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United States Department of Agriculture

Agricultural Research Service

Research Project: Identification of Host Factors and Immunopathogenesis of Pneumonia in Domestic and Bighorn Sheep

Location: Animal Disease Research

Project Number: 2090-32000-036-00-D
Project Type: In-House Appropriated

Start Date: Oct 18, 2016
End Date: Oct 17, 2021

The goals of this project are to decrease pathogen transmission and respiratory disease in domestic sheep and bighorn sheep through genetic and vaccine intervention strategies, and to fill scientific knowledge gaps in the immunopathogenesis of ovine respiratory disease by comparatively analyzing the innate and adaptive immune responses of domestic and bighorn sheep. Specifically, during the next five years we will focus on the following objectives: Objective 1: Identify the host factors associated with nasal shedding and pneumonia associated with Mycoplasma ovipneumoniae in domestic and bighorn sheep. Subobjective 1A: Identify genetic markers in domestic sheep for absence or reduced shedding of Mycoplasma ovipneumoniae, a respiratory pathogen of domestic and bighorn sheep. Subobjective 1B: Improve the accuracy of domestic sheep selection with genomic breeding values for absent or reduced shedding of Mycoplasma ovipneumoniae, a domestic and bighorn sheep respiratory pathogen. Objective 2: Determine the comparative innate and adaptive immune factors associated with susceptibility to Mycoplasma ovipneumoniae between domestic and bighorn sheep. Subobjective 2A: Characterize and compare innate immune responses of domestic and bighorn sheep leukocytes to Mycoplasma ovipneumoniae. Subobjective 2B: Characterize and compare adaptive immune responses and associated immunopathology of domestic and bighorn sheep infected with Mycoplasma ovipneumoniae. Subobjective 2C: Immunize naïve domestic and bighorn lambs with a developed intranasal adjuvanted killed Mycoplasma ovipneumoniae vaccine and compare immune response to that of experimentally infected domestic and bighorn sheep in Subobjective 2B.

Obj 1: Genome-wide association studies (GWAS) and whole genome re-sequencing will identify one or more genomic regions that are associated with probability and/or amount of M. ovipneumoniae shedding from domestic sheep (DS). Genomic selection will achieve selection accuracy of at least 40% (independent of pedigree information) for DS that have reduced probability and/or amount of M. ovipneumoniae shedding. Qualitative polymerase chain reaction (qPCR) will be used to determine presence/absence and quantify M. ovipneumoniae nasal shedding from DS and GWAS to identify localized genomic regions of interest for M. ovipneumoniae shedding phenotypes. Genotype DS with a high density array containing approximately 600,000 Single Nucleotide Polymorphism (SNP). Conduct causal mutation identification with fine mapping, whole genome re-sequencing, and genotype imputation. Conduct validation of identified markers in a different set of DS. Perform genomic selection calculations from the qPCR phenotypic and GWAS genotypic data by BayesR analysis. If the initial experimental designs are unsuccessful in evaluating the hypotheses, GeneSetEnrichmentAnalysis (GSEA) SNP methods will be employed and additional DS will be added. Obj 2: Perform quantifiable assessments to identify differences in innate immune responses of DS and bighorn sheep (BHS) leukocytes (LEU) exposed to M. ovipneumoniae. Compare adaptive immune responses and immunopathology of DS and BHS infected with M. ovipneumoniae in order to characterize immune corrects of protection. Develop an intranasal vaccine against M. ovipneumoniae that stimulates immune responses in DS and/or BHS comparable to the immune correlates of protection identified in the 2nd research goal. Expose isolated peripheral blood LEU to M. ovipneumoniae in vitro. Evaluate cellular responses using flow cytometry to determine phagocytosis and leukocyte differentiation molecule (LDM) abundances, and use commercially available kits to quantify cell activation. If cellular protein concentrations are below detectable levels for the enzyme-linked immunosorbent assay (ELISA) kits, Western blot analyses will be performed. Naïve DS and BHS will be infected with M. ovipneumoniae. Measure mucosal and systemic adaptive immune responses (antibody) utilizing bacteria growth inhibition, ELISA, and Western blot analyses. Measure cytokines and LEUs within pulmonary lavage fluid and blood pre- and post-infection by commercially available ELISA kits and characterized by flow cytometry. If too few cells are obtained from lavage, cells will be fixed on slides and analyzed by immunocytochemistry. Perform lymphocyte stimulation assays on peripheral blood mononuclear cell (PBMC) isolated post-infection. Analyze formalin-fixed paraffin-embedded archived lung tissue from naturally infected DS and BHS by immunohistochemistry to characterize the LEUs present during infection. Develop an immune stimulating complexes (ISCOM) adjuvanted intranasal M. ovipneumoniae vaccine and use it to immunize naïve lambs of each species. Immune response to immunization will be performed and compared to the measured responses of experimentally infected sheep.

Last Modified: 09/19/2017
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