Date of Graduation

5-2020

Document Type

Thesis

Degree Name

Bachelor of Science in Agricultural, Food and Life Sciences

Degree Level

Undergraduate

Department

Animal Science

Advisor/Mentor

Zhao, Jiangchao

Committee Member/Reader

Powell, Jeremy G.

Committee Member/Second Reader

Stenken, Julie

Abstract

Bovine respiratory disease (BRD) is one of the largest economic burdens facing United States beef producer’s modern day. The complexity in the onset and development of this multifactorial disease necessitates further insights into its functions to alleviate the tremendous consequences it generates for producers. The respiratory microbiome and their metabolomics play an important role to maintain health and predict BRD. However, due to low biomass, new technology is needed to increase the microbial concentration for omics (e.g. metatranscriptomics) research. In this study, a novel self-enrichment storage technique on the bovine nasal microbiome was performed and compared to a traditional nasal sample storage method to further develop microbial communities. To assess these methods, two nasal swab samples from 2 cohorts (10 calves each) at two different locations were collected at two different timepoints. Subsequently, one set of samples was subjected to the novel technique (room temperature culture for 7 days in Amies buffer then stored at -80C, RT), while the other set followed traditional storage protocol (stored into -80C directly, UL) as a control. The nasal microbiome was then characterized using 16S rRNA sequencing of the V4 region. The RT storage technique was characterized by a significant decrease in microbial diversity and richness when compared to UL storage samples for both locations and timepoints (p<0.05). Furthermore, RT samples showed distinct clustering from UL samples for both locations and timepoints when measured by both Jaccard and Bray-Curtis distances. Community structure between the storage techniques was also assessed at the genus level, characterized by a reduction in common airway genera such as Moraxella and Pasteurellaceae and an increase in common genera such as Enterococcus and Pseudomonas when the RT storage technique was compared to traditional UL storage. Random forest was found as an accurate model used to biomark and rank the most influential species differentiating the storage techniques. OTUs associated with BRD pathogens were identified as UL biomarkers, such as OTU53-Mycoplasma, OTU9-Moraxella and OTU35-Pasteurellaceae, while OTU1-Enterococcus and Otu18-Streptococcus were consistently observed to increase in RT, consistent with the corresponding genus shift. Finally, Procrustes analysis using Jaccard distance was used to determine the consistency in RT storage influence on the nasal microbiome among samples compared to UL storage. Consistent trend among samples was observed when comparing the RT storage technique to UL storage across both locations and timepoints. In summary, the novel self-enrichment room temperature storage technique was found to enrich specific microbiota but ultimately shifted the microbial structure of the “normal” respiratory community. Thus, future improvement and investigation into the novel self-enrichment technique is necessary to expand its uses for further analysis of the microbiomes function in the onset and development of BRD.

Keywords

Bovine Respiratory Disease; microbiome; Random forest; Procrustes

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