Document Type

Dissertation - Open Access

Award Date

2021

Degree Name

Doctor of Philosophy (PhD)

Department

Dairy Science

First Advisor

Johan Osorio

Keywords

Digital dermatitis, Gene Ontology, Lameness, RNA-Sequencing, Sole Ulcer, Transcriptomics

Abstract

This study aimed to evaluate the transcriptomic profile of both corium tissue from lactating dairy cows experiencing sole ulcers and skin lesions from lactating dairy cows diagnosed with digital dermatitis (DD). Hoof biopsies were performed in multiparous Holstein dairy cows selected based on their condition as clinically healthy or diagnosed with sole ulcers (n=7/group). Similarly, skin biopsies were taken from the center of active (M2/M4.1) DD lesions or non-active (M0/M4/M1) from multiparous Holstein dairy cows (n = 7/group) to assess the impact of DD on the skin transcriptome via RNA-seq analysis. All RNA samples were sequenced using Illumina, NovaSeq S4 at the University of Minnesota Genomics Center. Principal component analysis (PCA) and analysis of variance for each gene was calculated using the DESeq2 package in R to determine the differentially expressed (DE) genes, fold change (FC), and P-value based on the comparison of sole ulcer (SU) over healthy cows (HC) and digital dermatitis (DD) over apparently healthy (HDD). The DE genes with at least  2-fold change were retained for an enrichment pathway analysis using gene ontology (GO) terms and the enrichGO function in the Clusterprofiler package in R. The FDR cut-off value was set to < 0.01. The PCA analysis clearly discriminated the hoof transcriptomes between HC and SU cows. The number of DE genes in SU and HC was 10,274 ± 22 and 10,289 ± 36 (mean±SD), respectively. Several genes coding for keratins (e.g., KRT34 and KRT85) were downregulated (FC < -15) in SU cows, while the highest upregulated (FC = 10.9) gene in SU was IL6. The GO analysis showed significant downregulation of GO terms in SU cows compared to HC in the Cellular Component category, including intermediate and keratin filaments and intermediate filament cytoskeleton. Upregulated genes in SU cows resulted in a significant enrichment of GO terms in the Biological Process category, including extracellular matrix organization and vasculature development, and GO terms in the Cellular Component category such as extracellular matrix and cell surface. The GO terms enrichment across biological process, cellular components, and molecular function categories were highly involved in the keratinization process, inflammation, and transcriptional regulation. These processes are crucial for the development of sole ulcers causing lameness in dairy cows. Similarly, in the case of digital dermatitis also, the PCA analysis clearly discriminated the skin transcriptome between DD cows and NA. Genes coding for several keratins-associated proteins, including KRT34, KRTAP21-1, and KRTAP4-9, were downregulated in DD cows. The GO terms associated with downregulated genes in the Cellular Component category were mainly correlated with collagen-containing extracellular matrix and keratin filament, while GO terms in the Biological Process category were related to humoral immune response and biomineral tissue development. Upregulated genes were highly enriched in the Molecular Function GO terms of cell-adhesion and skin development as well as intermediate filament cytoskeleton in the Cellular Component category. Overall, this skin transcriptome evaluation comparing NA and DD cows identified key biological pathways in which there was a dysregulation in the skin extracellular matrix, encompassing the keratinization process, collagen binding, and immune response caused by DD lesions. This transcriptomic dataset captures the molecular adaptations in the development of sole ulcer and bovine DD. In the future, this information could be further utilized to generate nutritional and management strategies to reduce this infectious disease.

Number of Pages

120

Publisher

South Dakota State University

Rights

Copyright © 2021 the Author

Included in

Dairy Science Commons

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