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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: **Location:
                            Animal Disease Research** Project Number: 2090-32000-046-000-D
                        
                        Project Type: In-House Appropriated Start Date: Apr 6, 2023
                        
                        End Date: Apr 5, 2026 **Objective:**
                        
                        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.


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