Bovine respiratory disease (BRD) is a multi-factorial disease complex that involves interactions among stressors, management factors, and viral and bacterial pathogens. Mannheimia haemolytica and Histophilus somni are two important bacterial pathogens in BRD. Our laboratory has pursued several lines of investigation to understand better how these organisms cause pneumonia. One issue that has been relatively neglected is the ability of these bacteria to colonize healthy cattle. We hypothesize they do so at least in part by forming a biofilm in the upper respiratory tract. We find that M. haemolytica forms considerable biofilm in vitro that largely consists of protein and lesser amounts of carbohydrate and nucleic acid. Biofilm formation was partially inhibited by addition of anti-OmpA antibodies or certain monosaccharides (e.g. galactose). Currently we are investigating biofilm formation on bovine bronchial epithelial cells in vitro.
Concurrent viral infection predisposes to more severe BRD. We investigated this viral/bacterial synergism by assessing microarray gene expression in vitro by bovine bronchial epithelial cells exposed to bovine herpsesvirus-1 (BHV-1), M. haemolytica, or BHV-1 and M. haemolytica together. These analyses revealed different signaling pathways and complex interactions following exposure to M. haemolytica or BHV-1. Significantly greater inductions (>10 fold) of several inflammation associated genes (i.e. CXCL2, IL-6, IL-1α, e-selectin, and IL-8) were stimulated by M. haemolytica than BHV-1. Interactive pathway analysis revealed an overall similarity between the pro-inflammatory cytokine (IL-1b, TNF, LPS induction and NFkb) response to M. haemolytica alone, or M. haemolytica and BHV-1 together.
Finally, we investigated the ability of bovine neutrophils and macrophages to form extracellular traps in response to M. haemolytica and H. somni. M. haemolytica triggers trap formation largely by its leukotoxin (LKT). Both fully active LKT, and non-acylated pro-LKT released by lktC mutants of M. haemolytica, trigger trap formation by bovine PMNs and macrophages. These responses were blocked by antibodies against LKT or CD18 (the LKT receptor). In contrast, H. somni, which does not produce an RTX exotoxin,caused trap formation in part via outer membrane vesicles (OMVs) and associated proteins. A portion of the bacterial cells enmeshed within the DNA fibrils of extracellular traps appear to be killed. Although we do not have definitive proof for the role of NETs in BRD, we hypothesize that NETs contribute to the substantial amounts of extracellular DNA within the airways of cattle with pneumonia.