Location: Animal Disease Research
2019 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
In support of Objective 1, research continued to identify the host factors associated with nasal shedding of ovine respiratory pathogens. A project to identify the most important genes and gene regions for nasal shedding of Mycoplasma ovipneumoniae from domestic sheep through genome-wide association is ongoing. Since many traits such as host-pathogen interactions are controlled by a multitude of genes each making small contributions, we also began testing an approach to leverage many contributing genes simultaneously. Specifically, we examined genomic selection for nasal shedding of Mycoplasma ovipneumoniae. While larger numbers of animals would be beneficial to the project, the approach merits continued investigation.
In support of Sub-objective 2B, the retrospective study of utilizing archived paraffin-embedded tissues from naturally infected bighorn sheep and domestic sheep to compare and characterize the pulmonary immune response to Mycoplasma ovipneumoniae infection, is fully completed. A manuscript representing the findings of this study is completed and under current internal review by co-authors for submission to a peer reviewed journal. The immunologically naïve bighorn sheep and domestic sheep raised to complete Sub-objectives 2A and 2B were infected with Mycoplasma ovipneumoniae and are currently being monitored and samples (blood) collected weekly to perform experimental analyses on certain blood cells (neutrophils, lymphocytes, and monocytes); this data will be combined with pre-infection analyses that were completed prior to infection (Sub-objective 2A). Serum from pre-infection and early post-infection time points have been collected and are archived frozen, and post-infection serum samples continue to be collected weekly in order to perform interspecies comparative analyses on systemic immune response to Mycoplasma ovipneumoniae infection (Sub-objective 2B). Since the presence of mammalian cells is required to grow Mycoplasma ovipneumoniae that produces a capsule necessary for infectivity, the initial research plan (Sub-objective 2A) was to collect trachea from Mycoplasma-free domestic sheep. In order to reduce experimental animal numbers, an immortalized small ruminant cell line (sheep lung fibroblasts) has proven to be a successful means to cultivate large numbers of the infective form of Mycoplasma ovipneumoniae. Infectivity has been confirmed on repeat nasal swab samples collected from the bighorn sheep and domestic sheep infected (intranasal inoculation) with Mycoplasma ovipneumoniae that was cultured in this cell line system.
Mycoplasma ovipneumoniae was recently discovered in species outside of the Caprinae subfamily (sheep, goat, muskox), including deer family members (Capreolinae subfamily) and bison. Comparative genomic evaluation of Mycoplasma ovipneumoniae isolates from each mammalian host species is currently underway to determine relatedness or differences that may exist in the bacterium (bacterial subtypes) based on the host from which it originates. This is relevant in completing our epidemiological studies, as we have identified genetic differences in the Mycoplasma ovipneumoniae identified in domestic sheep and domestic goats, and differences between the “goat type” and “sheep type” Mycoplasma ovipneumoniae in ability to cause morbidity and mortality in bighorn sheep has been reported in the literature. By using the sheep fibroblast cell culture system, cultivation of large enough quantities of Mycoplasma ovipneumoniae for full-length genome sequencing is possible; this process has been validated in three strains of Mycoplasma ovipneumoniae, isolated from three different species (domestic sheep, bighorn sheep, and moose).
Accomplishments
1. Immortalized sheep fibroblast cell culture method allows cultivation of large quantities of infective Mycoplasma ovipneumoniae. Debate over potential transmission of Mycoplasma ovipneumoniae from domestic sheep to bighorn sheep threatens commercial sheep production in the U.S. through its impact on grazing allotment policy. Mycoplasma ovipneumoniae produces a capsule that is reported to be necessary for this bacterium to infect susceptible hosts. Capsule production is diminished by growing Mycoplasma ovipneumoniae by typical bacterial culture methods, in the absence of mammalian cells (host species cells). ARS researchers at Pullman, Washington, have developed a method using immortalized sheep lung fibroblast cells to provide the mammalian cell component for culturing Mycoplasma ovipneumoniae in its infective form. Key studies on the risks of and potential interventions against Mycoplasma ovipneumoniae depend on large-scale availability of active, infective organisms, and development of this advanced culture technique provides a critical tool empowering research into host-pathogen interaction at the interface of animal agriculture and wildlife disease.
Review Publications
Highland, M.A., Herndon, D.R., Bender, S., Hansen, L., Gerlach, R.F., Beckman, K.B. 2018. Identification of Mycoplasma ovipneumoniae in wildlife species beyond the subfamily caprinae. Emerging Infectious Diseases. 24(12):2384-2386. https://doi.org/10.3201/eid2412.180632.
Rovani, E.R., Beckman, K.B., Highland, M.A. 2019. Mycoplasma ovipneumoniae associated with polymicrobial pneumonia in a free-ranging yearling caribou (Rangifer tarandus tarandus) from Alaska, USA. Journal of Wildlife Diseases. 55(3):733-736. https://doi.org/10.7589/2018-08-188.
Murphy, B., Knowles, D., Highland, M.A. 2018. Visna-maedi. In: Coetzer, J.A.W., Thomson, G.R., Maclachlan, N.J., Penrith, M.L., editors. Anipedia-Infectious Diseases of Livestock. Anipedia. Available: https://www.anipedia.org/resources/visna-maedi/1035.
Casselli, T., Crowley, M.A., Highland, M.A., Tourand, Y., Bankhead, T. 2019. A small intergenic region of lp17 is required for evasion of adaptive immunity and induction of disease pathology by the Lyme disease spirochete. Cellular Microbiology. 21(7):e13029. https://doi.org/10.1111/cmi.13029.
Murphy, B., Knowles, D., Highland, M.A. 2018. Caprine arthritis-encephalitis. Anipedia-Infectious Diseases of Livestock. Available: https://www.anipedia.org/resources/caprine-arthritis-encephalitis/1027.
Cinar, M., Mousel, M.R., Herndon, M.K., Taylor, J.B., White, S.N. 2018. Tenascin-XB (TNXB) amino acid substitution E2004G is associated with mature weight and milk score in rambouillet, targhee, polypay, and suffolk sheep. Small Ruminant Research. 166:129-133. https://doi.org/10.1016/j.smallrumres.2018.06.013.
Kise, J.N., Neupane, M., White, S.N., Neibergs, H.N. 2018. Identification of genes associated with susceptibility to mycobacterium avium ssp. paratuberculosis (Map) tissue infection in Holstein cattle using gene set enrichment analysis-SNP. Mammalian Genome. 29(7-8):539-549. https://doi.org/10.1007/s00335-017-9725-4.
