Progress 06/01/17 to 05/31/20
Outputs
Target Audience:The products of this research project are of immediate value to those interested using genetics to increase immune fitness and disease resistance in chickens. This includes those interested in increasing food safety, minimizing the use of antimicrobials, and reducing the load of Campylobacter in poultry meat products for human consumption. Genotypes at the polymorphic MHC-B region are already know to a major factor in determining outcome of colonization of chicken populations by microbes. The influence of MHC-B genetics in determining the incidence of Marek's disease is a well-documented example of this. In this project we tested the hypothesis that the other polymorphic MHC gene cluster, the MHC-Y region, containing specialized MHC-Y class I molecules contributes to the significant influence of heritable genetics in the colonization of chickens by Campylobacter. Our findings support a link between MHC-Y genetics and colonization by Campylobacter.The evidence we report for the association of MHC-Y genotype with immune responses will also be of interest to geneticists and poultry breeders. The simple means for MHC-Y typing, developed as part of this project, will make further work on the contribution of MHC-Y to food safety and poultry health considerably easier to pursue. Changes/Problems:Because fewer Campylobacter trials were available than we anticipated, we had to change our direction somewhat. We decided that in order to continue to move forward with defining the function of MHC-Y gene polymorphism that we should look at a more general question of the relationship of MHC-Y genotype with immune responses. We were extremely fortunate to have the wonderful opportunity to work with Paul Siegel and Christa Honaker. We were also extremely fortunate to have access to DNA samples from lines in a similar set of selected lines at Wageningen University in a collaboration with Robert Taylor, Jr, University of West Virginia, and Henk K. Parmentier, Wageningen University. The work toward this modified goal progressed well. The findings firmly establish MHC-Y as a polymorphic gene region influencing immune responses. Overall this work defining an association of MHC-Y polymorphism with Campylobacter colonization and the more general role MHC-Y in guiding immune responses, provides a basis for poultry breeders to examine MHC-Y haplotypes as a possible means for improving poultry genetic stock through genetic selection targeting MHC-Y. What opportunities for training and professional development has the project provided?This project provided opportunities to Dr. Jibin Zhang, the post-doctoral investigator on this project, to learn the basics of immunology. This included not only learning within the lab on a daily basis, but also formal training in the AAI Immunology course at nearby UCLA in the summer of 2019. He learned new laboratory skills including the basics of tissue culture and some techniques of molecular biology with which he was not prveviously familiar. He gained experience in writing and presenting scientific reports. He had the experience of working in a research center largely focused on human health that is part of a medical center. How have the results been disseminated to communities of interest?We have disseminated the results of this work so faronly in one peer-reviewed publication. Several manuscripts are will soon be submitted. We presented our work at the PAG in San Diego during every year of this award. Wepresented at the NE-1834meeting in 2018. We presented at the AAI meeting in San Diego in May 2019. Dr. Zhang presented seminars and discussed his work with others in multiple online interviews. We alsodiscuss our findings periodically infomally with others interested in polymorphic gene regions. Had covid-19 not become a pandemic, this work would also have been presented by Marcia Millerin Tokyo on March 12, 2020 in a symposium entitled Evolution, Reproduction and Immune Recognition, a special symposium commemorating Dr. Susumu Ohno, in a talk entitled "Neofunctionalization of Major Histocompatibility Complex (MJC) Class I Genes in the Chicken." What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1) STR-based Typing Method. MHC-Y was discovered through heritable restriction fragment patterns revealed in Southern blots. For a very long time labor-intensive Southern blots were the only means of assigning MHC-Y genotypes to individual birds. It was possible to type only small numbers of birds at one time by restriction fragment patterns. To get beyond this barrier to work-flow, we developed a simpler method for MHC-Y typing based simple tandem repeat (STR) sequences. We were able to make this major advance as the result of having acquired sequence data for the MHC-Y gene region (see Part 3, below). A paper describing the STR typing method, deemed classic by one reviewer, is published (Zhang, Goto and Miller). 2) MHC-Y and Campylobacter Colonization. With STR-based typing method in hand, we typed birds in a study devoted to defining the genetics of Campylobacter colonization. These were birds in a study published by the Roslin Institute (Psifidi et al. 2016, BMC Genomics). In this work, backcross and intercross populations of Line 62 and Line N White Leghorn-derived inbred lines, known to show major differences in resistance to Campylobacter colonization, were challenged with Campylobacter and regions of the genome scored for their influence.Several quantitative trait loci were reported, but no major gene emerged. MHC-Y, for lack of a means to detect, was not in the array of regions tested. We first typed the birds the backcross population that weretested in three replicate challenges.DNA samples for these trials were sent to us by Androniki Psifidi and Mark Stevens at Roslin. One of the three replicate challenges provided very interesting results. In this small trial, we found a highly significant association of one MHC-Y haplotype with low levels of colonization. While the experiment needs to be replicated (the other two trials were experimentally flawed by titering issues in the inocula), this finding supports the postulated role of MHC-Y in Campylobacter colonization. To investigate further, we are typing of the Line 62 and Line N advanced intercross population. We will publish our findings when typing is complete (Zhang, Goto, Psifidi, Stevens, Miller). 3) MHC-Y and Immune Response. Because of limited access to Campylobacter trials (one promised sample set from another lab was not available because it could not be found), we expanded the study to look for evidence that MHC-Y contributes broadly in guiding immune responses. We looked at whether MHC-Y genotype could be correlated with vigor of immune responses in well-controlled experimental systems in which chickens were selected for high and low antibody lines. For this we collaborated with Paul Siegel and Christa Siegel at Virginia Tech to define the MHC-Y haplotypes segregating in chicken lines selected for over 40 generations for high and low antibody responses. While characterized for MHC-B, the MHC-Y haplotypes were unknown in these lines. We defined MHC-Y genotypes in pedigreed matings of chickens at the 44th and 45th generation for high (HAS) and low (LAS) antibody responses to a non-infectious experimental antigen, sheep red blood cells. The MHC-Y haplotypes in the HAS and LAS lines are distinctly different and different from companion lines in which selection for antibody responses was relaxed for over 20 generations ago. For a replicate experiment we looked at the MHC-Y genotypes in brown layer lines selected for antibody responses at Wageningen University. We found similarly skewed distributions of MHC-Y genotypes. It is unlikely that these skewed distributions of MHC-Y genotypes occurred by chance. These findings are consistent with our hypothesis that MHC-Y plays a role in immune activation with some haplotypes contributing to activation and others perhaps driving a quiescent state. A manuscript describing this work is nearly ready for submission (Zhang, Goto, Honaker, Siegel, Taylor, Parmentier, Miller). 4) MHC-Y polymorphism and function. To better define the polymorphism of MHC-Y we have made a significant effort to define the MHC-Y haplotype in the chicken reference genome (RJF Line 001, #256). With access to SMRT sequencing it was finally possible to obtain cohesive sequence for portions of MHC-Y cloned into BAC vectors. Because the region is highly repetitive it was impossible to assemble MHC-Y sequence data obtained by earlier methods. Although fully cohesive single sequence remains as a goal, current sequencing has provided long-read sequences for 694 kb within four contigsrevealing a large MHC-Y haplotype in the reference genome that includes 49 polymorphic, specialized MHC class I loci (some of which are matched with cDNA clones), 45 c-type-lectin-like loci, and members of three additional gene families. This sequence data is valuable in many ways. Not only did it allow us to develop STR typing for MHC-Y, it provides insights into how the region varies in size among haplotypes. It gave us a much better understanding of the polymorphic nature the region.A manuscript describing this work is nearly ready for submission (Goto, Warden, Glass, Hosomichi, Shiina, Zhang, Wu, Kang, Delany, Inoko, Miller). 5) Polymorphic features of MHC-Y class I (YF) molecules and ligand bound. We are completing an analysis of the distribution of polymorphic residues of MHC-Y class I molecules. MHC-Y class I amino acid sequence variability is comparable to that found in human HLA, but the positions of the polymorphic residues are different. We have data showing thecontents within the groove of these unusual molecules is not peptide. The polymorphic MHC-Y class I molecules are distinctly different from classical class I molecules that bind peptide antigens. A manuscript reporting these observations is being prepared (Miller, Goto, Gugui, Statmueller, Bjorkman). 6) Intergenic region sequence (IGS) variability in the nucleolar organizer region (NOR) in the chicken (RJF) genome. Among the BAC clones identified as containing MHC-Y gene sequences there were three clones that also contained portions of the NOR.These revealed that the MHC-Y region is separated from the NOR (only one in chickens) by only a few thousand bases. The sequence data covers four rDNA genes and adjacent of IGS regions. We are analyzing these sequences because of their value in advancing understanding of regulatory elements that reside in the IGS that control transcription of adjacent rDNA genes. A manuscript for this work is in the early stages of preparation (Delany, Goto, Warden, Miller).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Jibin Zhang, Ronald M Goto, Marcia M Miller. 2020. A simple means for MHC-Y genotyping in chickens using short tandem repeat sequences.
Immunogenetics 72(5):325-332. doi: 10.1007/s00251-020-01166-6. Epub 2020 May 25.
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Progress 06/01/18 to 05/31/19
Outputs
Target Audience:The scale and seriousness of antibiotic resistance in bacteria continues to grow. Alternatives to the use of antibiotics in food animals are needed to reduce the increasing frequency of antibiotic resistance in bacteria and to secure continued food safety and availability. Finding ways to raise chickens without antibiotics is important. "No antibiotics ever" products are increasingly sought out by consumers. This project is focused on exploring the possibility that genetic selection might be used as an alternative to antibiotics to provide healthier chickens that less frequently transmit harmful bacteria to humans. Safe-handling of poultry food products at processing and during meal preparation greatly reduces chances of foodborne illness, but instances of foodborne illness from poultry products persist. One of the most harmful bacteria transferred to humans in food is Campylobacter. In humans Campylobacter causes gastroenteritis and sometimes Guillain-Barré syndrome, a persistent autoimmune disease. Campylobacter, which typically causes no disease in chickens, grows in the gut of some chickens, but not in others. Genetic makeup appears to determine whether individual chickens harbor Campylobacter. Genes within the chicken highly polymorphic MHC-Y family of immune system genes are candidates for controlling Campylobacter colonization. This project is focused on learning whether this is true. Recent progress suggests that MHC-Y haplotype is indeed influencing Campylobacter colonization. Changes/Problems:We hope to be able to find additional disease challenge trials within which we can relate MHC-Y genotype to outcome. We believe these will be available, but they are not yet identified. What opportunities for training and professional development has the project provided?This project is providing training and professional development for a senior research fellow, Dr. Jibin Zhang. Participation of a student intern during the summer provided that student with an opportunity to problem-solve using scientific methods. How have the results been disseminated to communities of interest?Results have been presented at the NE1334 Meeting in Morgantown, WV in October 2018, at the NC-1170/NRSP-8 Meeting at PAG in San Diego, CA in January 2019, and at the America Association of Immunologists Meeting in San Diego in May 2019. What do you plan to do during the next reporting period to accomplish the goals?We will follow up on the observation linking MHC-Y haplotype to Campylobacter colonization. We will define additional MHC-Y haplotypes using the sequencer-based typing and identify additional challenge trials in which we can determine MHC-Y genotypes. We will look more closely at gene expression changes in response to immune challenge. We will complete several manuscripts for publication.
Impacts
What was accomplished under these goals?
Objective 1. MHC-Y genotype and resistance to colonization. Improvements in MHC-Y genotyping. Initially MHC-Y genotyping was dependent upon Southern blots that revealed heritable restriction fragment patterns. This definitive assay is labor intensive and not practical for genotyping large numbers of birds. We moved to genotyping based on PCR fragment-size polymorphisms associated with a microsatellite sequence and revealed these in agarose gels. The PCR fragment patterns are heritable and consistent with previous types assigned by Southern hybridization. For further increased efficiency and precision we are now using an ABI 3130 sequencer and LIZ500 standards to detect the size of the PCR fragments. When typing by gels, it has been difficult to resolve some highly similar PCR fragment patterns. Precise fragment sizes have helped to resolve previous ambiguities in assigning types. Switching to sequencer-based typing is a major advancement for the work necessary in Objective 1. Campylobacter Trials. We have assigned MHC-Y haplotypes, now through sequencer-based typing, for the birds used in a published study of the genetics of Campylobacter colonization (Psifidi et al. 2016, BMC Genomics). This study focused on a backcross of two White Leghorn-derived inbred lines [(61 X N) x N] known to differ in resistance to Campylobacter colonization. Several quantitative trait loci are identified in the published work, but no major gene emerged. Since the MHC-Y region was not in the panel of genes tested, its contribution was not included. Based on typing of the Lines 62 and N at the Avian Disease and Oncology Laboratory in East Lansing, Michigan, we anticipated that in the Psifidi study there would be two MHC-B types (MHC-B2 and MHC-B21) and four MHC-Y types (MHC-Y type 11 from Line 61 and MHC-Y types 5, 7 and 8 from Line N) segregating). As expected two MHC-B (2 and 21) haplotypes were found. But the MHC-Y types were not as anticipated. An additional and unexpected (a fifth) MHC-Y haplotype was found. The Psifidi backcross trial was conducted in three parts corresponding to three hatches. The colonization levels in only one hatch were broadly spread across colonization levels (not all low or all high), so that trial was most suitable for looking at associations between genotypes and colonization. Within this trial there is a highly significant association between one haplotype and this was most frequent in birds with low Campylobacter colonization. This is the first clear evidence supporting the postulated role of MHC-Y in Campylobacter colonization. Links between immune response and MHC-Y haplotype. In additional experiments, we asked a more general question about the link between MHC-Y and immune responses. Some evidence for this link was reported in Year 1. With the improved typing methods and the availability within the last year of selected fully-pedigreed families from the high and low antibody-response selected chicken lines (provided by Paul Siegel and Christa Siegel, our collaborators at Virginia Tech), we have been able to clearly associate individual MHC-Y haplotypes with antibody response. One haplotype is extremely common in the high antibody line and is likely behaving as a dominant allele. Others are much more common in the low antibody line. These data provide significant support for a link between MHC-Y haplotype and immune response. We wanted to see if we could find data that would help in settling uncertain about the possibility that the observed association occurred by chance rather than by selection during the development of high and low antibody response lines. To evaluate the possibility that such an association was coincidental, we identified another pair of lines selected for high and low antibody responses. These are lines that were similarly selected for 32 generations at Wageningen University. Typing revealed that a similar separation of MHC-Y haplotypes occurred in these lines. It seems unlikely that such results would happen twice simply by chance and so it is more likely that the separation of haplotypes observed in these two sets of high and low antibody lines is the result of a link between MHC-Y haplotype and immune response. Objective 2. To characterize polymorphic features of MHC-Y haplotypes focusing on the expressed polymorphic YF genes. We have completed an analysis of the distribution of polymorphic residues on MHC-Y class I molecules predicted from the sequence data we have achieved for the Chicken Reference Genome (Red Jungle Fowl) and additional sequences from several cDNA clones from other haplotypes. [A manuscript describing the sequence of the MHC-Y region (achieved finally through Single Molecule, Real-Time (SMRT) sequencing) is nearly ready for submission.] We found that the MHC-Y class I amino acid sequences are as variable as human HLA and chicken BF class I. But, the positions of the polymorphic residues are different. In contrast to the MHC class I molecules (HLA and BF) that bind peptide, where many of the polymorphic residues point into the groove and influence peptide binding, in the MHC-Y class I molecules, most of the polymorphic residues are not in the binding groove. In the MHC-Y class I molecules, most of the polymorphic residues are on the alpha1-helix and point up or away from the groove. These residues are candidates for conferring specificity to interactions between MHC-Y class I molecules and their cognate receptors. These interactions might be independent of ligands bound within the MHC-Y binding groove, which is mostly hydrophobic. Objective 3. To gain insight into the function of YF MHC class I-like molecules. Work on this objective is continuing.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
Goto RM, Gugiu G, Zhang J, Stadtmueller B, Bjorkman P, Miller MM. (2019) Polymorphism in chicken MHC-Y class I molecules that bind lipid ligands. 103rd Annual Meeting of The American Association of Immunologists. San Diego, CA.
Zhang J, Goto RM, Honaker CF, Siegel PB, Miller MM. (2019) Segregation of chicken MHC-Y haplotypes in high and low antibody selected lines provides evidence that MHC-Y contributes to the genetics of immune responses. 103rd Annual Meeting of The American Association of Immunologists. San Diego, CA. (Poster)
Miller MM, Zhang J, Warden C, Goto RM. (2019) Progress toward revealing MHC-Y diversity and function in chickens. Plant & Animal Genome Conference XXVII. San Diego, CA.
Zhang J, Goto RM, Honaker CF, Siegel PB, Miller MM. (2019) Distribution of haplotypes within selected chicken lines suggests MHC-Y contributes to the genetics underlying heritable high and low antibody responses. Plant & Animal Genome Conference XXVII. San Diego, CA. (Poster)
Mismash E, Zhang J, Goto RM, Miller MM. (2018) Study of the expression of genes for avian lipid-binding MHC-Y class I-like molecules. Eugene and Ruth Roberts Summer Student Academy Poster Session at City of Hope. Duarte, CA.
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Progress 06/01/17 to 05/31/18
Outputs
Target Audience:Campylobacter spp. infections are presently among the most common bacterial infections associated with human gastroenteritis worldwide. Poultry products are the major source of infection. With individual isolates increasingly found to be resistant to antimicrobials, Campylobacter jejuni is now classified as an antimicrobial threat. The major goal of this project is to find new means for reducing the presence of C. jejuni in poultry food products. Campylobacter bacteria colonize the chicken intestine (especially the caeca) often growing to high numbers while usually having little or no influence on bird health. Multiple studies have demonstrated that genetic lineage in chickens strongly affects colonization, but the responsible gene or genes are not clearly defined. The focus of this project is on defining the role of MHC-Y genetics in the observed differences in colonization of chickens by Campylobacter. The MHC-YF class I-like genes are candidate loci for affecting colonization. Changes in MHC-YF gene expression has been observed following Campylobacter colonization. These chicken YF MHC class I-like molecules are structurally most similar to the mammalian MR1 molecule that presents microbial riboflavin metabolites to innate-like mucosal-associated invariant T (MAIT) cells (Kjer-Nielsen et al. 2012, Nature). Chicken YF molecules may function in a similar manner although they bind a different ligand, lipids, including lipids of bacterial origin (Miller et al., manuscript in preparation). Chicken YF molecules, in contrast to MR1, are highly polymorphic. It could be that some isoforms are more effective than others in guiding host responses to Campylobacter and other bacteria. This project is focused on testing the hypothesis that MHC-Y genetics contributes to the early immune responses that occur in newly-hatched chicks and that MHC-Y has a role in determining colonization by Campylobacter and other bacteria. The supporting objectives are: 1. To investigate the contribution of MHC-Y in resistance to Campylobacter colonization correlating the level of colonization of individual chickens with MHC-Y genotype. 2. To characterize polymorphic features of MHC-Y haplotypes focusing on the expressed YF genes. 3. To gain insight into the function of YF MHC class I molecules using selected isoforms in a) molecular recognition assays to define the specificity of their recognition and b) binding assays to define the specificity of ligands bound. Changes/Problems:We have had the opportunity to expand our study to include chickens in defined lines (the HAS and LAS lines) that differ in immune responsiveness. Birds in these lines provide defined genetics that will speed efforts to define the contribution of MHC-Y to immunity including differential responses to Campylobacter ssp. and other bacteria. While our current method for typing has allowed us to make major advances in defining different MHC-Y genotypes, work will be expanded to develop a method to better define the number of expressed YF loci in different haplotypes and the sequences within them that define the YF MHC-fold (the structure formed by α1 and α2 domains of the YF proteins), the likely interface for signaling. What opportunities for training and professional development has the project provided?This project is providing training and professional development for a senior post-doctoral research fellow, Dr. Jibin Zhang. Participation of students during the summer provides opportunities for exposure to problem-solving using scientific methods. How have the results been disseminated to communities of interest?Results have been presented at the Plant and Animal Genome XXVI Meeting, January 13-17, 2018 in San Diego, California. This included a platform presentation by the Poultry Workshop (Abstract # 30662, Goto, Zhang, Fulton, and Miller) and a poster presentation (No. 0510, Zhang, Goto and Miller), which was also selected for a Lightning Talk in the Poultry Workshop. What do you plan to do during the next reporting period to accomplish the goals?We plan to add to the methods currently used for typing. We will examine how MHC-Y gene expression in different haplotypes changes in response to immunological challenges (including the non-infectious experimental antigen, sheep red blood cell and different strains of bacteria including strains of Campylobacter jejuni).
Impacts
What was accomplished under these goals?
Objective 1. MHC-Y genotype and resistance to colonization. We have focused most of our effort in the first year on defining the relationship between MHC-Y genotypes and the presence of Campylobacter and other bacteria in chickens. We currently use PCR reactions that produce products of varying length from a region within YF gene promoters that contains tracts of repetitive DNA of various lengths (microsatellite sequences). The products of PCR reactions provide heritable fingerprint patterns drawn from many or all the YF genes present in each haplotype to provide a pattern representing each MHC-Y genotype. We have PCR-fingerprinted samples in two sets sent to us by Androniki Psifidi and Mark Stevens at the Roslin Institute from a completed Campylobacter trial (Psifidi et al. 2016, BMC Genomics). These were from the [(61 X N) x N] backcross population portion of this large study. The first 100 samples were from the Roslin stocks having the most abundant DNA. These represented chickens from across the entire range of colonization scores. We found a greater number of patterns than expected. Earlier typing of the USA counterparts of Line 61 and N suggested that there might be only four haplotypes segregating in this backcross population. With four haplotypes segregating only twelve genotype patterns would be expected. We found more patterns and defined one additional haplotype frequently present. This first test did not provide convincing evidence for or against a link between MHC-Y genotype and Campylobacter colonization. Next we analyzed an additional 81 samples from the same trial. These samples were represented the chickens in the experimental tails - those with the lowest and highest colonization scores. This sample set provided seventeen genotype patterns. Three of these patterns were exclusively present in the birds in the low colonization cohort. We are now working to define the haplotypes contributing to the patterns and to see if we can gain insight into which haplotypes might be dominant and recessive so we can further evaluate the results. We are working to develop a 3rd generation typing method. We wish to move from microsatellite-based typing to typing based on the coding sequences of the expressed members of the YF genes family. MHC-Y haplotypes vary in complexity. The MHC-Y genomic region is apparently segmented. Some haplotypes are apparently small containing perhaps one or two segments and perhaps as little as two YF loci. Others, such as the haplotype in the RJF reference genome, are far larger with a much number of YF loci. Evidence emerging from our work on the reference genome sequence suggests only a fraction of the total number of YF loci in this physically large haplotype are actually intact, expressed genes that would directly affect immune cell interactions. So we are now working to design a typing a high-throughput sequencing method that focuses on intact loci and will provide gene sequence data. In addition we have nearly completed, with the existing method, the scoring of samples in an additional Campylobacter trial provided by Graham Plastow, U. Alberta and Janet Fulton, Hy-Line International. Also nearly complete is typing of birds in a trial by Doug Rhoades and Adnan Alrubaye, U Arkansas, investigating the role of Staphylococcus in causing lameness broiler chickens. While there are bits of evidence suggesting that MHC-Y contributes to immunity, evidence is still weak. To look for more evidence that MHC-Y gene region serves a role in immunity, as we posit, we looked at whether MHC-Y genotype and vigor of immune responses in chickens highly selected for high and low antibody responses could be correlated. With Paul Siegel and Christa Siegel at Virginia Tech, we defined the MHC-Y genotypes in chickens that have been under continual selection for 44 generations for high (HAS) and low (LAS) antibody responses to a non-infectious experimental antigen, sheep red blood cells. HAS and LAS have a common origin in the Cornell Random bred line but have diverged widely under continuous selection over decades. Typing of the 44th generation (189 birds) revealed ten MHC-Y fingerprint patterns. Four of these haplotype patterns were present only in the HAS line and three only in the LAS line. It is unlikely that distribution of patterns in this manner has occurred by chance. These findings are consistent with our hypothesis that MHC-Y plays a role in very early immune activation with some haplotypes contributing to activation and others perhaps driving a quiescent state. We will be conducting further tests with individual haplotypes in the HAS and LAS lines to learn how their presence affects responses to SRBC and the presence of bacteria including Campylobacter. These well-defined and well-maintained lines are a significant addition in this research effort. We expect that HAS and LAS lines will provide access to data helping to define how different MHC-Y haplotypes contribute to immunity to bacteria including Campylobacter. Objective 2. To characterize polymorphic features of MHC-Y haplotypes focusing on the expressed polymorphic YF genes. We have begun to gather additional sequence data from cDNA so as to be able to define the arrays of polymorphic residues displayed on the surface of different YF isoforms. Currently sequences for expressed YF genes one of the haplotypes in the high immune responsiveness HAS line are being determined. Sequence seterminations for additional YF genes from additional HAS and LAS haplotypes will soon follow. Objective 3. To gain insight into the function of YF MHC class I-like molecules. We have begun to investigate the expression of the MHC-YF class I genes in response to the presence of bacteria and will do more in the coming year. To start, we have performed a series of simple experiments using chicken cell lines and a laboratory strain of E. coli. We have looked at changes in YF expression by quantitative PCR in the chicken DF1, LMH and RP9 cell lines, each of which has a different MHC-Y genotype. We have compared the responsiveness of the three cell lines in short term exposures to E. coli. Each seems to respond somewhat differently. Overall it is clear that YF gene expression changes in response to presence of bacteria in the cultures. When exposed to lower concentrations (100-1000/ml) of E. coli, YF gene expression is quickly up-regulated. With exposure to higher concentrations (10,000/ml) MHC-Y gene activation is dampened. These in vitro experiments with cell lines provide a beginning framework for up-coming experiments with primary cells that we have collected from HAS and LAS birds with defined MHC-Y genotypes. Investigations to define the responding cells, perhaps equivalent to the human MAIT1 cells that recognize MR1, will follow.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
P0510 Variations in Genomic Organization, Gene Structure and Expression Among Chicken MHC-Y Haplotypes. Jibin Zhang, Ronald M. Goto and Marcia M. Miller, Beckman Research Institute, City of Hope, Duarte, CA
W836 Extensive Chicken MHC-Y Haplotype Diversity. Ronald M. Goto1, Jibin Zhang1, Janet E. Fulton2 and Marcia M. Miller1, (1)Beckman Research Institute, City of Hope, Duarte, CA, (2)Hy-Line International, Dallas Center, IA
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