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ARS Home Pacific West Area Pullman, Washington Animal Disease Research Research





- Piel, Lindsay
- Herndon, David
- Mousel, Michelle
- Schneider, David
- Chung, Chungwon
- Bastos, Reginaldo
- 2023
- Publications
- Animal Health (103)

Research Project: Understanding Host-Bacterial Interactions to Mitigate Disease in Small Ruminants **Location:
                            Animal Disease Research** 2023 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 progress for the first year of project 2090-32000-046-000D, titled, “Understanding Host-Bacterial Interactions to Mitigate Disease in Small Ruminants”, which continues research from the previous project 2090-32000-036-000D and bridge project 2090-32000-041-000D, titled “Identification of Host Factors and Immunopathogenesis of Pneumonia in Domestic and Bighorn Sheep”.

In support of Objective 1, researchers at Pullman, Washington, continued to sample a sheep flock in Dubois, Idaho, which is providing important data for all of the project sub-objectives. This sheep flock has historically been positive for Mycoplasma ovipneumoniae allowing scientists to analyze flock genetics in relation to nasal swab detection of the bacterium and characterize bacterial dynamics over longitudinal time. During FY2023, the Dubois, Idaho flock was sampled three times via nasal swab and blood collection to support Sub-objectives 1.A and 1.B. Work supporting Sub-objective 1.A has identified a variety of outcomes during preliminary analysis of bacterial strains present during wet nasal swab collection; this includes, single-strain carrying sheep, multi-strain carrying sheep, sheep with alternate Mycoplasma species presence, or sheep where mycoplasma was not detected. Within these results, single strain carrying ewes provide not only whole genome candidates for Sub-objective 1.A, but also support inoculating naïve lambs with collected field strains in Sub-objective 1.C. While the presence of multiple strains is not surprising, utilized traditional multi-locus sequence typing is not able to fully characterize strains carried by these animals. Therefore, in an effort to better understand the dynamics of multiple strain carrying sheep, scientists plan to pursue alternate methodology to be able to assess strain persistence, spread, or reduction over time. 

Inoculation studies in naïve lambs with a single strain of Mycoplasma ovipneumoniae continued in FY2023. As mentioned, sampling of the Dubois, Idaho maintained flock allowed for lambs to be inoculated with field strains. It was noted that animals inoculated with alternate bacterial strains had differing clinical symptoms and infection status, providing support for strain-specific virulence. 

Research for Objective 2 focused on C. burnetii, which is new to the Small Ruminant Respiratory Project. However, the FY2022 annual report described work focusing on predicting bacterial proteins that could be recognized by host or model species was taking place. During FY2023, scientists have expanded the host species which have had predicted interactions with bacterial proteins. Specifically, this saw inclusion of small ruminant species as they are imperative for the One Health initiative due to their role as a major reservoir for the bacterium of interest. Additional work to promote further inclusion of the caprine species has involved acquiring extramural funding from the Animal and Plant Health Inspection Service (APHIS). This upcoming work will broaden the characterization of goat breed immune systems present within the United States as preliminary analysis relied on present immune system sequences obtained predominantly from the Scottish Cashmere breed. 

Additional work within Objective 2 is the beginning of murine inoculation studies with Nine Mile Phase II (NMII) avirulent C. burnetii. Initial analysis suggests that gestating mice will have clinical symptoms following bacterial inoculation with the NMII avirulent strain of C. burnetii. Furthermore, scientists have determined that viable bacteria can be re-isolated from tissues following necropsy. **Accomplishments**


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