Location: Animal Disease Research Unit
2025 Annual Report
Objectives
Objective 1: Characterize host and bacterial factors contributing to disease pathogenesis in response to Mycoplasma ovipneumoniae (M. ovipneumoniae).
Sub-objective 1A: Characterize bacterial diversity in domestic sheep through longitudinal study of bacterial genomics and host antibody responses.
Sub-objective 1B: Validate host genomic regions associated with M. ovipneumoniae nasal shedding.
Sub-objective 1C: Characterize immune responses to M. ovipneumoniae in naïve lambs.
Objective 2: Working with an attenuated, BSL-2 strain of Coxiella burnetii (C. 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.
Sub-objective 2B: Assess immunogenicity of in silico predicted T-cell epitopes.
Sub-objective 2C: Develop a BSL-2 C. burnetii placental shedding model.
Approach
Objective 1 aimes to define pathogenesis of M. 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 (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, C. burnetii. Sub-objectives 2A and 2B will employ bioinformatic tools and multiplex cytokine/chomkine assays to assess immunogenicity of peptides of interest. Failure of peptids 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 C. 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 phosphate buffered saline (PBS) control mice are not detected, then alternate inoculation timelines will be tested.
Progress Report
This report documents the FY 2025 progress of project 2090-32000-046-000D, titled, “Understanding Host-Bacterial Interactions to Mitigate Disease in Small Ruminants”, which began in April of 2023.
In support of Objective 1, characterize host and bacterial factors contributing to disease pathogenesis in response to M. ovipneumoniae. Progress in fiscal year (FY) 2025 has seen completion of deep sequencing M. ovipneumoniae DNA from collected nasal swabs coming from a variety of yearling domestic sheep breeds for goal 1 Sub-objective 1A. Analysis of the resultant dataset is ongoing, but preliminary review suggests that certain sequenced genetic loci give a better summary of multi-strain presence. Goal 2 of Sub-objective 1A aims to characterize bacterial proteins recognized by host immune systems. Mass spectrometry data has been generated for four domestic sheep serum samples and one bighorn sheep serum sample. Initial analysis has noted the identification of several similar bacterial proteins being recognized by antibodies isolated from both of the aforementioned host species.
Sub-objective 1B has seen the continued serial sampling of a domestic sheep flock for validation of the genome-wide association study (GWAS) published in 2021, where nasal swab processing for bacterial quantification has begun. Lastly, inoculation studies for Sub-objective 1C have continued with transcriptional analysis being completed on the pilot study, consisting of six lambs. Notably, control and M. ovipneumoniae inoculated animals had distinct messenger RNA profiles identifying different immune cell populations in bronchoalveolar lavage.
In support of 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 2B was updated in FY24 and advancement in FY25 has included vaccination of cattle species with detergent solubilized avirulent C. burnetii lysate or with a whole cell bacterin that is fixed in formalin. Importantly, the bacterin is representative of the licensed products for C. burnetii in other countries. During vaccination, blood was collected to measure resultant immunologic changes through immune cell profiling, characterization of cellular responses to in vitro stimulation, and antibody analysis. Secondarily, data from murine inoculation studies with avirulent C. burnetii, Sub-objective 2C, has been analyzed. Analysis has determined different disease outcomes between inoculated gestational and non-gestational mice. Furthermore, inoculation of gestational mice resulted in altered hormonal and immune cytokine measurements compared to their mock counterparts, which may give insights into the pathogenesis of C. burnetii.
The lifetime of this project has seen advancement in the understanding of M. ovipneumoniae strain diversity within a production flock, identification of M. ovipneumoniae proteins which stimulate host antibody production, advancement of M. ovipneumoniae diagnostics, and measurement of host responses following inoculation with M. ovipneumoniae. Inoculation studies allowed for ARS researchers in Pullman, Washington, to identify alternate shedding characteristics between two isolated field strains of M. ovipneumoniae. Notably, this led to a diagnostic strategy in which the nasopharynx is swabbed for bacterial detection instead of the nasal cavity. In depth analysis of transcriptional data and flow cytometry data from domestic lamb inoculation studies continues but shows that immune stimulation following inoculation of domestic sheep lambs with M. ovipneumoniae did occur. Work on C. burnetii has progressed vaccine design for ruminant species. Initially, this was through in silico identification of proteins suggested to interact with ruminant major histocompatibility complexes. Currently, cells isolated from vaccinated cattle are being assessed in vitro for their ability to secrete interferon gamma, a cytokine associated with protective outcome during C. burnetii infection, following exposure to detergent solubilized lysate and whole cell bacterin.
Accomplishments
1. Differential immunological responses of adult domestic and bighorn sheep to inoculation with Mycoplasma ovipneumoniae type strain Y98. Bighorn and domestic sheep species respond to bacterial respiratory infections with different levels of morbidity and mortality. It is believed that M. ovipneumoniae infection of these animals initiates respiratory disease, but studies had not previously monitored for specific effects during monomicrobial infection with Mycoplasma ovipneumoniae. ARS researchers in Pullman, Washington, found that following inoculation with M. ovipneumoniae, bighorn sheep nasally shed more bacterial DNA, had mild upper respiratory symptoms, and mild lethargy as compared to domestic sheep which did not have clinical signs. Similarly, immune system activation was more prevalent in bighorn sheep. This work advances the understanding of why bighorn sheep may suffer more readily during M. ovipneumoniae introduction into herds than domestic sheep.
2. Upper respiratory tract detection of Mycoplasma ovipneumoniae employing nasopharyngeal swabs. The threat of spread of Mycoplasma ovipneumoniae, a bacterium that causes respiratory illness in ruminants, has limited the lands available for grazing by stakeholders and producers. The easiest test for monitoring the spread of this bacterium typically employs nasal swabs for direct detection of bacterial DNA. Unfortunately, nasal swabs tend to be fraught with inhibitors of polymerase chain reaction (PCR), lowering the diagnostic’s sensitivity. To enhance the effectiveness of this diagnostic tool, ARS researchers in Pullman, Washington, tested the sensitivity after swabbing the nasopharynx, or region above the larynx. It was determined that nasopharyngeal swabbing in concordance with altered real time quantitative PCR methodology increased diagnostic sensitivity by 2.5-fold within the domestic sheep species. This progress will promote better prevalence measures for this and potentially other bacteria of interest.
3. Genetic parameters for Mycoplasma ovipneumoniae nasal DNA copy number provide progress to promote domestic and bighorn sheep coexistence on public lands. Reducing the ability of domestic sheep to transmit M. ovipneumoniae, a bacteria that can cause respiratory illness, to bighorn sheep provides opportunity for a change in grazing land usage. ARS researcher in Pullman, Washington, in coordination with ARS researchers at Dubois, Idaho, calculated the estimated heritability of M. ovipneumoniae DNA nasal shedding from the Rambouillet sheep breed. Following heritability calculation, a breeding program selecting for domestic sheep which have lower nasal shedding of M. ovipneumoniae DNA can be employed.
Review Publications
Herndon, D.R., Grossman, P.C., Hwang, J.K., Piel, L.M. 2024. Upper respiratory tract detection of Mycoplasma ovipneumoniae employing nasopharyngeal swabs. BMC Veterinary Research. 20. Article 502. https://doi.org/10.1186/s12917-024-04342-y.
Wilson, C.S., Taylor, J.B., Mousel, M.R., White, S.N., Piel, L.M., Wilmer, H.N., Murdoch, B.M. 2024. Genetic parameters for Mycoplasma ovipneumoniae nasal DNA copy number provide progress to promote domestic and bighorn sheep coexistence on public lands. Small Ruminant Research. 240. Article 107382. https://doi.org/10.1016/j.smallrumres.2024.107382.
Arzik, Y., Kizilaslan, M., Behrem, S., Piel, L.M., White, S.N., Ulas Cinar, M. 2025. Exploring genetic factors associated with tapeworm resistance in Central Anatolian Merino sheep via GWAS approach. Animals. 15(6). Article 812. https://doi.org/10.3390/ani15060812.
Madsen-Bouterse, S.A., Herndon, D.R., Grossman, P.C., Rivolta, A.A., Fry, L.M., Murdoch, B.M., Piel, L.M. 2024. Differential immunological responses of adult domestic and bighorn sheep to inoculation with Mycoplasma ovipneumoniae type strain Y98. Microorganisms. 12(12). Article 2658. https://doi.org/10.3390/microorganisms12122658.
Akyuz, B., Hasan Sohel, M., Konca, Y., Arslan, K., Gurbulak, K., Abay, M., Kaliber, M., White, S.N., Cinar, M. 2024. Effects of low and high maternal protein intake on fetal skeletal muscle miRNAome in sheep. Animals. 14(11). Article 1594. https://doi.org/10.3390/ani14111594.
Mehmet, K., Arzik, Y., Behrem, S., White, S.N., Ulas Cinar, M. 2023. Comparative genomic characterization of indigenous fat-tailed Akkaraman sheep with local and transboundary sheep breeds. Food and Energy Security. 13(1). Article e508. https://doi.org/10.1002/fes3.508.
Kizilaslan, M., Arzik, Y., Behrem, S., Yavuz, E., White, S.N., Ulas Cinar, M. 2024. Unravelling the genetic architecture of serum biochemical indicators in sheep. Genes. 15(8). Article 990. https://doi.org/10.3390/genes15080990.