Date of Graduation

7-2021

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Biological Sciences

Advisor/Mentor

Douglas D. Rhoads

Committee Member

Adnan Alrubaye

Second Committee Member

Gisela F. Erf

Keywords

Bacterial pathogenesis, Directed Genome Evolution, Intracellular survival, Staphylococcus

Abstract

Bacterial Chondronecrosis with Osteomyelitis (BCO) is a debilitating infection that negatively impacts animal welfare and costs the broiler industry billions of dollars annually. We have previously isolated Staphylococcus agnetis 908 from BCO samples obtained from broilers at the University of Arkansas research farm. This isolate can induce BCO lameness at greater than 50% in broilers exposed to the pathogen in drinking water. We found that S. agnetis 908 is capable of surviving and escaping macrophages compared to a closely related cattle isolate,1379. Through Directed Genome Evolution (DGE) we identified that this difference is at least partially associated with an alanine to glutamate substitution for residue 164 of the enzyme deoxyribose phosphate aldolase (a.k.a. deoC, DERA). This study further explores whether A164E in deoC is responsible for enhanced survival and escape of S. agnetis 908 from macrophages. S. agnetis 1379 was transformed with the PCR products of the 908 deoC1 and deoC2 paralogs. The resulting transformants were cocultured with chicken macrophage-like cells in standard phagocytosis assays for DGE. The survivors were characterized for sequence changes in deoC through PCR sequencing. In addition, we investigated the effect of the A164E on host-cell damage by utilizing gentamicin protection assays in tandem with crystal violet staining. We evaluate the drawbacks of the gentamicin protection assay and evaluated an alternative enzyme protection assay. The crystal violet staining revealed that 908 and the transformant of 1379 inflicted more cellular damage. The enzyme protection assay was superior to gentamicin protection and indicated that there were no significant differences in the damage to HTCs caused by the different bacterial strains.

Identification of the bacterial virulence factors important to the infection process, and how these interact with the host immune responses are important in devising management plans for mitigation of BCO which would help the broiler industry control this important and costly disease.

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