Doctoral Dissertations

Date of Award

5-1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Animal Science

Major Professor

Stephen P. Oliver

Committee Members

Philip Bochsler, Judith Grizzle, Fred Hopkins, Alan Mathew, Kelly Robbins

Abstract

Streptococcus dysgalactiae accounts for a significant number of intramammary infections during lactation and the nonlactating period. In spite of its high prevalence, little information is available about factors that contribute to the virulence of S. dysgalactiae. This organism adheres to bovine mammary epithelial cells and to extracellular matrix proteins in vitro, and invades bovine mammary epithelial cells in culture; all of which can be potentially important pathogenic mechanisms. Experiments were conducted to characterize factors and mechanisms involved in adherence and invasion of S. dysgalactiae to bovine mammary epithelial cells in vitro.

Adherence of S. dysgalactiae was affected by both the mammary epithelial cell type and bacterial strain used. Bacterial adherence was mediated by saturable cell receptors, and reduction of bacterial adherence following cell fixation indicated that cell surface proteins played a major role in this process. Delineation of bacterial and epithelial cell factors influencing adherence of S. dysgalactiae to mammary epithelial cell monolayers resulted in development of an in vitro method for quantifying bacterial adherence. Presence of two bacterial factors, lipoteichoic acid (LTA) and M-like protein, potentially involved in adherence of S. dysgalactiae to mammary epithelial cells, was detected in strains of S. dysgalactiae isolated from bovine intramammary infections. Bacterial surface proteins played a major role in adherence of S. dysgalactiae to mammary epithelial cells. However, use of antibodies directed against streptococcal M24 protein did not affect adherence of S. dysgalactiae to mammary epithelial cells. LTA appeared to play a minor role in adherence of one strain of S. dysgalactiae to mammary epithelial cells but had no effect on another strain of S. dysgalactiae.

Streptococcus dysgalactiae invasion into mammary epithelial cells increased with inoculum size; however, number of intracellular bacteria was not proportional to inoculum size indicating that a finite number of organisms are capable of invading epithelial cells. No net increase of intracellular organisms was detected at several bacterial densities evaluated; however, S. dysgalactiae remained viable throughout the period of evaluation. In addition, S. dysgalactiae did not appear to cause cell injury at any bacterial density and time point evaluated. These data suggest that S. dysgalactiae can survive within mammary epithelial cells for extended periods of time without losing viability or damaging the eukaryotic cell. This feature may be associated with development of persistent infection, protection of organisms from antimicrobial drugs and host defense mechanisms, and can also provide a route for bacterial colonization of subepithelial tissues. Activity of eukaryotic cell tyrosine protein kinases, intact microfilaments and de novo eukaryotic protein synthesis were required for invasion of S. dysgalactiae into bovine mammary epithelial cells; a process that appeared to occur via receptor-mediated endocytosis. In contrast, de novo bacterial protein synthesis was not required for invasion of S. dysgalactiae into MAC-T cells.

Collectively, these studies provide insight towards a more fundamental understanding of early interactions between bovine mammary epithelial cells and S. dysgalactiae and should assist in further research directed to develop methods to minimize production losses associated with intramammary infections in dairy cows.

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