Location: Animal Disease Research
2018 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 FY18 to address both of our objectives, which fall under NP103 (04 D), Respiratory Diseases - Sheep.
Objective 1: Collection of nasal swab samples from domestic sheep was continued so that there were a minimum of 3 time points for each sheep, rather than a minimum of 2 (Sub-objective 1a). Data indicated that more time points were required to define an individual animal’s shedding phenotype (high versus low/no shedding of Mycoplasma ovipneumoniae), therefore additional samples were collected and collection is now completed for 897 animals, fulfilling Sub-objective 1a. Sampling this large number of sheep was made possible through collaboration with the Range Sheep Production Efficiency Research Unit, U.S. Sheep Experiment Station, in Dubois, Idaho. Blood was also collected during nasal swab collections and a subset of animals had complete blood counts performed to assess general health, as outlined in Objective 1. A sensitive (98.6% sensitivity) and highly specific (99.7% specificity) quantitative polymerase chain reaction (PCR) (qPCR) for Mycoplasma ovipneumoniae was developed and validated. Development of this qPCR was substantial and necessary to advance Objective 1. All the nasal swab DNA samples were tested during FY18 (3000+ samples), in triplicate, using the newly developed qPCR. High quality DNA was isolated from blood collected from each sheep. DNA samples from 500 of the sheep have been submitted for single nucleotide polymorphism (SNP) OvineHD BeadChip screening to perform the Genome Wide Association Study (GWAS). Data from the initial 500 will be analyzed for significant genetic associations to nasal shedding of M. ovipneumoniae. This data analysis will determine whether DNA from the remaining sheep having 3+ time points collected will be needed to identify significant genetic associations with high versus low/no M. ovipneumoniae shedding phenotype.
Objective 2: Significant progress was made in FY18 by completing the validation of a set of commercially available antibody for use in immunohistochemical staining of formalin fixed paraffin embedded lung tissues from domestic and bighorn sheep. This was a goal expected to be completed in FY20. Staining on tissues from domestic and bighorn sheep (M. ovipneumoniae infected, and uninfected from each species) was completed. Validated antibodies are markers for recognizing the type of immune cell that is present in the lung tissue. This is necessary for performing interspecies comparative analyses on the type of immune response that is stimulated in response to M. ovipneumoniae infection. This is a vital step in proceeding to vaccine development (Sub-objective 2c). Stained tissue slides have been sent to the Histology and Imaging Core at the University of Washington in Seattle, Washington, for scanning. Scanning the slides will allow for quantitative assessment and interpretation of the stained slides. By utilizing archived slides from bighorn sheep samples, made possible through collaboration with the Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, Washington, and obtaining lung tissue from domestic sheep from a local abattoir (Vandal Brand Meats, Meat Laboratory, University of Idaho, Moscow, Idaho), allows examination of naturally infected animals in addition to the planned experimental infection study. Prior to proceeding with experimental infection, ex vivo experiments using isolated peripheral blood leukocytes and serum from uninfected captive raised bighorn and domestic sheep must be completed. Substantial progress has been made and the ex vivo experiments for uninfected animals are currently ongoing, with isolated neutrophil stimulation assays scheduled for completion by end of FY18. Lymphocyte and monocyte assays are now scheduled for FY19.
Epidemiological studies investigating the host range and prevalence of M. ovipneumoniae continued and have expanded. These studies have been made possible through collaborations with USDA Animal and Plant Health Inspection Service (APHIS), USDA Forest Service (San Juan National Forest, Colorado), Montana Fish, Wildlife and Parks, Colorado Department of Agriculture, National Animal Disease Center-ARS-USDA, Alaska Department of Fish and Game, and Alaska Division of Environment Health. These collaborations have provided a large number of respiratory samples (nasal swabs, lung tissue) to date from various hooved/ungulate species, including wild sheep (bighorn and Dall’s) and goats, domestic sheep and goats, moose, caribou, bison, white-tailed and mule deer, musk ox, llama, alpaca, and camel. The majority of samples received have had DNA extracted and analyzed by PCR and sequencing to test for the presence of M. ovipneumoniae. Testing continues on samples already received and those that continue to arrive from our collaborators. While prematurely described as being host restricted to the subfamily Caprinae, this research has identified M. ovipneumoniae in multiple members of the Capreolinae taxonomic subfamily, including caribou, moose, white-tailed and mule deer, as well as a bison. M. ovipneumoniae in association with pneumonia in a caribou calf and captive white-tailed deer and in a captive mule deer exhibiting clinical respiratory illness was identified for the first time. Optimization of PCR assays and subsequent DNA sequencing have provided a means of highly specific detection of M. ovipneumoniae in samples. Sensitivity of testing by standard PCR was determined using known amounts (calculated copy numbers) of M. ovipneumoniae spiked into nasal swab samples taken from captive born and raised specific pathogen free (M. ovipneumoniae-free) domestic and bighorn sheep, as it is known that PCR inhibitors in mucous and dirt can carry through into the isolated DNA, lowering sensitivity as compared to detection of pure culture. This revealed a lower detection limit of 1000 copies of bacteria on the initial swab, whereas 10 bacterium can be detected from a pure culture.
Accomplishments
1. Mycoplasma ovipneumoniae identified in a widening array of animals. M. ovipneumoniae, the currently believed primary agent of pneumonia in bighorn sheep, has been prematurely described as having a host range restricted to Caprinae species (sheep, goats, musk ox) with domestic sheep and goats being implicated as the historic and continued source for introduction of this bacterium in bighorn sheep. Epidemiological studies were performed and identified M. ovipneumoniae in the following numbers of tested animals: 21 of 421 (5 percent) of caribou, 9 of 362 (2.5 percent) of moose, 23 of 184 (12.5 percent) of Dall’s (thinhorn) sheep, and 5 of 43 (12.5 percent) mountain goats. ARS scientists in Pullman, Washington, in collaborations with several veterinarians and a state agriculture department identified this bacterium in captive white-tailed and mule deer exhibiting respiratory illness, and in a bison (no history reported). Determining the true host range of this bacterium is of utmost importance to stakeholders, as there is a current push to remove small domestic ruminants from public lands (grazing, brush clearing, and recreational uses) and current social pressures to create M. ovipneumoniae-free flocks/herds on private lands.
2. Accurate assay for quantifying shedding of Mycoplasma ovipneumoniae in sheep developed. ARS scientists in Pullman, Washington, developed a quantitative polymerase chain reaction (qPCR) assay that improved the accuracy of detecting M. ovipneumoniae. Nasal swabs collected from 493 sheep and 3 separate herds and locations were used for validating this assay. Sheep tested included lambs and adult ewes from Dubois, Idaho, young adult rams from Bozeman, Montana, and adult ewes from Durango, Colorado. Validation of this assay was a major accomplishment as currently available published qPCR assays either lacked sensitivity (poor detection ability) or specificity (false positive results). Validation calculations indicate this assay to have a sensitivity of 98.6% and a specificity of 99.7%, which is based on test results using an in-house, “gold standard”, general PCR assay.
3. Significant domestic sheep breed differences in nasal shedding of Mycoplasma ovipneumoniae identified. ARS scientists in Pullman, Washington, collected 3 or more nasal samples from 897 sheep including 320 Polypay, 456 Rambouillet, and 118 Suffolk to study shedding of M. ovipneumoniae. Using quantitative polymerase chain reaction (qPCR) assays, a significant breed difference in shedding of this bacterium was found. There was no/low shedding for 14.7 percent of Polypay; 27.2% of Rambouillet, and 10.2% of the Suffolk sheep and high shedding in 20.3 percent of Polypay; 9.6 percent of Rambouillet, and 28 percent Suffolk. The remainder of the tested sheep were intermittent high and low to no shedders. Based on this data, significantly fewer Rambouillet are high shedders of this bacterium as compared to Polypay and Suffolk breeds. The results support the hypothesis of a genetic association with carriage and shedding of M. ovipneumoniae and indicates mitigation of shedding may be obtained through genetic control.
Review Publications
White, S.N., Oliveira, R.D., Mousel, M.R., Gonzalez, M.V., Highland, M.A., Herndon, M.K., Taylor, J.B., Knowles Jr, D.P. 2017. Underdominant KCC3b R31I association with blood sodium concentration in domestic sheep suggests role in oligomer function. Animal Genetics. 48(5):626-627. https://doi.org/10.1111/age.12585.
Highland, M.A., Berglund, A.K., Knowles, D.P. 2018. Total IgG concentrations in ewe sera and colostrum and kinetics in lambs following colostrum ingestion is similar in domestic sheep and bighorn sheep (ovis aries and ovis canadensis). Sheep and Goat Research Journal. 32:36-42.
Notter, D.R., Mousel, M.R., Lewis, G.S., Leymaster, K.A., Taylor, J.B. 2017. Evaluation of Rambouillet, Polypay, and Romanov-White Dorper x Rambouillet ewes mated to terminal sires in an extensive rangeland production system: Lamb production. Journal of Animal Science. 95:3851-3862.
Davenport, K.M., Duan, M., Hunter, S.S., New, D.D., Fagnan, M.W., Highland, M.A., Murdoch, B.M. 2018. Complete mitochondrial genome sequence of bighorn sheep. Genome Announcements. https://doi.org/10.1128/genomeA.00464-18.
