Date of Award
Doctor of Philosophy
Norman F. Cheville
Mark R. Ackermann
Serogroup A Pasteurella multocida is the major cause of fowl cholera in poultry. Resistance of P. multocida to phagocytosis in non-immunized birds is associated with the presence of a hyaluronic acid capsule. The studies presented in this dissertation were designed to (1) investigate the adhesive properties of a capsulated serotype A:3 strain P. multocida and its non-capsulated variant (-:3) to turkey air sac macrophages and peripheral blood monocytes, (2) to identify, and isolate the monocyte receptor implicated in bacterial adhesion, and (3) to produce polyclonal antibodies against the P. multocida receptor. Capsulated serotype A:3, unlike the non-capsulated variant, adhered in large numbers to the macrophages but were not internalized. Depolymerization of the bacterial capsule with hyaluronidase increased phagocytosis. Addition to the macrophage cultures of hyaluronic acid, sodium metaperiodate, or trypsin, suppressed bacterial binding. In contrast to the experiments performed with air sac macrophages, serotype A:3 strains were not adherent to freshly isolated peripheral blood monocytes. Following culture of blood monocytes for six days in chamber slides, adhesion of the bacteria increased gradually. Coating chamber slides with entactin-laminin-collagen IV attachment matrix or exposure to phorbol myristate acetate enhanced bacterial binding to the monocytes whereas exposure to monoclonal antibodies directed against the major receptor for hyaluronic acid (CD44) decreased binding. Collectively, these findings indicated that capsular hyaluronic acid promotes adhesion, but not internalization, of serotype A:3 strains to turkey air sac macrophages or "activated" blood monocytes. The findings additionally indicated that the bacterial polysaccharide recognizes CD44 glycoproteins on monocytes;Hyaluronic-acid Sepharose affinity chromatography was subsequently used to purify hyaluronic acid binding proteins from cultured monocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblots of the eluted material demonstrated three major bands (81, 101, and 118 kDa) which reacted with a biotinylated hyaluronic acid probe. The 101 and 118 kDa proteins were electroeluted from the acrylamide gels by reverse polarity and used to hyperimmunize rabbits. Preinoculation serum reacted with several turkey monocyte antigens, including the 101 and 118 kDa hyaluronic acid binding proteins. Immunization did not result in higher titers or increased specificity.
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Ingrid Mireille Pruimboom
Pruimboom, Ingrid Mireille, "Characterization, identification, and purification of the bacterial receptor expressed by turkey peripheral blood monocytes for serogroup A:3 of Pasteurella multocida " (1998). Retrospective Theses and Dissertations. 11638.