Location: Animal Disease Research
2017 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
We have made substantial progress during fiscal year 2017 to address both of our objectives.
For objective 1, through our collaboration with ARS scientists at the Range Sheep Production Efficiency Research Unit, U.S. Sheep Experiment Station, in Dubois, Idaho, we collected nasal swab and blood samples from 1000 adult domestic sheep at 2+ time points. DNA, from all nasal swabs and blood samples, has been isolated for quantifying nasal shedding of Mycoplasma ovipneumoniae (M. ovipneumoniae) and to identify host genetic markers associated with nasal shedding of this bacteria, respectively. In order to accurately and quantitatively test for M. ovipneumoniae shedding we are currently developing and validating a quantitative polymerase chain reaction (qPCR) to improve the specificity, without losing sensitivity, of the previously published qPCR. This is necessary, as we discovered early in the year that the published qPCR was not specific for M. ovipneumoniae, but rather also detected a yet uncharacterized Mycoplasma spp. that can be carried and shed by a number of domestic and wildlife hooved/ungulate species, resulting in concern for false positive results or falsely increased quantified values in our analyses. Additionally, we have tested and/or are continuing to test sheep for evidence of concurrent illness by performing complete blood cell counts and for co-infections with agents such as Mycoplasma ovis, a blood-borne bacteria, and ovine progressive pneumonia virus (OPPV).
Epidemiological and immunology studies are in progress and made possible through collaborations with USDA Animal and Plant Health Inspection Service (APHIS), Montana State University, USDA Forest Service (San Juan National Forest, Colorado), Montana Fish, Wildlife and Parks, Alaska Department of Fish and Game, and Alaska Division of Environmental Health. We have received samples (nasal swabs, blood, and feces) from 71 bighorn sheep (3 different herds) and hundreds of privately owned small domestic ruminants (sheep and goats). Establishment of the described multiagency collaborations and receipt of these samples are substantial progress for objective 2, as these samples are necessary for comparing the adaptive systemic immune responses of domestic and bighorn sheep to natural (in-field) infection with M. ovipneumoniae. In addition to domestic and wild small ruminant samples, we have received several hundred nasal swab samples from farmed and wild white-tailed deer, mule deer, and elk. These nasal swab samples are being tested for the presence M. ovipneumoniae and other Mycoplasma species (spp.) that may either interfere with sample analyses and/or contribute to respiratory disease in wild and domestic sheep. In order to test nasal swabs from species other than wild and domestic sheep for the presence of M. ovipneumoniae and other Mycoplasma spp., we have developed more sensitive standard (non-quantitative) PCR techniques based on published primers and primers that we developed and are currently validating at ARS in Pullman, Washington. The goal and importance of this work, in relation to the objectives set forth in this project plan, is to ensure that we identify reservoirs of M. ovipneumoniae in order to fully understand sources of transmission of this bacterium. Additionally, and as importantly, we must be able to specifically identify the bacteria that are present in association with wild and domestic sheep pneumonia.
Additional progress has been made for objective 2 by developing an in vitro technique to grow M. ovipneumoniae in the presence of an immortalized small ruminant cell line (goat synovial cells). This is being developed as an alternative to reduce experimental animal numbers, as the current protocol requires obtaining either fetal ovine trachea collected by Caesarian section or tracheal epithelium collected from Mycoplasma-free sheep at the time of slaughter/euthanasia. This will be of great value in growing M. ovipneumoniae, as the presence of mammalian cells is required for the bacterium to produce capsule which is necessary for the bacterium to be infectious. Formation of the capsule is necessary for performing relevant in vitro or ex vivo immunology experiments, for experimental infection of captive domestic and bighorn sheep, and in vaccine development, as the capsule of Mycoplasma spp. is reported to be a virulence factor, required for attachment of the bacteria to host cells.
Substantial progress has been made in the validation of monoclonal antibodies for use in immunohistochemical staining of formalin fixed paraffin embedded lung tissues from domestic and bighorn sheep infected with M. ovipneumoniae. This research will allow for comparative characterization of the pulmonary immune responses in bighorn and domestic sheep naturally and experimentally infected with M. ovipneumoniae. We have currently validated 9 of the 13 antibodies that we aim to use for fulfilling this component of objective 2 (subobjective 2C). Additionally, through collaborative efforts with the Washington Animal Disease Diagnostic Laboratory, we have identified archived bighorn sheep tissues needed to complete our analysis, and we are currently collecting lung samples from sheep/lambs at a local abattoir that have evidence of natural M. ovipneumoniae infection. Characterizing the immune response in the lungs of infected bighorn and domestic sheep will advance our knowledge in understanding the basic mechanisms underlying the apparent susceptibility difference reported in these two closely related species. This data is required for development of a M. ovipneumoniae vaccine (subobjective 2C) that is specifically designed to stimulate a protective immune response in the host.
Accomplishments
1. Completed testing for Mycoplasma ovipneumoniae in privately owned domestic goats from across the Western U.S. In addition to the ongoing removal of shared use public lands for domestic sheep production use, removal of shared use is also impacting goat owners for the very same reason; the perceived risk of transmission of M. ovipneumoniae to wild sheep. In response to stakeholder concerns and in collaboration with USDA Animal and Plant Health Inspection Service (APHIS), ARS researchers at Pullman, Washington, completed testing on 3 time point nasal swab samples collected from 576 goats from 83 premises in 13 states. Prevalence of M. ovipneumoniae in the domestic goats tested in this study was quite low at 6.6 percent overall with goats testing positive on repeat sampling from just 6.0 percent of the premises. While similar prevalence analysis has been reported for domestic sheep, this is the first large scale surveillance study of this kind in domestic goats and is vital to understanding the reservoirs and prevalence of M. ovipneumoniae, a primary infectious agent of small ruminant pneumonia. In addition, this information is needed by regulatory agencies to science-based public lands management decisions.
Review Publications
Tuggle, C.K., Giuffra, E., White, S.N., Clarke, L., Zhou, H., Ross, P.J., Acloque, H., Reecy, J.M., Archibald, A., Boichard, M., Chamberlain, A., Cheng, H.H., Crooijmans, R., Delany, M., Groenen, M.A., Hayes, B., Lunney, J.K., Plastow, G.S., Silverstein, J., Song, J., Watson, M. 2016. GO-FAANG meeting: A gathering on functional annotation of animal genomes. Animal Genetics. 47(5):528-533.
Cinar, M., Mousel, M.R., Herrmann-Hoesing, L.M., Taylor, J.B., White, S.N. 2016. Ovar-DRB1 haplotypes *2001 and *0301 are associated with sheep growth and ewe lifetime prolificacy. Gene. 595(2):187-192.
Batra, S.A., Shanthalingam, S., Donofrio, G., Haldorson, G.J., Chowdhury, S., White, S.N., Srikumaran, S. 2017. Immunization of bighorn sheep against mannheimia haemolytica with a bovine herpesvirus 1-vectored vaccine. Vaccine. 35(12):1630-1636.
Highland, M.A. 2017. Small ruminant lentiviruses: Strain variation, viral tropism, and host genetics influence pathogenesis. Veterinary Pathology. 54(3):353-354.
