Project : USDA ARS

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

2021 Annual Report

Objectives
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.

Approach
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.

Progress Report
This is the final report for the project 2090-32000-036-000D which will terminate in October 2021. The new project, titled “Genetic Approaches and Tools to Prevent, Control, and Eradicate Transmissible Spongiform Encephalopathies” is currently undergoing NP103 OSQR review. A summary of results for all the expiring project sub-objectives are described below. In support of Objective 1, researchers in Pullman, Washington, identified host factors through multiple means. Approximately 650 sheep were included in genomic analysis to identify genes and gene regions associated with M. ovipneumoniae nasal carriage. Genetic regions identified include genes that influence immune function and gene regulation, and this work has been published in a peer reviewed journal. Work is ongoing to increase the number of sheep with nasal shedding data to validate our findings. In addition, we are working to narrow the genomic regions associated with nasal shedding of Mycoplasma ovipneumoniae to identify causal mutation(s). Additional host factors were identified through genome annotation for sheep,other ruminants, and livestock species as part of the Functional Annotation of Animal Genomes project (FAANG). Researchers in Pullman, Washington, also reported the first mitochondrial genome sequence of bighorn sheep. Furthermore, researchers in Pullman, Wasington, contributed to the development of high throughput phenotyping and big data analytics for livestock. In support of Objective 2, researchers in Pullman, Washington, determined multiple factors in immune responses and laid foundations for vaccine interventions. A notable contribution was a vaccine tested against respiratory pathogen M. haemolytica for bighorn sheep using a BHV-1 vector. IgG kinetics for domestic and bighorn sheep lambs as a component of comparative immunity were determined to be similar, as well as identification of a much higher prevalence of M. ovis in domestic sheep than previously appreciated, and comparison of currently available diagnostic tests for M. ovipneumoniae. Researchers in Pullman, Washington, compared immunological responses to M. ovipneumoniae between adult domestic and bighorn sheep. This provides the foundation for understanding comparative immunology at younger ages by clarifying potential maternal and flock contributions to immune status. Researchers in Pullman, Washington, also produced a new Mycoplasma genome sequence, supporting development of microbial genomics as a contributor to vaccine development in future work. Finally, researchers in Pullman, Washington, identified M. ovipneumoniae in wildlife outside caprinae, including caribou, suggesting that control of M. ovipneumoniae in domestic sheep will not be a full solution to bighorn sheep epizootics under field conditions.

Accomplishments
1. Proteome-wide epitope prediction to enhance rational vaccine design for Coxiella burnetii. Coxiella burnetii is a bacterial pathogen that can affect both livestock and human beings. A major challenge for C. burnetii research has been prediction of useful epitopes for inclusion in next generation vaccines. ARS researchers in Pullman, Washington, screened approximately 2000 C. burnetii proteins for epitopes to stimulate T-cell immune responses, including both intracellular pathogen-specific responses and broad immune responses. This study utilized artificial intelligence to predict epitope presentation across a wide range of hosts (including cattle, human, and mouse) and a broad swath of genetic variation within each host species. The results identified epitopes common to these host species and new to specific vaccine design. These data suggest ways to efficiently advance rationally designed, broadly useful next-generation vaccines across multiple species in future research.

2. First genome-wide association study for Mycoplasma ovipneumoniae shedding. Immune genes associate with nasal shedding of Mycoplasma ovipneumoniae in domestic sheep. A bacterium, Mycoplasma ovipneumoniae, can cause pneumonia in domestic and Bighorn sheep leading to reduced production and potentially death. ARS scientists in Pullman, Washington, conducted an experiment and identified regions of the domestic sheep genome which were associated with nasal shedding of Mycoplasma ovipneumoniae. The identified genetic regions influence immune function and gene regulation. Understanding genetic differences between domestic sheep that shed Mycoplasma ovipneumoniae and sheep that do not, will allow for selective breeding to eliminate shedding of this bacteria and reduce incidence of pneumoniae.

3. First genome-wide association study for Coxiella burnetii susceptibility. Coxiella burnetii is a bacterium that can cause Q Fever in humans and coxiellosis (including abortions and abortion storms) in livestock. Livestock have been blamed for many human Q Fever outbreaks, which can include fever, pneumonia, hepatitis, pregnancy complications, and potentially fatal swelling of the heart. ARS researchers in Pullman, Washington, in collaboration with scientists from Washington State University, performed the first genome-wide association study of C. burnetii susceptibility. They used a fruit fly model system to identify and validate roles for 15 genes, with many participating in immune responses. Clear equivalence of these genes with livestock and human genes suggests new avenues for research. Further work will be required to better understand C. burnetii infection in both livestock and human beings.

4. Enabling improvement in accuracy for detection of Mycoplasma ovipneumoniae. A major challenge for sheep is the potential transmission of Mycoplasma ovipneumoniae. Detection of M. ovipneumoniae from domestic and wild animals is most commonly performed by polymerase chain reaction (PCR). The presence of previously undescribed bacteria with similarity to M. ovipneumoniae makes the development, implementation, and interpretation of PCR detection assays difficult. ARS researchers in Pullman, Washington, in collaboration with researchers at the Alaska Department of Fish and Game and Kansas State University, discovered, sequenced, and released the genome and gene annotation for a novel species of Mycoplasma. This resource allows for an improvement in the accuracy of detection of this bacterium by offering the ability to increase the specificity of detection assays.

U.S. DEPARTMENT OF AGRICULTURE

Animal Disease Research: Pullman, WA