Interactions of haemophilus somnus with bovine macrophages

Abstract

The objective of this study was to investigate the effect of H. somnus on the phagocytic function and bactericidal activity of bovine mononuclear phagocytes (BMP) using in vitro and ex vivo assays. Using a flow cytometric phagocytosis assay, it was found that logarithmically growing H. somnus significantly inhibited the phagocytosis of opsonized S. aureus by bovine alveolar macrophages (BAM) obtained both from healthy calves and from cattle experimentally infected with H. somnus. However, neither heat- or formalin-killed, log-phase H. somnus nor in vitro passaged H. somnus showed any effect on the phagocytic activity of these cells. In contrast to BAM, bovine blood monocytes (BBM) had a significant increase in their phagocytic activity following in vitro exposure to logarithmically growing H. somnus. The bactericidal ability of bovine mononuclear phagocytes in interaction with H. somnus was studied using two in vitro assay systems measuring nitric oxide (NO) production and the chemiluminescence response. H. somnus rapidly inhibited the Luminol-dependent chemiluminescence (LDCL) of BBM and BAM costimulated with opsonized S. aureus. Inhibition of the LDCL response of BBM and BAM was abrogated with either opsonized or killed H. somnus. In contrast to inhibited LDCL of BMP, both BBM and BAM infected with H. somnus had stimulated production of NO. Using a colorimetric bactericidal assay, it was found that: (1) H. somnus was able to survive within BBM in vitro and the kinetics of its survival were similar to that seen in BBM isolated from experimentally infected cattle; (2) treatment of BBM with varying concentrations of rBoIFN-ã, rBoTNF-á, rBoIL-1â, rBoGM-CSF and E. coli LPS had no effect on the survival of H. somnus. Moreover, using ultrastructural studies, and 3H. uracil incorporation into nucleic acids, it was possible to demonstrate the survival of H. somnus in BMP. With in vitro addition of H. somnus to whole blood cultures prepared from cattle experimentally infected with H. somnus, TNF production was not detectable during the 24 h incubation period while IFN-ã and IL-6 were detected. These results indicate that the ability of H. somnus to modulate microbicidal activity of BMP could, in turn, assist the intracellular survival and contribute to pathogenesis of bovine haemophilosis.