Location: Animal Disease Research Unit
2024 Annual Report
Objectives
Objective 1: Characterize host and bacterial factors contributing to disease pathogenesis in response to M. ovipneumoniae.
Sub-objective 1A: Characterize bacterial diversity in domestic sheep through longitudinal study of bacterial genomics and host antibody responses. (Herndon, Madsen-Bouterse, Piel, Vacant Geneticist, Vacant Microbiologist)
Sub-objective 1B: Validate host genomic regions associated with M. ovipneumoniae nasal shedding. (Mousel, Herndon, Piel, Vacant Geneticist)
Sub-objective 1C: Characterize immune responses to M. ovipneumoniae in naïve lambs. (Piel, Madsen-Bouterse, Mousel, Vacant Immunologist, Vacant VMO Research Supervisor)
Objective 2: Working with an attenuated, BSL-2 strain of Coxiella burnetii, Nine Mile Phase II, begin animal model development to advance vaccine efforts against C. burnetii.
Sub-objective 2A: Extend in silico C. burnetii proteome-wide epitope prediction to small ruminants. (Piel, Schneider)
Sub-objective 2B: Assess immunogenicity of in silico predicted T-cell epitopes. (Piel, Fry, Vacant Geneticist)
Sub-objective 2C: Develop a BSL-2 C. burnetii placental shedding model. (Piel, Vacant Microbiologist)
Approach
Objective 1 aims to define the pathogenesis of Mycoplasma ovipneumoniae within the domestic sheep. This goal will be attained by examining pathogen diversity, the role of host genetics, and the interaction between host and pathogen. Experimental approaches to assess pathogen diversity (Sub-objective 1A) include whole genome sequencing of field isolates as well as mass spectrometry to identify immunogenic proteins which illicit an antibody response. If there are multi-strain infections, then deep amplicon sequencing will be employed to determine ratios of present strains and assess strain dynamics. Sub-objective 1B will assess which host genetic regions play a role in the detection of M. ovipneumoniae DNA on nasal swabs. This methodology is a genome wide association study (GWAS), which requires genotypic and phenotypic data. Genotypic data will be attained through SNP-Chip technology and the quantity of M. ovipneumoniae DNA will be measured via real-time PCR analysis. If associations fail to be made between the genotype and phenotype, then whole genome sequencing of hosts that exemplify the highest and lowest detection of the bacterium will be completed. Lastly, host responses to monomicrobial infection with M. ovipneumoniae are to be measured during inoculation studies in Sub-objective 1C. The initial inoculation study plans to characterize the peripheral and localized immune cells through flow cytometry and available domestic sheep targeting antibodies. Collection of localized, lower airway, immune cells during inoculation requires that the broncoalveolar lavage (BAL) technique be used. Bacterial presence will be measured by employing PCR modalities on collected nasal swabs and postmortem tissues. Secondary inoculation studies aim to characterize alterations in respiratory mRNA profiles during infection. The employed methodology will be single cell RNA-sequencing. Importantly, if immune or mRNA profiles remain unaltered during single-strain monomicrobial infection with M. ovipneumoniae, then substitute inoculation protocols will be attempted. These include multi-strain infection with M. ovipneumoniae or polymicrobial disease, which maintains the presence of Pasteurellaceae species.
Objective 2 focuses on identifying immunogenic proteins within C. burnetii and developing a murine model of placental shedding using an avirulent, BSL2, Coxiella burnetii. Sub-objectives 2A and 2B will employ bioinformatic tools and multiplex cytokine/chemokine assays to assess immunogenicity of peptides of interest. Failure of peptides to illicit immune responses during in vivo exposure will indicate use of other available adjuvants or immunization with whole proteins of interest. Sub-objective 2C will determine if gestating mice shed avirulent Coxiella burnetii via their placenta following intraperitoneal inoculation. Disease progression will be measured through temperature, weight gain, spleen percent body weight, bacterial colony forming units, immunohistology, real-time PCR, and multiplex cytokine/chemokine assays. If differences between the C. burnetii inoculated and PBS control mice are not detected, then alternate inoculation timelines will be tested.
Progress Report
This report documents FY 2024 progress for project 2090-32000-046-000D, “Understanding Host-Bacterial Interactions to Mitigate Disease in Small Ruminants”, which began in April 2023.
In support of Objective 1, research has continued to characterize collected bacterial field isolates from prior sampling of a historically positive sheep flock in Dubois, Idaho. In support of Sub-objective 1.A, approximately 20 single strains of M. ovipneumoniae have been identified, and all have been sent for whole genome sequencing. As documented in FY2023, there were several sheep that carried multiple strains of M. ovipneumoniae when sampled in the nasal cavity. Efforts to delineate the chronological progression of bacterial isolates within the domestic sheep nasal cavity will promote knowledge of the dynamics of carriage of M. ovipneumoniae. In FY 2024, serial sampling of the domestic sheep flock was completed to assess longitudinal strain dynamics. Furthermore, methodology was generated to promote sequencing of the longitudinally collected samples. Subordinate efforts to characterize the genomic diversity of M. ovipneumoniae included the release of two genomes on the National Center for Biotechnology Information (NCBI). Along with characterizing the genomic diversity of this bacterium, protein purification from field isolates will allow for the assessment of protein diversity and host reactivity, using host serum collected in conjunction with nasal swabs. Optimization of the protein purification protocol is currently underway.
Sub-objective 1.B requires serial sampling of the same Dubois, Idaho, sheep flock, where three more sampling time points have been collected in FY 2024. This sub-objective will use a collected numerical phenotype for nasal shedding of the bacterium and a host genotype to assess what host factors may be responsible for alternate shedding characteristics between sheep breeds.
Assessment of the interplay between M. ovipneumoniae and the domestic sheep has employed inoculation studies. Data analysis from lamb inoculation studies continues, where FY 2024 assessed the differences in cytokine profiles following inoculation of lambs with either one of two collected field strains or the type strain of M. ovipneumoniae. FY 2023 documented alternate infectivity of collected field strains and cytokine profiles agree that there is a strain-specific difference in host responses between the two employed field strains. A supplementary outcome from Sub-objective 1.C goal one is the observation that M. ovipneumoniae could be more reliably isolated from nasopharyngeal (deep nasal cavity) swabs as compared to nasal (middle nasal cavity) swabs. This led to sampling of a secondary domestic sheep flock to determine if nasopharyngeal swabs would generate a higher sensitivity during diagnostics and ultimately confirmed the initial observation within research animals. Goal two of Sub-objective 1.C is currently underway.
FY 2023 included the small ruminant species when assessing C. burnetii peptides that might stimulate host immune systems (T-cell epitopes). In FY 2024, a manuscript detailing predicted T-cell epitopes was published. Together these efforts have led to a list of peptides predicted to interact with ruminant, human, and mouse species for Sub-objective 2.A. Sub-objective 2.B focuses on the ruminant’s, specifically cattle, ability to react to T-cell epitopes predicted with bioinformatic software. FY 2024 saw the preparation for study start in FY 2025.
Lastly, FY 2024 saw the completion of mouse inoculation studies with avirulent Coxiella burnetii during the gestational timeframe. Analysis to characterize the outcome of inoculation during gestation and the effect of different genetic backgrounds is ongoing.
Accomplishments
1. Expanded C. burnetii proteome-wide T-cell epitope predictions to include small ruminant species. Ruminants are considered the reservoir of Coxiella burnetii and are therefore considered a spillover species for this zoonotic bacterium to humans. Vaccines for either humans or agricultural animals against C. burnetii are not present within the United States. Therefore, ARS researchers in Pullman, Washington, employed bioinformatic tools to define in silico interactions between bacterial peptides and small ruminant (goat and sheep) immune systems. This will promote progress towards vaccine generation.
2. Database publication of two M. ovipneumoniae genomes on the National Center for Biotechnology and Information. ARS researchers in Pullman, Wasthington, published rwo Mycoplasma ovipneumoniae genomes isolated from bighorn sheep under accession numbers JBAHVZ000000000 and JBAISL000000000. These strains of bacteria came from animals located in the United States and will help to determine the bacterial diversity within the country.
Review Publications
Grossman, P.C., Schneider, D.A., Kirkpatrick, R., White, S.N., Piel, L.M. 2023. Expansion of proteome-wide Coxiella burnetii comparative T-cell epitope prediction to include small ruminant hosts. Cellular Immunology. 5(5):143-161. https://doi.org/10.33696/immunology.5.180.
Vargas Jurado, N., Notter, D.R., Taylor, J.B., Brown, D.J., Mousel, M.R., Lewis, R.M. 2023. Combined purebred and crossbred genetic evaluation of Columbia, Suffolk, and crossbred lamb birth and weaning weights: Systematic effects and heterogenous variances. Journal of Animal Science. 102. Article skad410. https://doi.org/10.1093/jas/skad410.