Progress 02/01/15 to 01/31/20
Outputs
Target Audience:The target audience of this project, in both the US and UK, includes researchers with a focus on animal health or animal genomics, veterinarians, poultry breeders/producers, other professionals linked to the poultry industry, students, the USDA, the Biotechnology and Biological Sciences Research Council (BBSRC), and other US and UK government agencies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Initially two lab members from the US trained with the UK co-investigators and brought techniques back to the US for application in the project. Training of US lab members on protocols for pathogen-challenge studies and bacterial enumeration techniques included 2 postdocs, a research associate, 5 graduate students, and 2 undergraduate students. Training of UK lab members on protocols for pathogen-challenge studies and the use of transgenic chicken model with gfp-expressing APEC to visualize bacteria-host phagocyte interaction included 3 postdocs and other lab members. Dr. Monson (postdoc funded by this project at ISU) received additional training on protocols for RNA-sequencing cDNA library construction and experience with analysis of high-throughput data from genotyping and sequencing, including co-expression and allele specific expression analyses. Dr. Monson participated into multiple scientific conferences to increase knowledge, network and discuss this project as well as a grant-writing workshop. Dr. Bryson (postdoc funded by this project at Roslin) adapted protocols for use in chickens, participated in the Avian Personal License course, and attended multiple symposia to extend her knowledge and share project results. The PD and coPDs received several recognitions during this project, including invited keynote speaker presentations, Fellow of the International Society of Animal Genetics (PD Dr. Lamont), Fellow of the American Association for the Advancement of Science (PD Lamont), Fellow of the Royal Society of Biology (coPD Stevens) and member of the scientific board for the World's Veterinary Poultry Association (coPD Vervelde). How have the results been disseminated to communities of interest?Papers have been published in peer-reviewed journals. Abstracts, posters and oral presentations have been presented at multiple scientific conferences and seminars, including Plant and Animal Genomes conferences, Avian Immunology Research Group meetings, an International Society of Animal Genetics conference, the Avian Genetics and Immunity Symposium, the Pirbright Institute Seminar series, the Edinburgh Infectious Disease Symposium, the Edinburgh Immunology Group Symposium, the British Society for Immunology Congress, and multiple seminars at Iowa State University. Public engagement during this project includes the participation of Drs. Bryson and Vervelde in the Midlothian Science Festival/Roslin Institute Open Day, raising awareness of bacterial infections including APEC, and through direct interaction of Drs. Monson and Lamont with poultry industry representatives at the National Breeders Roundtable meetings and the Iowa Egg Industry Symposium. A YouTube video was also jointly created and used to disseminate information about this project: https://www.youtube.com/watch?v=qDKbwVHPwZo&. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Overall impact statement: Avian pathogenic E. coli (APEC) causes colibacillosis, an important bacterial disease in poultry. Our studies represent one of the first efforts to identify genomic regions associated with resistance to APEC colonization in chickens. Predicted heritabilities for APEC colonization levels after inter-airsac infection were low, suggesting limited feasibility of genetic improvement based on the identified candidate SNPs. However, associations were detected with bacterial level in the inoculated lung, illustrating the importance of the local immune response. Changes in the splenic transcriptome after challenge with APEC revealed responses in many immune-related genes and pathways, including in an allele specific manner, and provided insight into the molecular mechanisms of the early systemic immune response to bacterial infection. Our novel use of transgenic chickens with fluorescent mononuclear phagocytic cells allowed an unprecedented opportunity to identify host cell and pathogen interactions. The validation of the precision-cut lung slice model sets a foundation for examining early host-pathogen interactions in respiratory diseases such as APEC. Precision-cut lung slices will have a significant impact on the field, as it can reduce the number of birds used in vivo and it is more cost efficient. The greater understanding of the host-pathogen interaction and its genetic control generated through this project will pave the way for breeding and immunological control of this important poultry disease. Objective 1. Characterize interaction of E. coli with antigen-presenting cells (APC) in the bronchus-associated lymphoid tissue (BALT). Two pilot studies at Roslin determined that inbred chicken lines 72 and 15I would not be suitable to dissect the immune responses underlying avian differential resistance to APEC, as 15I birds did not survive 10E7 CFU APEC O1 and lower doses (10E5 or 10E6) did not generate differential responses in lines 72 and 15I. Subsequent studies employed CSF1R-reporter transgenic chickens that express a fluorescent reporter (mApple) in all mononuclear phagocyte cells (MPCs) and challenged with gfp-expressing APEC O1 to enable visualization of the interaction between the MPCs (red) and APEC (green). Flow cytometric analysis determined that at 1 day-post-inoculation (dpi), more than 50% of APEC was co-localized with lung leukocytes of which over 70% were CSF1R+ MPCs. Lung MPCs from APEC infected and PBS treated transgenic chickens were FACS fractionated into gfp-APEC positive and gfp-APEC negative cells and used for RNA-seq, flow cytometry and histology. Non-MPC gfp-APEC positive cells were also isolated and used in these same analyses. A new model for examining host-pathogen interactions in the chicken lung was developed using precision cut lung slices (PCLS). The PCLS provide an ideal model for early host-pathogen interactions as these are difficult to examine in vivo and the influence of tissue architecture is lost in vitro. Real-time dynamic interaction of reporter MPCs with gfp-APEC has been observed in viable PCLS from the CSF1R-reporter transgenic chickens. Objective 2. Determine cell-type-specific adaptive immune response to colibacillosis. As little is known about the role of the adaptive immune response in colibacilosis, the contribution of lymphocyte subsets during APEC infection was investigated using adoptive transfer experiments. This technique transfers primed lymphocytes to naïve recipients and can confer resistance to other avian pathogens. Preliminary experiments used MHC-matched chickens and adoptively transferred fluorescently labelled spleen- or lung-derived lymphocytes in the presence or absence of LPS stimulation (E. coli derived). Transferred lymphocytes were detectable by flow cytometry in recipient tissues including blood, lung and spleen at 24 and 48 hours post transfer, validating this technique for in vivo APEC challenges. These pilot studies also showed an increased proportion of lung-derived donor lymphocytes in the recipient lung relative to spleen-derived donor cells. This preferential homing to the tissue of origin, suggests a mechanism of tissue imprinting in the avian immune response, as seen in mammalian lymphocytes. Using these techniques for an APEC challenge experiment, CD8+ T lymphocytes were isolated from APEC primed birds and adoptively transferred into naïve recipients prior to APEC challenge. Following primed CD8+ T cell transfer, a trend toward lower bacterial titers was detected, but no statistical significance was reached. Further experimentation is still needed to determine whether protection against APEC can be transferred and which cell population is responsible. Objective 3. Map host genomic elements controlling responses to APEC. Three replicates of a pathogen-challenge trial were performed in the US using chickens from a specialized genetic line (advanced intercross of broiler x Fayoumi) challenged with 10E7 CFU of APEC O1. Five tissues were collected and used to determine bacterial loads as an estimate of relative resistance/susceptibility of the birds. Genotypes were determined for over 300 chickens using the Affymetrix Axiom 600K Chicken Genotyping Array, of which approximately 190,000 SNPs were high quality and segregating. Genetic control of resistance to tissue colonization was investigated using the bacterial loads as phenotypes for heritability estimation and genome-wide association study (GWAS). Heritability estimates were performed using a genomic-relationship matrix and multiple transformations of the bacterial load data; heritabilities were found to be very low, suggesting that bacterial load may be influenced by many genes and by non-genetic factors (such as lab batch effects). Single SNP associations were performed for GWAS; 20 SNPs located on the microchromosomes reached genome-wide significant for the right lung, illustrating the importance of the local immune response despite the low heritability of the phenotypes. Objective 4. Identify cis-acting elements affecting host resistance. Two replicates of a pathogen-challenge trial were performed in the US using chickens from a reciprocal F1 cross of broiler x Fayoumi lines. Samples from reciprocal crosses of inbred Leghorn x Fayoumi lines challenged alongside the broiler crosses have been saved for future work. Birds were challenged with 10E6 CFU APEC O1 and tissues collected 1, 2 and 6 days-post-inoculation (dpi) to determine bacterial colonization levels and/or to archive for RNA isolation. To detect early systemic immune gene responses to APEC, RNA-seq was performed for the spleen using samples from the reciprocal broiler crosses for the APEC-infected and non-infected groups at 1 dpi and 2 dpi. Libraries were sequenced on the Illumina HiSeq 3000, resulting reads mapped to the chicken genome (Galgal6a), and used for differential expression, co-expression and allele specific expression analyses. Over 600 genes had significant responses to APEC, with the majority of response at 1 dpi and the greatest magnitude of expression change in innate immune genes such asIL22,IL17A, andPTX3. Co-expression patterns identified 10 significant genes modules, which included immune response and cell division genes. Allele specific expression (ASE) provided markers forcis-acting regulatory elements impacted by APEC infection, including APEC-specific ASE within genes with significant DE. These early immune expression responses to APEC could be investigated for markers for resistance or for mechanisms to reduce infection. Objective 5. Evaluate potential to translate results into commercial layer lines. Because few significant SNPs were identified in Objective 3, ASE results from Objective 4 provide potential markers for investigation in commercial layer lines. Allele frequencies for these SNPs and associations with production traits would need to be determined.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Alber A, Morris KM, Bryson KJ, Sutton KM, Monson MS, Chintoan-Uta C, Borowska D, Lamont SJ, Schouler C, Kaiser P, Stevens MP and Vervelde L. (2020). Avian pathogenic Escherichia coli (APEC) strain-dependent immunomodulation of respiratory granulocytes and mononuclear phagocytes in CSF1R-reporter transgenic chickens. Front. Immunol. 10:3055.
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Monson MS, Kaiser MG, Bryson KJ, Alber A, Wolc A, Vervelde L, Stevens MP and Lamont SJ. (In prep). Rapid immune gene and allele specific responses to infection with avian pathogenic Escherichia coli (APEC) in the chicken spleen transcriptome.
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Monson MS, Kaiser MG, Wolc A, Chintoan-Uta C, Vervelde L, Stevens MP and Lamont SJ. (In prep). Investigating genetic control of tissue colonization by avian pathogenic Escherichia coli in an advanced intercross chicken line.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Monson MS, Kaiser MG, Wolc A and Lamont SJ. Differential, allele specific, and co-expression in the chicken spleen transcriptome under avian pathogenic Escherichia coli infection. Plant and Animal Genomes Conference XXVIII. San Diego, CA. January 11-15, 2020. Abstract W876. Oral presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Monson MS, Kaiser MG, Wolc A and Lamont SJ. Allele specific expression in the chicken spleen transcriptome under APEC infection: US-UK collaborative research. 100th Conference of Research Workers in Animal Diseases. Chicago, IL. November 2-5, 2019. Abstract P205.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Monson MS, Kaiser MG, Wolc A and Lamont SJ. Allele specific and differential expression in the chicken splenic transcriptome in response to avian pathogenic Escherichia coli. 37th International Society for Animal Genetics Conference. Lleida, ESP. July 7-12, 2019. Abstract P220.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Monson MS and Lamont SJ. Co-expression in the response to avian pathogenic Escherichia coli in the chicken spleen transcriptome. Plant and Animal Genomes Conference XXVII. San Diego, CA. January 12-16, 2019. Abstract PE0438.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Monson MS, Kaiser MG, Bryson KJ, Vervelde L, Stevens MP, Wolc A and Lamont SJ. Rapid transcriptome responses in chicken spleen to avian pathogenic Escherichia coli infection: US-UK collaborative research. 99th Conference of Research Workers in Animal Diseases. Chicago, IL. December 2-4, 2018. Abstract 253. Oral presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Monson MS, Kaiser MG and Lamont SJ. Gene expression reveals chicken spleen responses to E. coli infections. 2018 Iowa Egg Industry Symposium. Ames, IA. November 13, 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Monson MS, Kaiser MG, Vervelde L, Stevens MP and Lamont SJ. Responses to avian pathogenic Escherichia coli infections revealed by the chicken spleen transcriptome. XV Avian Immunology Research Group Meeting. Oxford, UK. September 5-7, 2018. Abstract 48.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Monson MS, Kaiser MG, Vervelde L, Stevens MP, Wolc A and Lamont SJ. Identifying host responses to avian pathogenic Escherichia coli using the chicken splenic transcriptome. Plant and Animal Genomes Conference XXVI. San Diego, CA. January 13-17, 2018. Abstract P0528.
|
Progress 02/01/18 to 01/31/19
Outputs
Target Audience:The target audience of this project, in both the US and UK, includes researchers with a focus on animal health or animal genomics, veterinarians, poultry breeders/producers, other professionals linked to the poultry industry, students, the USDA, the Biotechnology and Biological Sciences Research Council (BBSRC), and other US and UK government agencies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The postdoctoral trainees continued to refine their expertise in research protocols, and also increased their knowledge and scientific network by attending professional conferences. The PD received recognitions in the past year. PD Lamont was invited to give keynote talks at the International Society of Animal Functional Genomics (Australia) and the Keystone Symposium on Genetic Diversity and Disease (Uganda). PD Lamont was selected to receive the Outstanding Achievements in International Agriculture Award, College of Agriculture and Life Sciences, Iowa State University How have the results been disseminated to communities of interest?Papers have been published in peer-reviewed journals. Abstracts, posters and oral presentations were presented at several scientific conferences, including the Avian Immunology Research Group, the Plant and Animal Genome conference, the National Breeders Roundtable and the Iowa Egg Industry Symposium. Two public seminars were given at Iowa State University. What do you plan to do during the next reporting period to accomplish the goals?For all objectives, results will continue to be publically presented at national and international conferences. 1. Characterize interaction of E. coli with antigen-presenting cells (APC) in the bronchus-associated lymphoid tissue (BALT). Nothing, objective completed. 2. Determine cell-type-specific adaptive immune response to colibacillosis. Nothing, objective completed. 3. Map host genomic elements controlling responses to APEC. A manuscript will be prepared to submit for publication in peer-reviewed journal. 4. Identify cis-acting elements affecting host resistance. Bioinformatic processing and mapping of the broiler F1 right lung RNA seq data, and detection of allele specific expression (ASE) in both the splenic and lung datasets. This analysis will consider ASE in response to APEC challenge, post-infection time, direction of reciprocal cross, and the interaction of these factors. A manuscript will be prepared to submit for publication in peer-reviewed journal. 5. Evaluate potential to translate results into commercial layer lines. Using associated SNPs identified in Objective 3 and the ASE results of Objective 4, SNPs will be genotyped to determine allele frequencies in multiple generations of 8 genetics lines from a major layer breeding company and associations with production traits determined.
Impacts
What was accomplished under these goals?
Overall impact statement: Avian pathogenic E. coli (APEC) causes colibacillosis, an important bacterial disease in poultry. Our studies represent one of the first efforts to identify genomic regions associated with resistance to APEC colonization in chickens.Predicted heritabilities for APEC colonization levels after inter-airsac infection were low, suggesting limited feasibility of genetic improvement based on the identified candidate SNPs. However, associations were detected with the bacterial level in the inoculated lung, suggesting heritability of the local immune response. Changes in the splenic transcriptome after challenge with APEC revealed responses in many immune-related genes and pathways, providing insight into the molecular mechanisms of the early systemic immune response to bacterial infection.Our novel use of transgenic chickens with fluorescent mononuclear phagocytic cells allowed an unprecedented opportunity to identify host cell and pathogen interactions. The development of lymphoid structures in the avian respiratory tract was visualized using transgenic chickens and showed earlier organization than previously thought which will facilitate research to improve vaccine responses. The validation of the new precision-cut lung slice model sets a foundation for examining early host-pathogen interactions in respiratory diseases. Precision cut lung slice from chickens will have a significant impact in the field, reducing the numbers birds used in vivo. This model therefore satisfies the principles of 3R's, with increased ethical pressure on the scientific community to reduce in vivo work, as well as having the added benefit of greater cost efficiency.The greater understanding generated through this project of the host-pathogen interaction and its genetic control will pave the way for breeding and immunological control of this important poultry disease. Objective 1. Characterize interaction of E. coli with antigen-presenting cells (APC) in the bronchus-associated lymphoid tissue (BALT). A manuscript was prepared and submitted on studies using CSF1R-reporter transgenic chickens to study the mononuclear phagocytic cells of the respiratory tract. This method gives new insight into host-pathogen interactions in situ. Studies on use of the lung-slice model to study host-pathogen interactions in chickens continued. Objective 2. Determine cell-type-specific adaptive immune response to colibacillosis. CD8+ T cells were isolated from APEC primed birds and adoptively transferred into naïve recipients prior to APEC challenge. Following the primed CD8+ T cell transfer a trend toward lower bacterial titres was detected, but no statistical significance was reached. A repeat of the CD8+ T cell transfer was delayed due to issues with bird fertility. Objective 3. Map host genomic elements controlling responses to APEC. Using genotypes collected with the Affymetrix Axiom 600k Chicken Genotyping Array (in 2016), genetic control of resistance to APEC was investigated in 324 chickens (advanced intercross line derived from broiler x Fayoumi). Because the phenotypes measured had very low heritability, various transformations of the bacterial load data were explored. The bacterial load in the lung adjacent to the site of inoculation showed the greatest heritability and will be the major focus in the genome-wide association analysis. Objective 4. Identify cis-acting elements affecting host resistance. Progress toward this objective continued, with the past year focusing on analysis of RNA-sequencing (RNA-seq) was used to characterize transcriptome responses to APEC infection in the spleen from progeny of reciprocal crosses between broiler (disease-susceptible) and Fayoumi (disease-resistant) lines. Ten gene clusters were correlated with APEC infection using co-expression analysis, in which many of the driver genes most strongly associated with APEC were also significantly differentially expressed including CTLA4, CCL20, FABP5, MMP7, and HBAD. Detection of allele specific expression (ASE) in these RNA-seq datasets is ongoing. The splenic transcriptome revealed innate immune pathways that could be potential targets to modulate resistance to APEC. A manuscript was prepared and submitted to a peer-reviewed journal. Objective 5. Evaluate potential to translate results into commercial layer lines. Nothing to report.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Alber A, Costa T, Chintoan-Uta C, Bryson K, Kaiser P, Stevens M, Vervelde L. Dose dependent differential resistance of inbred chicken lines to avian pathogenic Escherichia coli. Avian Pathology, 2018, DOI:10.1080/03079457.2018.1562154
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Sutton K, Costa T, Alber A, Bryson K, Borowska D, Balic A, Kaiser P, Stevens M, Vervelde L. Visualisation and Characterisation of mononuclear phagocytes in the chicken respiratory tract using CSF1R-transgenic chickens. Veterinary Research, 2018, 49: 104
|
Progress 02/01/17 to 01/31/18
Outputs
Target Audience:The target audience of this project, in both the US and UK, includes researchers with a focus on animal health or animal genomics, veterinarians, poultry breeders/producers, other professionals linked to the poultry industry, students, the USDA, the Biotechnology and Biological Sciences Research Council (BBSRC), and other US and UK government agencies. Changes/Problems:The identified low heritabilities of APEC load in the various tissues in the APEC challenge experiment has caused us to reassess whether the genome-wide association study is the best source of data to select the SNPs to test in Objective 5, or whether other types of data generated in this project would be a better source. This has taken additional time to discuss and evaluate the possibilities, and the data sources (RNA-seq) will take additional time to generate, thus delaying the work on Objective 5. What opportunities for training and professional development has the project provided?Additional training of lab members in the US (especially Dr. Monson, postdoc funded by this project) was performed for protocols for RNA-sequencing cDNA library construction and for analysis of high-throughput data from genotyping and sequencing.Dr. Monson participated in multiple scientific conferences to increase knowledge, network and discuss this project. She also participated in a day-long grant-writing workshop.Dr. Bryson (postdoc funded by this project in Roslin) participated in the Avian Personal Licence course and attended three symposia (Antimicrobial resistance on 22/3/2017; Edinburgh Immunology Group on 15/6/17 and Edinburgh Infectious Diseases on 01/6/17). The PD and coPD had received recognitions in the past year. How have the results been disseminated to communities of interest?An abstract, poster and oral presentation were presented at the International Society of Animal Genetics Conference in July 2017 in Ireland (Objective 3 and 4). Oral presentations have been given at the Avian Genetics and Immunity symposium, June 2017, and The Pirbright Institute Seminar series in Sept 2017. Abstracts and posters have been prepared for The Edinburgh Infectious disease symposium, June 2017, The Edinburgh Immunology Group symposium, June 2017, and abstracts submitted to the British Society for Immunology Congress, September 2017. Public engagement in the past year was significant. Drs. Bryson and Vervelde participated in The Midlothian science festival/The Roslin Institute open day October 2017. They engaged the public (~500 visitors) and raised awareness of bacterial infections including APEC. To have global dissemination of information about this project in popular media, a U-Tube video was jointly created and is hosted on the USDA website, with links on the Roslin and Iowa State University sites: https://www.youtube.com/watch?v=qDKbwVHPwZo&feature=youtu.be PD Lamont was invited to give keynote talks on genetics of disease resistance in poultry at the World Veterinary Poultry Association (WVPA), and the Animal Genetics and Disease conference at the Wellcome Genome Campus. coPD Stevens was selected to give the Avian Pathology lecture at the WVPA conference. What do you plan to do during the next reporting period to accomplish the goals?For all objectives, results will continue to be publically presented at national and international conferences and will be submitted for publication in peer-reviewed scientific journals. 1. Characterize interaction of E. coli with antigen-presenting cells (APC) in the bronchus-associated lymphoid tissue (BALT). We will analyze the transcriptome data from the sorted infected and uninfected macrophages. We will follow up on our adoptive transfer experiments, examining the specific phenotype of the important lymphocyte subsets involved during APEC infection e.g. Th1, Th2, Treg, and cytotoxic phenotypes. Data generation and analysis will be conducted on the precision cut lung slice model to determine early events in host-pathogen interactions. We will continue to examine the uptake and interaction of APEC with macrophages in lung slices, and compare the interactions of different strains of APEC with these cells 2. Determine cell-type-specific adaptive immune response to colibacillosis. Following on from pilot studies, an in vivo study is underway to examine the role of CD4+ and CD8+ T cell subsets in APEC infections. Primed CD4+ and CD8+ T cells will be isolated from APEC challenged birds and transferred into naïve recipients 24 hours prior to APEC challenge of the recipient birds. The capacity of the transferred primed T cell subsets to protect naïve chickens against APEC challenge will be examined during the infection in terms of clinical symptoms, pathology and bacteriology. 3. Map host genomic elements controlling responses to APEC. Additional analyses (such as window-based and Bayesian methods) for GWAS will be tested. Associated regions or SNPs will be further investigated for proximity to candidate genes that could contribute to resistance or susceptibility to bacterial colonization. 4. Identify cis-acting elements affecting host resistance. Next steps include sequencing of the broiler F1 cross right lung libraries, bioinformatic processing and mapping of the resulting reads, and detection of allele specific expression (ASE) in both the splenic and lung datasets. This analysis will consider ASE in response to APEC challenge, post-infection time, direction of reciprocal cross, and the interaction of these factors. Spleen and lung will also be investigated in the Leghorn x Fayoumi reciprocal crosses, including RNA-sequencing and DE and ASE analyses. Use of the additional reciprocal crosses could identify ASE from the Leghorn and epistatic effects of Fayoumi-derived ASE. 5. Evaluate potential to translate results into commercial layer lines. Using associated SNPs identified in Objective 3 and the ASE results in Objective 4, up to 100 SNPs will be genotyped to determine allele frequencies in multiple generations of 8 lines from a major layer breeding company. Within these commercially relevant lines, candidate variants/genes will be investigated for significant impacts on production traits and could provide information for their commercial products.
Impacts
What was accomplished under these goals?
Overall impact statement: Avian pathogenic E. coli (APEC) is the major cause of colibacillosis, an important bacterial disease, in poultry. Our studies provide one of the first efforts to identify genomic regions associated with APEC resistance in chickens.Predicted heritabilities for APEC colonization levels after inter-airsac infection were low, suggesting limited feasibility of genetic improvement based on the identified candidate SNPs. However, changes in the splenic transcriptome after challenge with APEC revealed responses in many immune-related genes and pathways, providing valuable insight into the molecular mechanisms of the early systemic immune response to bacterial infection.Our novel use of transgenic chickens with fluorescent mononuclear phagocytic cells allowed an unprecedented opportunity to identify host cell and pathogen interactions. The validation of the new precision-cut lung slice model sets a foundation for examining early host-pathogen interactions in respiratory diseases. The greater understanding of the host-pathogen interaction and its genetic control will pave the way for breeding and immunological control of this important poultry disease. Objective 1. Characterize interaction of E. coli with antigen-presenting cells (APC) in the bronchus-associated lymphoid tissue (BALT). Mononuclear phagocytic cells (MPC) were isolated from the lung of infected and PBS treated transgenic chickens, expressing fluorescent reporter proteins in the mononuclear phagocytic lineage, under the control of the CSF-1R promotor, then MPC were separated by FACS into fractions of cells positive or negative for the presence of intracellular GFP expressing APEC. A fraction of non-MPC positive for the presence of intracellular APEC was also isolated. RNA isolated from these cell fractions was subjected to RNAsequenced (awaiting data). To compliment this work, the populations involved have been examined by flow cytometry and histology. Populations of cells expressing differential levels of CSF-1R have been identified, with distinct capacities for APEC uptake. We have also established and validated a new model for examining host-pathogen interactions in the chicken lung. Precision cut lung slices (PCLS) were generated from transgenic chickens expressing fluorescent reporter proteins (either mApple [red] or Green fluorescent protein) in the mononuclear phagocytic lineage, as a platform to examine the early interactions. These PCLS provide an ideal model for examining the early events in host-pathogen interactions, as early interactions would be extremely difficult to examine in vivo, and using in vitro cultures would negate the influence of the tissue architecture on these interaction. Real-time, dynamic interaction of macrophages with the fluorescently labelled APEC have been observed in viable PCLS from fluorescent CSFR-1 reporter chicken. Objective 2. Determine cell-type-specific adaptive immune response to colibacillosis. As little is known about the role of the adaptive immune response in colibacillosis we will investigate the contribution of lymphocyte subsets during APEC infection. Adoptive transfers are the bases of many elegant studies examining the role of the adaptive immune response. The technique has been applied to avian models, with transfer of primed lymphocytes conferring protection to a variety of pathogenic challenges including infectious bronchitis virus (Seo et al., 2000) and influenza virus (Seo et al., 2001). However, the difference in the behaviour of the transferred cells due to the vast physiological differences from mammals, including the lack of lymph nodes in the avian host, has not been addressed. We therefore carried out a series of pilot studies in MHC-matched chickens, adoptively transferring fluorescently labelled spleen- or lung-derived lymphocytes, in the presence and absence of E.coli derived LPS stimulation. The transferred fluorescently labelled lymphocytes were identified by flow cytometry in a number of recipient tissues including the blood, lung and spleen at 24 hours and 48 hours post transfer, validating the use of this technique in in vivo APEC challenges. In these pilot studies an increased proportion of lung-derived donor lymphocytes were detected in the recipient lung, relative to the proportion of spleen-derived donor lymphocytes. This preferential homing to the tissue of origin suggests that a mechanism of imprinting occurs in the avian immune response, as has been described for mammalian lymphocytes. Objective 3. Map host genomic elements controlling responses to APEC. Using genotypes collected with the Affymetrix Axiom 600k Chicken Genotyping Array (in 2016), genetic control of resistance to APEC was investigated in 324 chickens (advanced intercross line derived from broiler x Fayoumi). As part of the live-bird experiments (in 2015), bacterial loads after challenge with APEC were measured in five tissues.These data were used as an indicator of the relative resistance of each individual and provided phenotypes for heritability estimation and genome-wide association study (GWAS). Approximately 190,000 SNPs (after filtering for 95% call rate, 2% MAF, etc.) were used for analyses in ASReml and GenABEL.Heritability estimates were refined using a genomic-relationship matrix (rather than pedigree-based as performed in 2016) and found to be very low.This suggests that bacterial load may be influenced by many genes and by non-genetic factors (such as batch variation in the laboratory processing).Single SNP associations were tested for each tissue in GenABEL; 20 SNPs located on the microchromosomes reached genome-wide significance for the right lung and warrant further investigation. Signalling lymphocyte activation molecule family (SLAMF) are a group of receptors that modulate cells of both the innate and adaptive immune system and have been reported to be involved in the uptake of gram negative bacteria. We designed primers and carried out qPCRs to examine the levels of SLAMF1, SLAMF2, SLAMF4 and SLAMF8 in lung tissue isolated from infected and PBS treated birds. No significant effects have been noted at the tissue level. Objective 4. Identify cis-acting elements affecting host resistance. Progress toward this objective continued, based upon study of tissue samples collected in 2016 from two replicates of the APEC challenge experiment, incorporating birds from four reciprocal F1 crosses (broiler x Fayoumi and Leghorn x Fayoumi).Initial experiments have focused on the broiler reciprocal crosses.Total RNA was isolated from two tissues (spleen and right lung) to provide an assessment of the host expression response both at an infection site (lung) and within an immune response organ (spleen).Samples for RNA isolation were selected to provide full siblings in the APEC-infected and non-infected groups, to incorporate two time points (1 DPI and 2 DPI) and to equally represent both replicates of the challenge (A and C).In total, 96 RNA samples were produced (n = 3 samples/F1 cross/DPI/treatment/replicate/tissue) and used for cDNA library construction.Spleen libraries (n = 48) were sequenced on the Illumina HiSeq 3000, producing 100bp paired-end reads.After quality control steps, the reads were mapped to the chicken reference genome (Galgal5) and the read counts per gene were used for differential expression (DE) analysis.Significant DE between treatments and/or across DPI was detected in the spleen for more than 400 genes.Exposure to APEC induced the greatest response at 1 DPI, with DE of genes involved in leukocyte activation, phagocyte differentiation and migration, and complement, suggesting the initiation of an early immune response. Fewer DE genes identified at 2 DPI, which is consistent with the decrease in bacterial load in the spleen over time determined during the challenge experiments). Objective5. Evaluate potential to translate results into commercial layer lines. Not started yet.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Monson, M.S., Kaiser, M.G., Wolc, A., Lamont, S.J. Investigating genetic control of resistance to avian pathogenic Escherichia coli colonization in chickens. 36th International Society of Animal Genetics Conference; July 16-21, 2017; Dublin, IRL. Abstract MT172.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
L. Vervelde. Fluorescent chickens to lighten up your research application of CSF1R-transgenic chickens to study host-pathogen interactions The Pirbright Institute, 20th September 2017, Pirbright, UK.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Andreas Alber, Taiana Costa, Cosmin Chintoan-Uta, Karen J. Bryson, Mark P. Stevens and Lonneke Vervelde. Differential response of chicken lines 15I and 72 to high but not medium or low dose APEC infection. The Edinburgh Infectious disease symposium, 1st June 2017, Edinburgh, UK
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Andreas Alber, Taiana Costa, Cosmin Chintoan-Uta, Karen J. Bryson, Mark P. Stevens and Lonneke Vervelde. Differential response of chicken lines 15I and 72 to high but not medium or low dose APEC infection. The Edinburgh Infectious disease symposium, 15th June 2017, Edinburgh, UK
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Andreas Alber, Taiana Costa, Cosmin Chintoan-Uta, Karen J. Bryson, Mark P. Stevens and Lonneke Vervelde. Differential response of chicken lines 15I and 72 to high but not medium or low dose APEC infection. Avian Genetics and Immunity, 19th June 2017 Guilford, UK.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Monson, M.S., Kaiser, M.G., Wolc, A., Lamont, S.J. Investigating genetic control of resistance to avian pathogenic Escherichia coli colonization in chickens. 36th International Society of Animal Genetics Conference; July 16-21, 2017; Dublin, IRL. Oral presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
L. Vervelde. Fluorescent chickens to lighten up your research application of CSF1R-transgene chickens in immunological studies. Avian Genetics and Immunity, 19th June 2017, Guilford, UK.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
L. Vervelde. Fluorescent chickens to lighten up your research application of CSF1R-transgenic chickens in immunological studies. XXth World Veterinary Poultry Association Congress, 4th Sept 2017, Edinburgh, UK.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Bryson K, Esposito M., Lamont S., Stevens M., McLaughlan G. and Vervelde L. Precision cut lung slices: A platform for examining host-pathogen interactions in an avian model. Accepted, The British Society for Immunology, December 4-7th, Brighton, UK
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Bryson K, Alber A, Ellis S, Borowska D, Stevens M, Lamont S and Vervelde L. The adaptive immune system in an avian model. Accepted, The British Society for Immunology, December 4-7th, Brighton, UK
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Progress 02/01/16 to 01/31/17
Outputs
Target Audience:The target audiences of this US-UK project includes, in both the US and the UK, researchers and practitioners in animal health and animal genomics, commercial breeders of poultry, poultry farmers/producers, vaccine developers, students, the USDA and other US government agencies and the Biotechnology and Biological Sciences Research Council (BBSRC) and other UK government agencies. Changes/Problems:The studies to date have provided valuable information about needed modifications in protocols to successfully continue the research. All the original objectives remain unchanged. The differential response of inbred chicken lines 72 and 15I to combined challenge with infectious bronchitis virus and APEC, reported decades earlier, was demonstrated to hold true at high but not at low doses of APEC alone. This clearly established that this specific animal model will not be appropriate for use to study adaptive immunity as proposed, because the survival time at high doses is insufficient to capture the needed measurements and samples. This information helps to appropriately redirect the next steps of the current project, and also alerts other researchers about limitations in this experimental animal model of 72 and 15I chicken lines. The work at Roslin is expanded in a different direction to achieve the objective, taking full advantage of new technologies and availability of unique genetic material, by planning to do whole-mount imaging of the lung, trachea and air sac of APEC-infected birds. The ability to examine APEC response in multiple genetic lines has been addressed by adding an additional reciprocol-cross pair to the allele-specific expression studies at Iowa State University. What opportunities for training and professional development has the project provided?New and additional training of lab members in the US was conducted on protocols for the pathogen-challenge studies and bacterial colony enumeration. Melissa Monson (postdoc - funded by this project), Michael Kaiser (research associate), Jibin Zhang (postdoc), Damarius Fleming (PhD student), Angelica Bjorkquist (PhD student), Melissa Herrmann (PhD student), Kaylee Rowland (PhD student), John Hsieh (PhD student), Zach Adams (undergraduate student). New and additional training of lab members at the Roslin Institute, University of Edinburgh, was conducted on protocols for pathogen-challenge studies, and for use of transgenic chicken model with gfp-expressing APEC to visualize bacteria-host phagocyte interaction. The PD and coPDs received several recognitions in the past year. PD Lamont was named a Fellow of the International Society of Animal Genetics, and also won the Iowa State University College of Agriculture and Life Sciences Diversity Award. coPD Stevens was elected a Fellow of the Royal Society of Biology. coPD Vervelde was appointed a member of the scientific board for the World's Veterinary Poultry Association for the 2017 meeting. How have the results been disseminated to communities of interest?Abstracts and posters have been presented at the Plant and Animal Genome meeting in January 2016 (Objective 3) and at the Avian Immunology Research Group meeting in September 2016 (Objective 1). What do you plan to do during the next reporting period to accomplish the goals?For all objectives, as soon as research studies are completed, the results will be promptly and publically presented at conferences, and prepared as manuscripts for submission for publication in peer-reviewed scientific journals. 1. Characterize interaction of E. coli with antigen-presenting cells (APC) in the bronchus-associated lymphoid tissue (BALT). MacRed chickens will be inoculated with APEC-gfp, the lungs will be harvested at various times post-infection, from which the antigen-presenting cells (APCs) will be isolated by flow cytometry. Then, RNAseq will be used to assess the responses of APCs. Whole-mount imaging of the lung, trachea and air sac will be conducted to evaluate the interaction of the bacteria with host cells. 2. Determine cell-type-specific adaptive immune response to colibacillosis. From the birds tested in Objective 1 studies, the lung and spleen will be used to study the adaptive immune response by specific cell subsets, using immunohistochemistry. Expression of signature cytokines will be measured by qRT-PCR. 3. Map host genomic elements controlling responses to APEC. The GWAS will be completed using the GenSel program to identify genomic regions associated with resistance and susceptibility to bacterial colonization in the advanced intercross line (of Fayoumi and broiler founder lines). 4. Identify cis-acting elements affecting host resistance. Two tissues (spleen and right lung) from four different crosses of lines will be used for the expression experiments. The next steps are library construction and RNA-sequencing of these samples, which will ultimately allow examination of differential expression (DE) and ASE in response to factors including APEC challenge, post-infection time, and direction of reciprocal cross in each pair of crosses, plus the interactions of these factors. 5. Evaluate potential to translate results into commercial layer lines. Allele frequencies and effects on production traits in commercially relevant populations will be determined for the top candidate genes identified in the study. From the results of Objective 3, and additionally informed by the gene-expression study results, about 100 selected SNPs will be genotyped on several generations of 8 major lines of birds contributing to the commercial products of a major layer breeding company.
Impacts
What was accomplished under these goals?
OVERALL IMPACTS The differential response of inbred chicken lines 72and 15I to combined challenge with infectious bronchitis virus and APEC, reported decades earlier, was demonstrated to hold true at high but not at low doses of APEC alone. This clearly establishes that this specific animal model will not be appropriate for use to study adaptive immunity, because the survival time at high doses is insufficient to capture the needed measurements and samples. This information helps to appropriately redirect the next steps of the current project, and also alerts other researchers about limitations in this experimental animal model of 72and 15I chicken lines. Thegfp-expressing and wild-type APEC 01 strains were found to replicate at same rate in various tissues, validating the use of thegfp-expressing strain as a method to characterize interaction of bacteria and host cells in the CSF-1R transgenic chicken. The studies provided the first heritabilities of APEC bacterial colonization in several tissues, which helps to predict the feasibility of identifying genetic elements controlling the host response and using them in genetic improvement programs to enhance bacterial resistance. 1. Characterize interaction of E. coli with antigen-presenting cells (APC) in the bronchus-associated lymphoid tissue (BALT). Three pilot studies were performed to (i) set up an animal model to dissect host-pathogen interactions during avian colibacillosis, with particular reference to immune responses underlying pathology and protection; (ii) determine if inbred chicken lines line 72 and 15I respectively exhibit resistance or susceptibility to APEC, as reported in the literature following co-infection with Infectious Bronchitis Virus and APEC; (iii) develop techniques to study the interaction of gfp-expressing APEC O1 with antigen presenting cells (APC) in vivo using transgenic birds that express a fluorescent protein in all myeloid cells. Having optimised the inoculation route and strain, in two separate studies we showed that line 15I birds are highly susceptible to a challenge with 107 APEC O1. The clinical response of line 72 birds to the same strain and dose was less marked, however bacterial counts in the lungs and visceral organs were not significantly different. After challenge of the lines with a lower dose (106 and 105) we did not detect any differential resistance to colonisation at a range of sites. Because the high dose is lethal (within 24 hrs) for 15I birds it was concluded that these lines would not be suitable to dissect the immune responses underlying avian differential resistance to APEC. In a subsequent study, transgenic chickens expressing a fluorescent reporter protein (mApple) in all cells of the mononuclear phagocyte lineage (CSF-1R transgenic) were challenged with gfp-expressing APEC O1 to enable visualisation of the interaction between mononuclear phagocytes (red) and APEC (green) by flow cytometric analysis and histology ex vivo. In this study, the gfp-expressing APEC O1 were constructed and validated by comparison with the wild type APEC O1 and it was demonstrated that both strains replicated to similar levels in the lungs and visceral organs. Co-localisation of APEC and mononuclear phagocytes was studied in the lungs, spleen and peripheral blood. Flow cytometric analysis demonstrated that more than 50% of the bacteria was detected associated with leukocytes in the lung of which the majority (over 70%) was taken up by CSF-1R+ mononuclear phagocytes at 1 day post inoculation. Although APEC could be cultured from spleen and blood, the detection level was too low to study their interaction with phagocytes in this study. 2. Determine cell-type-specific adaptive immune response to colibacillosis. Not started yet. 3. Map host genomic elements controlling responses to APEC. Using blood collected from over 300 chickens (advanced intercross line derived from broiler x Fayoumi), we obtained genotype data for each individual using the Affymetrix Axiom 600k Chicken Genotyping Array. All samples genotyped successfully and after filtering for quality control (QC) parameters (95% call rate, 2% MAF, etc.), approximately 200,000 SNPs were found to be segregating in the AIL and thus usable for genome-wide association study (GWAS). The bacterial loads after challenge with avian pathogenic E. coli (APEC) were measured in five tissues during the live-bird studies conducted in the previous year to provide the phenotype data that will be used for estimating relative resistance and susceptibility of individual birds for the GWAS analysis. Heritabilities for bacterial colonization levels were estimated using these phenotypes; most were low but measurable, suggesting complex genetic control and influence of non-genetic factors. 4. Identify cis-acting elements affecting host resistance. Two replicate pathogen challenges of reciprocal F1 crosses of broiler x Fayoumi were performed during the past year. Two additional reciprocal crosses of inbred Leghorn x Fayoumi were challenged alongside the broiler crosses to allow the opportunity to determine epistatic interactions on Fayoumi-derived allele specific expression (ASE). Birds were challenged with APEC using a lower dose and other protocol refinements developed in the previous year's studies. Tissues were collected 1, 2 and 6 days after APEC inoculation and used to determine bacterial colonization levels in multiple tissues of each bird. Samples from eight tissues were collected and archived for future isolation of RNA. Two tissues (spleen and right lung) will be used for the initial expression experiments. These will allow assessment of a target tissue (lung) and an immune response organ (spleen) to assess host response at various stages. 5. Evaluate potential to translate results into commercial layer lines. Not started yet.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Monson, M.S., Kaiser, M.K., Lamont, S.J. Investigating host resistance to avian pathogenic Escherichia coli using colonization levels in a chicken advanced intercross line. Plant and Animal Genomes Conference XXIV; January 9-13, 2016; San Diego, CA. Abstract P0669.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Alber, A., Costa, T., Chintoan-Uta, C., Stevens, M., Vervelde, L. Innate immunity in the respiratory tract of chickens during avian pathogenic E. coli infection. XIV Avian Immunology Research Group Meeting; September 7-10, 2016; Herrsching am Ammersee, DEU. Abstract P35.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Costa, T., Alber, A., Balic, A., Stevens, M., Vervelde, L. Characterisation of the developing immune tissues in the chicken respiratory tract. XIV Avian Immunology Research Group Meeting; September 7-10, 2016; Herrsching am Ammersee, DEU. Oral presentation.
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Progress 02/01/15 to 01/31/16
Outputs
Target Audience:Animal health researchers, professionals, and students Changes/Problems:Training of US personnel with the UK group introduced the US researchers to some detailed and time-saving protocols, which were then applied in the US studies. What opportunities for training and professional development has the project provided?Two researchers from the US trained with the UK co-investigators, and brought the new knowledge back to the US for application in the project. Several lab members in the US were trained in protocols in the conduct of the pathogen-challenge studies. How have the results been disseminated to communities of interest?An abstract has been submitted (and accepted) for the Plant and Animal Genome meeting in January 2016. What do you plan to do during the next reporting period to accomplish the goals?The blood collected from the APEC-challenge experiment will be used to isolate DNA that will be genotyped on the 600K SNP panel. The genotype and phenotype data will be used to conduct genome-wide association studies to identify regions controlling APEC infection. An APEC-challenge study with an F1 cross will be conducted, to harvest samples to study allele-specific expression. Results of completed studies will be presented at conferences and prepared for manuscript submission.
Impacts
What was accomplished under these goals?
The long-term goal of this joint US-UK project is to reduce the negative impact of respiratory avian pathogenic Escherichia coli (APEC) on the poultry industry through development of complementary veterinary (vaccines and biologicals) and breeding (genetic selection for resistant alleles) control strategies that are based on detailed understanding of the host functional response to E. coli infection. Avian colibacillosis caused by APEC is responsible for mortality in poultry flocks as well as other important symptoms, yet there is little research on avian airway responses to APEC and the basis of heritable host resistance. Impacts of the past year included use of unique inbred chicken lines from Iowa State University that differ in resistance to APEC to initiate studies on heritability of APEC resistance, generating quantitative phenotypes of bacterial load in several tissues. We collected blood for isolation of genomic DNA for future genotyping. We trained US and UK personnel in relevant techniques, and coordinated and refined protocols by conducting a joint pilot study. These activities of developing human resources, refining and coordinating protocols, and collecting biological samples and quantitative phenotypes form the foundation for future studies on host functional response to APEC. Objective 1) Characterize interaction of E. coli with antigen-presenting cells (APC) in the bronchus-associated lymphoid tissue (BALT). No activities in the past year. Objective 2) Determine cell-type-specific adaptive immune response to colibacillosis. No activities in the past year. Objective 3) Map host genomic elements controlling responses to APEC. Two major phases of work were accomplished in the past year. In the first phase, two researchers from the US travelled to the UK partners' site to participate in a pilot challenge study. The purposes of this study were to acquire and refine information for the protocols before conducting the larger-scale animal trials in the UK, and also to provide training in specific procedures to the US researchers so that protocols would be standardized between the two countries' studies. The results of the pilot were that pathology was not expressed at the level anticipated, and therefore refinements in future protocols will be made. The trained US researchers returned to the US and applied the information and procedures learned. In the second phase, a large-scale pathogen-challenge trial was conducted in the US. Over 400 chickens of specialized genetic lines (advanced intercross of Fayoumi X broiler) were challenged with avian pathogenic E. coli (E. coli). Tissues were harvested from these birds, and used to quantify the bacterial load in several tissues, to estimate the relative resistance/susceptibility of the birds. This information will serve as the phenotypes to enter into the next phase of work, genome-wide association analysis, after the 600K SNP data are received. Objective 4) Identify cis-acting elements affecting host resistance. No activities in the past year. Objective 5) Evaluate potential to translate results into commercial layer lines. No activities in the past year.
Publications
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