Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev K. Narayanan / Fusobacterium necrophorum is a Gram-negative, anaerobic, and rod-shaped to pleomorphic bacterium. It is frequently associated with necrotic infections of animals and humans. It is a major bovine pathogen and causes hepatic abscesses, foot rot, and necrotic laryngitis (calf-diphtheria). Liver abscesses in feedlot cattle and foot rot in beef and dairy cattle are of significant economic importance to the cattle industry. Fusobacterium necrophorum is classified into two subspecies, subsp. necrophorum and subsp. funduliforme. The subsp. necrophorum is more virulent and isolated more frequently from bovine hepatic abscesses than subsp. funduliforme. Outer membrane proteins (OMPs) of Gram-negative bacteria play an important role in their adhesion to host eukaryotic cells and hence, help in the establishment of infection and disease. Our objectives were to characterize OMPs of the two subspecies of F. necrophorum and assess their role in adhesion to bovine cells. Electrophoretic separation of extracted OMPs of subsp. necrophorum showed a total of 19 bands. Four bands of 38, 40, 60 and 74 kDa were more prominent than others. The OMPs of subsp. funduliforme showed a total of 20 proteins bands, of which, five were prominent (37.5, 58, 70, 140 and 150 kDa). The 40 kDa band was prominent in subsp. necrophorum while 37.5 kDa band was prominent in subsp. funduliforme. The human strains of F. necrophorum subsp. funduliforme had more heterogeneous banding patterns than the bovine strains of subsp. funduliforme. The role of OMPs in adhesion was studied using bovine endothelial cell line (EJG cells). A significant decrease in the attachment of subsp. necrophorum and subsp. funduliforme to bovine endothelial cell line (EJG cells) was observed when the cell line was preincubated with the OMPs of each subspecies. Treatment of the bacterial cells with trypsin also decreased their binding. In addition, when each subspecies was incubated with the polyclonal antibody produced against their OMPs before adding them to endothelial cells, there was a significant reduction in the bacterial attachment and the inhibition was subspecies specific. A 40 kDa OMP of subsp. necrophorum was identified that binds to the bovine endothelial cells with high affinity. The protein when preincubated with the endothelial cells, lead to a significant decrease in the bacterial binding to the endothelial cells. The N-terminal sequencing of the protein indicated similarity to FomA, an outer membrane protein of Fusobacterium nucleatum, an oral pathogen of humans. In summary, OMPs of F. necrophorum subsp. necrophorum and subsp. funduliforme differ from each other and they play a significant role in binding to bovine endothelial cells. We identified a 40 kDa OMP in subsp. necrophorum that binds to the bovine endothelial cells with high affinity and have a potential role as adhesin.

Master of Science / Department of Animal Sciences and Industry / Evan C. Titgemeyer / Seven ruminally cannulated heifers approximately 225 kg initial BW were used in a 7 × 4 Youden square design to determine the effects of different levels of limonene on ruminal Fusobacterium necrophorum populations. Treatments included: 1) control, 2) limonene at10 mg/kg diet DM, 3) limonene at 20 mg/kg diet DM, 4) limonene at 40 mg/kg diet DM, 5) limonene at 80 mg/kg diet DM, 6) CRINA-L (a blend of essential oil components) at 180 mg/kg diet DM, 7) tylosin at 12 mg/kg diet DM. Each period included 11 d with 10 d washouts between periods. Samples were collected on d 0 (before treatment initiation), 4, 7, and 10 for measuring F. necrophorum by most probable number (MPN) method using selective culture medium. Results indicate that CRINA-L (P = 0.52) and tylosin (P = 0.19) did not affect ruminal F. necrophorum populations. Limonene linearly decreased (P = 0.03) F. necrophorum populations, and the optimal dietary concentration for limonene was 40 mg/kg DM. Limonene did not affect ruminal degradation rate of lysine, NH3 concentration, or VFA profiles in ruminal fluid. Limonene was useful for reducing ruminal concentrations of F. necrophorum. It may have potential to control liver abscesses, although further research will be needed to assess the effect of limonene under feedlot conditions.