.2015.10.024 (PMID: 26597494)

Shi, S., Y. Shen, S. Zhang, Z. Zhao, Z. Hou, H. Zhou, J. Zou, and Y. Guo. 2017. Combinatory evaluation of transcriptome and metabolome profiles of low temperature-induced resistant ascites syndrome in broiler chickens. Sci Rep 7(1):2389. doi: 10.1038/s41598-017-02492-8.

Slawinska, A., J. C.-F. Hsieh, C. Schmidt, and S. J. Lamont. 2016. Heat stress and lipopolysaccharide stimulation of chicken HD11 cell line activates expression of distinct sets of genes. PLOS ONE. doi:10.1371/journal.pone.0164575

Stepicheva, N. A., and J. L. Song. 2016. Function and regulation of microRNA-31 in development and disease. Mol Reprod Dev 83:654-674. doi 10.1002/mrd.22678

Sullivan K. A., and G. F. Erf. 2017. Leukocyte infiltration profiles during the cutaneous phytohemagglutinin response. Poult. Sci. 96:3574-3580.

Sun, H., P. Liu, L. K. Nolan, and S. J. Lamont. 2016. Thymus transcriptome reveals novel pathways in response to avian pathogenic Escherichia coli infection. Poult. Sci. 95:2803–2814.  doi: 10.3382/ps/pew202

Sun, H., R. Bi, P. Liu, L. K. Nolan, and S. J. Lamont. 2016. Combined analysis of primary lymphoid tissues' transcriptomic response to extra-intestinal Escherichia coli (ExPEC) infection. Dev Comp Immunol 57: 99–106

Swaggerty, C. L., I. Y. Pevzner, H. He, K. J. Genovese, and M. H. Kogut. 2017. Selection for pro-inflammatory mediators produces chickens more resistant to Campylobacter jejuni. Poult. Sci. 96:1623-1627.

Swaggerty, C. L., J. L. McReynolds, J. A. Byrd, I. Y. Pevzner, S. E. Duke, K. J. Genovese, H. He, and M. H. Kogut. 2016. Selection for pro-inflammatory mediators produces chickens more resistant to Clostridium perfringens-induced necrotic enteritis. Poult. Sci. 95:370-374.

Swaggerty, C. L., Kogut, M. H., He, H., Genovese, K. J., Johnson, C., and Arsenault, R. J. 2017. Differential levels of cecal colonization by Salmonella enteritidis in chickens triggers distinct immune kinome profiles.  Front. Vet. Sci. | doi: 10.3389/fvets.2017.00214 

Taylor, R. L., Jr.  2016. Letter to the Editor – A publication experiment.  Poult. Sci. 95:227 doi:10.3382/ps/pev451

Taylor, R. L., Jr. 2016.  Nunc Dimitis - W. Elwood Briles. Poult. Sci. 95:2477 doi:10.3382/ps/pew176

Taylor, R. L., Jr. 2017.  Renew the priority for manuscript review.  Poult. Sci. 96:4133 doi 10.3382/ps/pex267

Taylor, R. L., Jr., Z. Medarova, and W. E. Briles. 2016.  Immune effects of chicken non-Mhc alloantigens. Poult. Sci. 95:447-457 doi:10.3382/ps/pev331 (review)

Tilley, J. E. N., J. L. Grimes, M. D. Koci, R. A. Ali, C. R. Stark, P. K. Nighot, T. F. Middleton, and A. C. Fahrenholz. 2017. Efficacy of feed additives to reduce the effect of naturally occurring mycotoxins fed to turkey hen poults reared to 6 weeks of age. Poult Sci. doi 10.3382/ps/pex214

Tuggle, C. K., E. Giuffra, S. N. White, L. Clarke, H. Zhou, P. J. Ross, H. Acloque, J. M. Reecy, A. Archibald, R. R. Bellone, M. Boichard, A. Chamberlain, H. Cheng, R. P. Crooijmans, M. E. Delany, C. J. Finno, M. A. Groenen, B. Hayes, J. K. Lunney, J. L. Petersen, G. S. Plastow, C. J. Schmidt, J. Song, and M. Watson. 2016. GO-FAANG meeting: a Gathering On Functional Annotation of Animal Genomes. Anim Genet 47:528-533. doi 10.1111/age.12466

Tuggle, C., E. Giuffra, S. N. White, L. Clarke, H. Zhou, P. J. Ross, H. Acloque, J. M. Reecy, A. Archibald, R. R. Bellone, M. Boichard, A. Chamberlain, H. Cheng, R. P.M.A. Crooijmans, M. E. Delany, C. J. Finno, M. A. M. Groenen, B. Hayes, J. K. Lunney, J. L. Petersen, G. S. Plastow, C. J. Schmidt, J. Song, and M. Watson. 2016. “GO-FAANG meeting: a Gathering on functional annotation of animal genomes” Animal Genet DOI: 10.1111/age.12466.

Tuo, W., L. Li, Y. Lv, J. Carrillo, D. Brown, W. C. Davis, J. Song, D. Zarlenga, and Z. Xiao. 2016. Abomasal mucosal immune responses of cattle with limited or continuous exposure to pasture-borne gastrointestinal nematode parasite infection. Vet Parasitol 229:118-125. doi 10.1016/j.vetpar.2016.10.005

Van Goor, A., A. Slawinska, C. J. Schmidt, and S. J. Lamont. 2016. Distinct functional responses to stressors of bone marrow derived dendritic cells from diverse inbred chicken lines. Dev. Comp. Immunol. 63: 96–110

Van Goor, A., C. M. Ashwell, M. E. Persia, M. F. Rothschild, C. J. Schmidt, and S. J. Lamont. 2017. Unique genetic responses revealed in RNA-seq of the spleen of chickens stimulated with lipopolysaccharide and heat. PLOS ONE 12(2): e0171414. doi:10.1371/journal.pone.0171414

Vuong, C. N., W.-K. Chou, V. A. Kuttappan, B. M. Hargis, L. R. Bielke, and L. R. Berghman. 2017. A Fast and Inexpensive Protocol for Empirical Verification of Neutralizing Epitopes in Microbial Toxins and Enzymes. Front. Vet. Sci. 4 Available at https://www.frontiersin.org/articles/10.3389/fvets.2017.00091/full

Wang, X., J. Liu, G. Zhou, J. Guo, H. Yan, Y. Niu, Y. Li, C. Yuan, R. Geng, X. Lan, X. An, X. Tian, H. Zhou, J. Song, Y. Jiang, and Y. Chen. 2016. Whole-genome sequencing of eight goat populations for the detection of selection signatures underlying production and adaptive traits. Sci Rep 6:38932. doi 10.1038/srep38932

Warren, W.C., L.W. Hillier, C. Tomlinson, P. Minx, M. Kremitzki, T. Graves, C. Markovic, N. Bouk, K. Pruitt, F. Thibaud-Nissen, V. Schneider. T. Mansour, C.T. Brown, A. Zimin, R. Hawken, A.B. Pyrkosz, M. Morisson, V. Fallon, A. Vignal, W. Chow, K. Howe, J.E. Fulton, M.M. Miller, P.I. Lovell, C. Mello, M. Wirthlin, A.S. Mason, R. Kuo, D.W. Burt, J.B Dodgson and H.H. Cheng, 2017.  A new chicken genome assembly provides insight into avian genome structure. G3: Genes, Genomes, Genetics, 7 (1) p 109-117.  doi:10.1534/g3.116.035923

Weng, Z., A. Wolc, X. Shen, R.L. Fernando, J.C.M. Dekkers. J. Arango. P. Settar, J.E. Fulton, N.P. O’Sullivan, and D.J. Garrick, 2016.  Effects of number of training generations on genomic prediction for various traits in a layer chicken population.  Genetics Selection Evolution 48:22, DOI 10.1186/s12711-016-0198-9.

Wolc, A., A. Kranis, J. Arango, P. Settar, J.E. Fulton, N.P. O’Sullivan, A. Avendano, K.A. Watson, J.M. Hickey, G. de los Campos, R.L. Fernando, D.J. Garrick and J.C.M. Dekkers, 2016.  Implementation of genomic selection in the poultry industry.  Animal Frontiers 6: 23-31.

Wolc, A., J. Arango, P. Settar, J.E. Fulton, N.P. O’Sullivan, J.C.M. Deckers, R. Fernando and D.J. Garrick, 2016.  Mixture models detect large effect QTL better than GBLUP and result in more accurate and persistent predictions.  J. Anim. Sci. Biotech. doi 10.1186/s40104-016-0066-z

Xu, L., R. J. Haasl, J. Sun, Y. Zhou, D. M. Bickhart, J. Li, J. Song, T. S. Sonstegard, C. P. Van Tassell, H. A. Lewin, and G. E. Liu. 2017. Systematic Profiling of Short Tandem Repeats in the Cattle Genome. Genome Biol Evol 9:20-31. doi 10.1093/gbe/evw256

Xu, L., Y. He, Y. Ding, G. Sun, J. A. Carrillo, Y. Li, M. M. Ghaly, L. Ma, H. Zhang, G. E. Liu, and J. Song. 2017. Characterization of Copy Number Variation's Potential Role in Marek's Disease. Int J Mol Sci 18. doi 10.3390/ijms18051020

Xu, L., Y. Hou, D. M. Bickhart, Y. Zhou, H. A. Hay el, J. Song, T. S. Sonstegard, C. P. Van Tassell, and G. E. Liu. 2016. Population-genetic properties of differentiated copy number variations in cattle. Sci Rep 6:23161. doi 10.1038/srep23161

Zar Mon, K. K., P. Saelao, M. M. Halstead, G. Chanthavixay, H.-C. Chang, L. Garas, E. A Maga, and H. Zhou. 2016. Salmonella enterica serovars Enteritidis infection alters the indigenous microbiota diversity in young layer chicks. Front. Vet. Sci. - Veterinary Infectious Diseases. 2:61. doi: 10.3389/fvets.2015.00061.

Zhang, C., M. Wang, N. He, M. F. Ahmed, Y. Wang, R. Zhao, X. Yu, J. Jin, J. Song, Q. Zuo, Y. Zhang, and B. Li. 2018. 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.