Bovine respiratory syncytial virus infection enhances Pasteurella multocida adherence on respiratory epithelial cells

Abstract

Primary infection with bovine respiratory syncytial virus (BRSV) predisposes cattle to secondary infection with bacteria that cause bovine respiratory disease complex (BRDC). However, the interaction between BRSV and bacteria is unclear. This in vitro study examined the adherence of Pasteurella multocida (PM) to BRSV-infected cells was assessed in colony forming unit assays, by flow cytometry analysis, and by indirect immunofluorescence analysis (IFA) of epithelial cells (A549, HEp-2, and MDBK). An in vitro model based on infection of BRSV-infected epithelial cells revealed that PM adherence to BRSV-infected cells was 2- to 8-fold higher than uninfected cells. This was confirmed by flow cytometry analysis and IFA. Epithelial cell expression of mRNA encoding cytokines and chemokines increased after exposure to PM, but increased further after co-infection with BRSV and PM. BRSV-mediated adherence of PM to epithelial cells may underlie the serious symptoms of BRDC.

Keywords: Adherent bacteria; BRDC; BRSV; Co-infection.

Fig. 1

Adhesion of Pasteurella multocida (PM) to respiratory epithelial cells depends on the time for which cells are exposed to the bovine respiratory syncytial virus (BRSV) at MOI 1. (A) The number of PM cells adhering to BRSV-infected A549 HEp-2 and MDBK at 48 and 72 h (straight line) post-infection was significantly higher than the number adhering to uninfected cells (dash line) at the same times. Data are expressed as the mean ± SEM (n = 4); * p < 0.01. (B) Flow cytometry analysis confirmed that the number of PM cells adhering to BRSV-infected A549 cells increased with virus infection time: [1] gray layer, number of BRSV-infected cells; [2] black layer, number of PM adhering to infected cells significantly enhance from uninfected cell (open layer). The graph shows mean relative fluorescence intensity ± SEM (n = 3); * p < 0.05. (C) Fluorescence microscopy analysis of FITC-labeled PM (PM-FITC). The results confirm the BRSV-staining with phycoerythrin (BRSV-PE) infection time-dependent increase in adherence of PM-FITC cells to BRSV-infected A549 cells (compared with uninfected A549 cells).

Fig. 2

Adherence of Pasteurella multocida (PM) to bovine respiratory syncytial virus (BRSV)-infected epithelial cells is MOI-dependent (closed bars). A549, HEp-2 and MDBK cells (were infected with virus at a MOI of 0.1 and 1). This was not the case for uninfected cells (open bars) and cells infected with UV-inactivated BRSV (UV-BRSV; patterned bars). Data are expressed as the mean ± SEM (n = 4); * p < 0.05 and ** p < 0.01.

Fig. 3

Expression of mRNA encoding proinflammatory cytokines and chemokines by respiratory epithelial cells co-infected with bovine respiratory syncytial virus (BRSV) and Pasteurella multocida (PM). A549 cells were infected with BRSV (MOI = 1) and then exposed to PM at a MOI of 100. The cells were then washed, and total mRNA was analyzed by quantitative RT-PCR. Expression of IL-1β, IL-6, and IL-8 by PM-infected or BRSV plus PM-infected A549 cells is shown. Expression of MCP-1 and RANTES increased only upon co-infection with BRSV and PM. Data are expressed as mean total mRNA expression normalized to GAPDH (±SEM) (n = 4); * p < 0.01.