Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Title: Genome sequencing of novel Clostridial isolates with probiotic potential Primarily academic with knowledge transfer to industry. Title: Macrophage priming affects disease resistance and susceptibility in response to viral pathogens. Academic Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Title: Genome sequencing of novel Clostridial isolates with probiotic potential Training of lab technicians in bacterial culture and DNA extraction protocols. Title: Macrophage priming affects disease resistance and susceptibility in response to viral pathogens. Mr. Jory Clark, who started his Master Thesis on this project is on target to finish his experiments in the early summer of 2016, graduating by fall. He has successfully mastered various laboratory techniques during this project, such as isolation of mononuclear cells, cell culture and differentiation, RNA isolation, cDNA synthesis, RealTime PCR in addition to data analysis and has already been included in the submission of one of our manuscripts (under review). Mr. Clark also successfully applied to Western University's Veterinary College and will start his DVM studies fall of 2016. How have the results been disseminated to communities of interest?Title: Genome sequencing of novel Clostridial isolates with probiotic potential Deliverables Swift SM, Seal BS, Garrish JK, Oakley BB, Hiett K, Yeh HY, Woolsey R, Schegg KM, Line JE, Donovan DM. A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability. Viruses 2015, 7(6):3019-3034. Oakley BB, Kogut M, Donovan DD, Cox NA, Berrang ME, Chalghoumi R, Seal, BS. Managing the Gut Microbiome of Food Animals With Tools of Microbial Ecology. Poster presentation at IPC2015 meeting, Budapest, Hungary, June 21-23 2015. Title: Macrophage priming affects disease resistance and susceptibility in response to viral pathogens. Manuscript submitted: PLOS ONE: Irizarry KJ,*, Downs E, Bryden R, Clark J , Griggs L, Kopulos R, Boettger CM, Carr TJ Jr. , Keeler CL, Collisson E, Drechsler Y,**: RNA Sequencing Demonstrates Large-Scale Temporal Dysregulation of Gene Expression in Stimulated Macrophages Derived from MHC-Defined Chicken Haplotypes Abstract submitted: AAI 2016: Drechsler Y, Irizarry KJ, Downs E, Bryden R, Clark J , Griggs L, Kopulos R, Boettger CM, Carr TJ Jr. , Keeler CL, Collisson E: Global dysregulation of gene expression in macrophages from different haplotypes correlates with disease resistance Grants planned: USDA NIFA RFA 2016 (to be announced) What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The two below projects represent the funded and completed projects during the 2014-2015 NIFA period per the goals of the overall project. Title: Genome sequencing of novel Clostridial isolates with probiotic potential PI: Brian Oakley Co-I(s): Dr. Raja Chalghoumi, Assistant Professor, Animal Science Department, University of Carthage, Tunisia Background The rationale for this project was based on recent data by the PI and others showing that bacteria belonging to the Clostridiales order are dominant members of the healthy chicken cecal microbiome. Members of our research team had previously embarked on a systematic approach to isolate and characterize novel strains of Clostridia occurring naturally with the healthy chicken GI tract for potential use as probiotics. A Fulbright fellow (Dr. Chalghoumi) visiting the PI's previous lab successfully brought into cultivation various members of the Clostridia from chicken ceca. The funding requested here was for two main purposes: 1) whole-genome sequencing of these strains, and 2) travel to an international scientific congress (IPC2015) on probiotics. As part of a larger research program by the PI to develop tools and strategies for managing the microbiota, this research met the mandates of Section 1433 funds by promoting general welfare through improved health and productivity of domestic poultry and reducing reliance on antibiotics as growth promoters. The genomic data made possible by this project is enabling characterization of these strains to facilitate their potential use as probiotics. Travel to the IPC2015 congress helped promote the project via interactions with the scientific community and presentation of results. Results Bacterial isolates previously obtained from the mid-gut and ceca of commercial broiler chickens were grown from freezer stocks and genomic DNA preparations sent for whole-genome sequencing at Retrogen. Genomic DNA was sequenced on an Illumina MiSeq instrument with 300 bp paired-end reads using the V3 reagant kit. Approximately 10.8 Gb of high-quality data (>=Q30) was obtained from the sequencing run. Following sequencing, data were further processed using standard genomics tools including trimmomatic, the fastx toolkit, flash, assembly with Velvet and Spades, and annotation with RAST. Most analyses are still underway, but we have obtained some preliminary results. Of the 48 isolates sequenced, at least two appear to represent novel species within Clostridium Cluster I based on preliminary genomic analyses. Genome sequencing of these two strains (N50 values of 129255 kb and 470109 kb respectively) has revealed several genes that may have commercial value as alternative antimicrobials, particularly several bacteriophage holin and endolysin genes. The sequences of these genes has not yet been published, but was used for comparative genomics in a related project with a peer-reviewed paper in 2015 as listed below. With many analyses of the large genomic data set still underway, we are targeting submission of a more comprehensive genomics manuscript in the 2016 calendar year. Title: Macrophage priming affects disease resistance and susceptibility in response to viral pathogens. PI: Drechsler, Yvonne Background and justification: Although the ultimate strategy for controlling viral and bacterial infections is to develop genetically resistant animals, the underlying molecular mechanisms conferring disease resistance are poorly understood. We have previously demonstrated that B2 haplotype birds resolved infectious bronchitis virus (IBV) infection in lungs, kidneys and trachea more readily than B19 infected chicks and showed less clinical illness (Banat et al. 2013). At least part of this difference can be explained by the consistently more vigorous responses of B2 macrophages to Poly IC and IFNg stimulation than responses of B19 derived macrophages (Dawes et al. 2014). Recent studies in mammalian and avian species have shown that priming or polarization of macrophages is a critical factor in shaping the immune response towards inflammation or tolerance/healing. Thus investigating the underlying mechanisms will significantly improve our understanding of the role of macrophages in disease resistance. Objectives and hypotheses: The overriding hypothesis is that polarization of macrophages is involved in disease resistance of chicken haplotypes in response to pathogens. Our overall goal is to identify unique markers or genes that are involved in polarization of chicken macrophages and determine the role of these markers in the "training" or priming of macrophages on disease resistance. Specific Objectives: Polarize macrophages in vitro and identify differential gene expression in B2 versus B19 macrophages Approach: The proposed goals will build on our recent and ongoing studies characterizing differences in peripheral macrophages in different B haplotype chickens. In the context of identifying mechanisms between resistance and susceptibility in birds, Macrophages will be stimulated in vitro with IFNg and IL-4 to polarize. Pathways including TLR signaling and adenosine receptor signaling will be further investigated with PCR. These genes were selected based on results obtained by RNA sequencing of stimulated macrophages from different B haplotypes. Potential impact: The proposed studies will be directly applicable to our capacity to identify, select and breed poultry on the basis of their resistance to respiratory viruses and defining mechanisms associated with polarization of macrophages may lead to better strategies to control and prevent disease and provide innovative, critical immunological tools. Results He et al. have shown that HD11 avian macrophage cells can be stimulated differentially by Th1 and Th2 cytokines IFNg, IL 4 and both cytokines were able to stimulate the chicken macrophage cell line HD-11 to produce nitric oxide. However, there are no studies currently published on characterization of M1 and M2 polarization of primary avian macrophages. We are currently establishing which genes and markers are upregulated when primed differentially in peripheral monocytes in response to Th1 and Th2 cytokines. We have stimulated macrophages from B2 and B19 haplotype chickens with IFNg and IL-4. While HD-11 controls showed some nitric oxide release after stimulation with both cytokines as shown by He et al., we have found that only IFNg stimulates the release of nitric oxide from B2 as well as B19 primary macrophages. IL-4 did not induce nitric oxide in any of the primary macrophages, which is more in line with the function of IL-4 as M2 cytokine. Macrophages have both iNOS and arginase enzymes that can convert arginine to NO or ornithine, respectively. Typically IFNg as M1 cytokine will induce NO production while IL-4 does not as previously shown in human, mouse and other macrophages. RNA has been collected from B2 and B19 macrophages stimulated with either IFNg or IL-4 for 0, 2 or 4 hours. Primers characterizing M1 vs M2 phenotypes have been established (IL1, IL6, IL12, HES-1, SOCS3, CD163, TGFb, IL-10, YM-1 and Mannose Receptor 1, VEGF) and Real-Time PCR performed. Data analysis is currently being done on preliminary results, with still more birds ordered to increase the sample size.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Swift SM, Seal BS, Garrish JK, Oakley BB, Hiett K, Yeh HY, Woolsey R, Schegg KM, Line JE, Donovan DM. A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability. Viruses 2015, 7(6):3019-3034.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Oakley BB, Kogut M, Donovan DD, Cox NA, Berrang ME, Chalghoumi R, Seal, BS. Managing the Gut Microbiome of Food Animals With Tools of Microbial Ecology. Poster presentation at IPC2015 meeting, Budapest, Hungary, June 21-23 2015.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
PLOS ONE: Irizarry KJ,*, Downs E, Bryden R, Clark J , Griggs L, Kopulos R, Boettger CM, Carr TJ Jr. , Keeler CL, Collisson E, Drechsler Y,**: RNA Sequencing Demonstrates Large-Scale Temporal Dysregulation of Gene Expression in Stimulated Macrophages Derived from MHC-Defined Chicken Haplotypes
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2015
Citation:
Abstract submitted AAI 2016:
Drechsler Y, Irizarry KJ, Downs E, Bryden R, Clark J , Griggs L, Kopulos R, Boettger CM, Carr TJ Jr. , Keeler CL, Collisson E: Global dysregulation of gene expression in macrophages from different haplotypes correlates with disease resistance
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