Masters Theses
Date of Award
12-2021
Degree Type
Thesis
Degree Name
Master of Science
Major
Animal Science
Major Professor
Gina M. Pighetti
Committee Members
Daniel Mathew, Yongming Sang
Abstract
Streptococcus uberis is an environmental pathogen that can internalize into mammary epithelial cells (MECs), evade degradation within cells, and cause mastitis. Our objective was to investigate interactions between S. uberis and MEC function relative to RAB4a (Ras-related protein RAB4a), HECTD4 (HECT Domain E3 Ubiquitin Protein Ligase 4), and PSAP (Prosaposin coding gene) which are involved with intracellular trafficking and degradation and were located next to SNPs associated with in vivo mastitis phenotypes. A common MEC-line (MACT) and a primary line (BOMEC) were inoculated with S. uberis strains isolated from a chronic (UT888 CFU Mean=4.5x107) or acute (UT366 CFU Mean=2.4x108) mastitis case. Candidate gene expression, cell viability, and intracellular survival of bacteria were evaluated at 0, 3, 9, and 27 hours for infected and non-infected cells using a mixed model ANOVA (SAS 9.4, Cary, NC). The fixed effects were cell type, strain, infection status, and time, with a random effect of time (cell type*strain). Cell viability was not associated with any fixed effects, with mean viability remaining above 88% at all time points. Percent of internalized UT366 (LSMean=0.006, SE=0.0016) was higher than UT888 (LSMean=0.0023, SE=0.0016) (p=0.0026). Percent internalized for both strains peaked at 3 hours (LSMean=0.012, SE=0.0018), decreased at 9 hours (LSMean=0.0007, SE=0.0018), and decreased again at 27 hours (LSMean=-0.0002, SE=0.0018) (pS. uberis and candidate genes, small interfering RNA was used to reduce expression in MACT prior to infection with UT888. The number of live cells collected was reduced with knockdown of HECTD4 (Control: Mean=1.1x106 cells/mL SE=1.2x105, KD: Mean=2.3x105 cells/mL SE=4.6x104, p6 cells/mL SE=8.4x104, KD: Mean=9.2x105 cells/mL SE=1.2x105, p=0.0008), but not with PSAP (Control: Mean=1.2x106 cells/mL SE=6.8x104, KD: Mean=7.0x105 cells/mL SE=1.1x105, p=0.13). Reduction in cell viability also was observed after HECTD4 (Control: 90% SE=7.9, KD: 56% SE=6.8, pS. uberis indicate some potential relevance to intracellular survival of S. uberis.
Recommended Citation
Malcomson, Hannah Lacy Martin, "Interactions of Candidate Gene Expression and in vitro Infection with Streptococcus uberis in Bovine Mammary Epithelial Cells. " Master's Thesis, University of Tennessee, 2021.
https://trace.tennessee.edu/utk_gradthes/6304
Included in
Animal Diseases Commons, Animals Commons, Bacteria Commons, Bacterial Infections and Mycoses Commons, Bacteriology Commons, Cell Biology Commons, Dairy Science Commons, Immunology of Infectious Disease Commons, Immunopathology Commons, Large or Food Animal and Equine Medicine Commons, Veterinary Microbiology and Immunobiology Commons