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ARS Home » Pacific West Area » Pullman, Washington » Animal Disease Research » Research » Research Project #441237

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

Location: Animal Disease Research

2022 Annual Report

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.

Progress Report
This bridging project began October 18, 2021 and continues research from expired project 2090-32000-036-000D, which terminated in October 2021 and is currently undergoing NP103 Ad Hoc Office of Scientific Quality Review. Continued work on Objective 1 has focused on validating the prior results by increasing animal numbers and including a new sheep breed of interest. ARS researchers aim to sample an additional 600 sheep while including the Targhee sheep breed. During FY22, ARS researchers in Pullman, Washington, collected three sample time points and has focused on optimizing the assay assessing copies of M. ovipneumoniae DNA isolated by nasal swab. In support of Objective 2, ARS researchers are in the process of completing preliminary inoculation studies of M. ovipneumoniae in lambs. Prior work focused on the innate and adaptive immune responses of adult domestic and bighorn sheep species. However, it is unknown how younger animals will respond to infection and how their immune responses compare to those of adult animals.

1. Increased detection specificity of Mycoplasma ovipneumoniae on nasal swabs. M. ovipneumoniae is a bacterium that plays a role in chronic non-progressive pneumonia in domestic sheep populations. While this disease tends to be mild in domestic sheep, it causes high mortality rates in populations of bighorn sheep. As a result, grazing lands on which the domestic sheep feed have had access restricted to limit transmission between the two sheep species. Our team has designed a quantitative PCR method which limited off-target detection of other closely related Mycoplasma species from nasal swabs. This improvement allows for more accurate prevalence data on M. ovipneumoniae to be gathered and increases future testing accuracy.

2. Associated candidate genetic regions and monocyte immune cell numbers from complete blood counts. Monocytes are a circulating blood cell that can populate tissues with dendritic cells and macrophages, two cell types which are important in innate immunity. Veterinarians use monocyte numbers to determine when an animal is undergoing a period of prolonged stress or if there is a present infection. Association of complete monocyte numbers from sheep blood identified two significant genetic regions and ten suggested significant genetic regions. Assessment of these regions determined the presence of immune associated genes which could affect monocyte differentiation and proliferation. Establishing genes or mutations which alter monocyte numbers may help define sheep which are more susceptible to certain diseases like Coxiella burnetii and small ruminant lentivirus.

3. Summarized a new methodology to identify vaccine candidates. Reverse vaccinology is a method that uses artificial intelligence or matrices to predict what proteins of a pathogen could interact with the immune system of a prospective host. Prior to 2021, few researchers had completed full proteome assessment of bacterial agents. A manuscript published by the project research team led to this commentary which explains how reverse vaccinology can be used broadly to predict vaccine candidates which are suggested to interact with the immune system in multiple capacities. Overall, this work promotes vaccine development for more complex organisms, like bacteria or protozoa.

Review Publications
Lieske, C.L., Herndon, D.R., Highland, M.A., Beckmen, K.F. 2022. Laboratory concordance study for the molecular detection of Mycoplasma ovipneumoniae. Journal of Wildlife Diseases. 58(2):257-268.
Mousel, M.R., White, S.N., Herndon, M.K., Herndon, D.R., Taylor, J.B., Becker, G.M., Murdoch, B.M. 2021. Genes involved in immune, gene translation and chromatin organization pathways associated with Mycoplasma ovipneumoniae presence in nasal secretions of domestic sheep. PLoS ONE. 16(7). Article e0247209.
Noll, L.W., Highland, M.A., Hamill, V.A., Tsui, W., Porter, E.P., Lu, N., Sebhatu, T., Brown, S., Herndon, D.R., Grossman, P.C., Bai, J. 2022. Development of a real-time PCR assay for detection and differentiation of Mycoplasma ovipneumoniae and a novel respiratory-associated Mycoplasma species in domestic sheep and goats. Transboundary and Emerging Diseases.
Davenport, K.M., Massa, A.T., Bhattarai, S., McKay, S.D., Mousel, M.R., Herndon, M.K., White, S.N., Cockett, N.E., Smith, T.P., Murdoch, B.M. 2021. Characterizing genetic regulatory elements in ovine tissues. Frontiers in Genetics. 12. Article 628849.
Massa, A.T., Mousel, M.R., Durfee, C.J., Herndon, M.K., Hemmerling, K.M., Taylor, J.B., Neibergs, H.L., White, S.N. 2021. A DNA regulatory element haplotype at zinc finger genes is associated with host resilience to small ruminant lentivirus in two sheep populations. Animals. 11(7). Article 1907.
Oliveira, R.D., Mousel, M.R., Gonzalez, M.V., Durfee, C.J., Davenport, K.M., Murdoch, B.M., Taylor, J.B., Neibergs, H.L., White, S.N. 2022. A high-density genome-wide association with absolute blood monocyte count in domestic sheep identifies novel loci. PLoS ONE. 17(5). Article e0266748.
Guzman, R.M., Howard, Z.P., Liu, Z., Oliveira, R.D., Massa, A.T., Omsland, A., White, S.N., Goodman, A.G. 2021. Natural genetic variation in Drosophila melanogaster reveals genes associated with Coxiella burnetii infection. Genetics. 217(3). Article iyab005.
Vargas Jurado, N., Notter, D.R., Taylor, J.B., Brown, D.J., Mousel, M.R., Lewis, R.M. 2022. Model definition for genetic evaluation of purebred and crossbred lambs including heterosis. Journal of Animal Science. 100:1-14.