Intracellular survival ofHaemophilus somnusin bovine blood monocytes and alveolar macrophages

The mechanisms used byHaemophilus somnusto survive and multiply within bovine mononuclear phagocytes are not fully understood. In order to study the interaction between bovine mononuclear phagocytes andH. somnus, a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was developed to assess the survival ofH. somnuswithin cultured bovine blood monocytes (BBM). Using this system, it was found thatH. somnuswas able to survive within BMMin vitro, and the kinetics of its survival were similar to that seen in BBM isolated from experimentally infected cattle. Using ultrastructural studies, it was possible to demonstrate the survival ofH. somnusin freshly isolated bovine mononuclear phagocytes in membrane-bound vacuoles. To determine if activation of macrophage function would result in elimination of intracellularH. somnus, BBM were treated withE. colilipopolysaccharide (LPS) or recombinant bovine (rBo) cytokines, interferon-γ(IFN-γ), granulocyte macrophage colony stimulating factor (GM-CSF), tumour necrosis factor-α (TNF-α) or interleukin-1β (IL-1β). Treatment of BBM with rBoIFN-γ, rBoGM-CSF orE. coliLPS resulted in decreased intracellular survival ofH. somnusat 18 and 48 h, whereas BBM treated with rBoTNF-α or rBoIL-1β had reduced intracellular survival ofH. somnusonly at 18 h. However, none of these treatments resulted in complete elimination of the intracellular bacteria. The ability ofH. somnusto survive and multiply in both freshly isolated and cytokine-treated cultured BBM demonstrated the capability ofH. somnusto escape from macrophage killing mechanisms. This capability may play a role in the dissemination ofH. somnusinfection in the body.