Date of Award

1991

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Animal Science (Animal, Dairy, and Poultry Sciences)

First Advisor

Theron G. Snider, III

Abstract

Fractions exhibiting activity detrimental to the killing function of activated bovine monocyte/macrophages towards Candida albicans were obtained from Mycobacterium paratuberculosis. Active fractions were initially derived by ultrasonication of bacterial samples followed by ammonium sulfate precipitation, ion-exchange chromatography, gel filtration chromatography, heat precipitation, acid precipitation and alkali treatment. Two different fractions that exhibited marked inhibition of macrophage killing ability were derived and partially characterized. Novel methodology was developed to accomplish the task of lysis and fractionation of mycobacteria utilizing the Matrix Solid Phase Dispersion (MSPD) technique that appeared to be much more efficient and is certainly less time consuming and less expensive than classical methodologies. Fractionation of cellular components were confirmed by analysis of the solvent extracts by SDS-PAGE, Markwell protein determination, High Pressure Liquid Chromatography (HPLC) and Gas Chromatography/Mass Spectrometry (GC/MS). By making minor modifications in the MSPD technique both genomic and plasmid DNA were obtained in partial purity. The DNA samples were further purified using classical methodology to obtain DNA that was amenable to restriction endonuclease digestion with yields comparable to those of classical means. By utilizing the MSPD technique for lysis and fractionation of mycobacteria, two M. paratuberculosis strain 18 MSPD fractions demonstrating marked inhibitory activity towards the killing function of macrophages were obtained (ACN and H$\sb2$O MSPD extracts). Further fractionation by centrifugal filtration and by Concanavalin A affinity chromatography demonstrated as many as three compounds that show macrophage inhibitory activity. All of the active fractions appear to be combinations of carbohydrate and lipid (glycolipids) with proteinaceous components either being minimal or absent. Preliminary plasma desorption mass spectral data suggests that one fraction may be a mixture of mycolic acids, while the other two fractions contained mannosyl and/or glucosyl functional groups.

Pages

231

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