Location: Animal Disease Research
2020 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
Progress continues on Objective 1, focusing on additional sheep in which their Mycoplasma ovipneumoniae shedding data has beengenotyped and error checked. Shedding data with large variation in the triplicate measurements was rerun to improve the accuracy of the data. Genome-wide association analyses was also conducted with additional samples and the corrected data contributed to a comprehensive system for nomenclature of bighorn sheep Major Histocompatibility Complex (MHC) alleles in collaboration with Moredun Research Institute of Scotland. The MHC is key for immune responses, and prior work had demonstrated novel bighorn sheep MHC alleles, including one with an E52Q substitution that could result in defects in DR dimerization, epitope presentation, and T-cell activation.
In support of Objective 2, the immunologically naïve bighorn sheep and domestic sheep inoculated with Mycoplasma ovipneumoniae were monitored for the remainder of the post-inoculation period and then culled. Substantial progress on the interspecies comparative analyses of systemic immune responses to M. ovipnuemoniae has been made using archived samples (serum) and data collected from blood cells (neutrophils, monocytes, and lymphocytes) during the post-inoculation period. In support of Sub-objectives 2A and 2B, analyses of samples collected at the post-inoculation endpoint (cull) are nearing completion. This includes immunohistochemical analyses of fixed and frozen tissues (lung and lymphoid), molecular detection of the pathogen in lung fluid and tissue, and analyses of immune cell types present in lung fluid (bronchoalveolar lavage leukocytes). Additional progress in support of Sub-objective 2B included a retrospective study using archived paraffin-embedded tissues from bighorn sheep and domestic sheep naturally infected with M. ovipneumoniae. A manuscript describing and comparing the pulmonary immune response to infection observed in these two species is undergoing revision following initial external review.
Accomplishments
1. Comparing methods of quantifying Mycoplasma ovipneumoniae, a respiratory bacterium of domestic and bighorn sheep. A major issue for the domestic sheep industry has been potential for transmission of M. ovipneumoniae from domestic to bighorn sheep. Limitations on grazing opportunities in historical ranges based on potential transmission threaten the continuity of stakeholder businesses and way of life. ARS researchers in Pullman, Washington, in collaboration with researchers at Washington State University compared methods of quantifying M. ovipneumoniae, including flow cytometry, quantitative polymerase chain reaction (PCR), and color changing unit (CCU) assays. While CCU assay was the gold standard with the widest range of detection and the ability to distinguish viable bacteria from dead cells, quantitative PCR had a wide range of detection and was capable of generating data in days rather than weeks, providing a substantial advantage in detecting and quantifying M. ovipneumoniae. These data suggest quantitative PCR may provide a useful method for rapidly quantifying M. ovipneumoniae bacteria while providing substantially better sensitivity than flow cytometry.
2. The first U.S. national survey of Mycoplasma ovis in domestic sheep operations. M. ovis is a blood pathogen in sheep, goats, and deer. While its presence has been documented in the United States, there had been no large-scale study documenting its prevalence and geographic range among sheep. ARS researchers in Pullman, Washington, in collaboration with researchers at USDA Animal and Plant Health Inspection Service performed the first national survey of M. ovis in U.S. sheep. Samples were collected from more than 34,000 U.S. sheep as part of National Animal Health Monitoring Surveys. M. ovis was present in the great majority of U.S. sheep operations (73.3%). Furthermore, when present, M. ovis occurred in 23.3% of sampled individuals within the flock. These data demonstrate M. ovis is more common than previously believed in the United States and may contribute to anemia, jaundice, and ill-thrift for many sheep.
Review Publications
Koltes, J.E., Cole, J.B., Clemmens, R., Dilger, R.N., Kramer, L.M., Lunney, J.K., Mccue, M.E., Mckay, S., Mateescu, R., Murdoch, B.M., Reuter, R., Rexroad III, C.E., Rosa, G.J.M., Serao, N.V.L., White, S.N., Woodward Greene, M.J., Worku, M., Zhang, H., Reecy, J.M., editors. 2019. A vision for development and utilization of high-throughput phenotyping and big data analytics in livestock. Frontiers in Genetics. 10:1197. https://doi.org/10.3389/fgene.2019.01197.
Urie, N.J., Highland, M.A., Knowles, D.P., Brannan, M.A., Herndon, D.R., Marshall, K.L. 2019. Mycoplasma ovis infection in domestic sheep (Ovis aries) in the United States: prevalence, distribution, associated risk factors, and associated outcomes. Preventive Veterinary Medicine. 171. https://doi.org/10.1016/j.prevetmed.2019.104750.
Ackerman, M., Schneider, D.A., Baker, K.N., Besser, T.E. 2019. Comparison of three methods of enumeration for Mycoplasma ovipneumoniae. Journal of Microbiological Methods. 165. https://doi.org/10.1016/j.mimet.2019.105700.
