The role of phagocytic cells in enhanced susceptibility of broilers to colibacillosis after Infectious Bronchitis Virus infection

Abstract

Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. We investigated whether IBV affects recruitment and function of phagocytic cells and examined NO production, phagocytic and bactericidal activity, and kinetics of peripheral blood mononuclear cells (PBMC) and splenocytes. Moreover, we measured cytokine mRNA expression in lung and spleen samples. Broilers were inoculated with IBV H120 vaccine or virulent M41 and challenged 5 days later with E. coli 506. A PBS control and E. coli group without previous virus inoculation were also included. Birds were sacrificed at various time points after inoculation (h/dpi). Inoculation with IBV induced extended and more severe colibacillosis than with E. coli alone. At 4dpi, the number of KUL-01(+) PBMC in all E. coli-inoculated groups was significantly higher than in PBS-inoculated birds, which correlated with lesion scores. From 1 to 4dpi, NO production by PBMC from all E. coli-inoculated animals was elevated compared to PBS birds. Bactericidal activity of PBMC in IBV-inoculated animals at 7dpi was lower than in PBS- and E. coli-inoculated birds, but phagocytic capacity and recruitment were not severely impaired. In spleen samples of IBV-infected animals reduced expression of IL-1beta, IL-6, IL-8, IL-10, IL-18 and IFN-gamma mRNA was found 1dpi. Our results suggest that enhanced colibacillosis after IBV infection or vaccination is caused at least by altered innate immunity and less by impairment of phagocytic cell function.

Fig. 1

Colibacillosis lesion scores at different time points after E. coli inoculation. Groups H120 and M41 were inoculated with their respective IBV strains 5 days before E. coli inoculation, whereas the PBS group received no IBV or E. coli. Per treatment group, five birds were sampled at each time point. Each dot represents the total lesion score of an individual bird with a maximum score of 12. The horizontal lines indicate the mean scores of each treatment group. Birds with systemic signs of colibacillosis, characterized by lesions in liver and/or pericardium, are designated with ‘s’. Groups with different letters are significantly different (P < 0.05).

Fig. 2

Percentage ± S.D. of monocytes/macrophages (KUL-01) in PBMC and splenocytes at different time points after E. coli inoculation. Groups H120 and M41 were inoculated with their respective IBV strains 5 days before E. coli inoculation, whereas the PBS group received no IBV or E. coli. Bars represent the average frequency of labeled cells per treatment group (n = 5), as a percentage of total live cells. Groups with different letters are significantly different (P < 0.05).

Fig. 3

Means ± S.E.M. of E. coli-induced NO production in response to heat-killed E. coli 506 by PBMC per group, at various time points after E. coli inoculation. Groups H120 and M41 were inoculated with their respective IBV strains 5 days before E. coli inoculation, whereas the PBS group received no IBV or E. coli. NO production by cells of individual chickens is calculated as the total NO production after stimulation with heat-killed E. coli or LPS, minus the background NO production of unstimulated cells. Groups with different letters are significantly different (P < 0.05).

Fig. 4

Real-time quantification of cytokine mRNA expression by cells isolated from (A) splenocytes and (B) lung tissue samples of PBS chickens (white bars), chickens inoculated with E. coli only (black bars), IBV H120 + E. coli (hatched bars) and IBV M41 + E. coli (grey bars). Data are expressed as mean relative fold increase at different time points after E. coli inoculation, compared to samples of PBS birds. Error bars show S.E.M. for triplicate samples of five birds per treatment group. Groups with different letters are significantly different (P < 0.05).