Bradley W. Fenwick

Growth-condition dependent expression of Pasteurella haemolytica A1 outer membrane proteins, capsule, and leukotoxin☆

Pasteurella haemolytica, strain P1148 (biotype A, serotype 1) was grown under iron-rich and iron-restricted conditions both with and without serum, and the outer membrane protein (OMP), capsule, and leukotoxin production studied. OMPs were evaluated by SDS-PAGE and examined by immunoblot to identify antigens recognized by sera from P. haemolytica A1 convalescent and vaccinated cattle. Capsule production was evaluated using fluorescent antibody staining and rapid plate agglutination reaction. Leukotoxin production was measured by neutrophil 51Cr-release assay. Expression of specific OMPs, amount and antigenic character of capsule, and quantity of leukotoxin produced by P. haemolytica A1 varied in response to alterations in the growth media. Immunoblots indicated the immune response of convalescent calves differs from vaccinated calves, and convalescent calves produce antibodies to novel OMPs induced by growth in iron-restricted conditions.

Normal Anatomical and Histochemical Characteristics of the Lacrimal Glands in the American Bison and Cattle

Dorsal lacrimal glands, superior glands of the third eyelid and Harderian glands (deep gland of the third eyelid) from 19 bison and 18 cattle free of apparent ocular disease were examined to compare the normal anatomical properties of these glands. All glands were characterized and measured (length and width). The gross anatomy of the dorsal lacrimal glands was similar, with the exception of a bipartite gland in cattle. The bisons superior gland of the third eyelid and Harderian gland was longer as compared with cattle. A subset of the bison and cattle samples (five bison and five cattle) was sectioned for histological and histochemical analysis. The histology of the dorsal lacrimal and superior gland of the third eyelid revealed tubuloalveolar cells with basophilic vacuolated cytoplasm in bison and eosinophilic granular cytoplasm in cattle. The Harderian glands consisted of a tubuloalveolar anterior part combined with large lumens acini lined with cuboidal epithelium in the posterior part; the posterior part of the bison Harderian gland was more predominant than in cattle samples. Mucosubstance histochemistry revealed acidic and neutral glycoproteins with similar staining patterns in all glands of both species.

Actin polymerization enhances Pasteurella haemolytica leukotoxicity

Pasteurella haemolytica leukotoxin is cytotoxic to bovine leukocytes, causing increased cell membrane permeability, osmotic swelling, release of cytosolic proteins and cell lysis. These studies were designed to test if leukotoxin causes release of the cytoskeletal protein, actin, from bovine leukemia cells and if purified actin-influenced bacterial growth or leukotoxin production. Culture supernatants caused a 7-fold decrease in viability of bovine leukemia cells and increased cell permeability that was accompanied by release of beta-actin into the cell culture supernatant. Exposing P. haemolytica to purified actin solutions induced the conversion of monomeric G-actin to polymerized F-actin. This conversion was partially inhibited by bovine P. haemolytica immune, but not pre-immune, serum. Loss of streptomycin resistance following treatment of the organism with acridine orange ablated the polymerizing activity. Incubation of P. haemolytica in the presence of purified F-actin did not affect growth but resulted in culture supernatant that had 3.0-3.9-fold greater leukotoxicity compared to medium alone or medium containing G-actin, heat-denatured actin or albumin. The effect of actin on leukotoxicity was concentration-dependent and directly associated with increases in secreted leukotoxin. The interaction between P. haemolytica and actin is potentially detrimental to the host by inducing polymerization of actin into insoluble filaments and by enhancing leukotoxicity.