Subcutaneous inoculation of Escherichia coli in broiler chickens causes cellulitis and elicits innate and specific immune responses

Abstract

Background: Cellulitis caused by Escherichia coli is a common cause of condemnation of broiler chickens at slaughter worldwide and is associated with economic losses and a possible negative impact on animal welfare. The study objective was to monitor clinical signs and immune responses after subcutaneous E. coli inoculation (1.1-1.8 × 10⁷ CFU), aiming to induce cellulitis. Three groups of broiler chickens (n = 15/group) were inoculated with well-characterized E. coli strains (group A: ECA18 O24:H4/ST117 and group B: ECB11 O153:H9/ST38) or with saline (control) at 22 days-of-age. Clinical signs of disease, body weight and immune parameters were monitored until euthanasia 12-14 days after inoculation followed by post-mortem examination.

Results: The daily weight gain of the inoculated chickens was significantly lower one day after inoculation compared to the controls. Seven (23%) of the inoculated chickens displayed clinical signs: ruffled feathers, mild weakness, open-beak breathing and/or reluctance to stand, of which two birds were euthanized and one bird died. Five chickens in group B were observed with bacteraemia, which lasted up to three days after inoculation for two chickens. A transient increase in chicken mannose receptor MRC1L-B expression on circulating monocytes was observed one day after inoculation in both E. coli inoculated groups, with a more pronounced increase in group B. On day 7 after inoculation, the in vitro adherence of heterophils, monocytes and thrombocytes to the inoculated strain was increased in group B. Antibody titers to the inoculation strains were increased in some chickens in both groups on days 7 and 14 after inoculation, with the highest titers in group B. Seven (47%) and 13 (87%) of the chickens in group A and B, respectively, were diagnosed with cellulitis at post-mortem examination. In most birds, lesions consisted of plaque-like material embedded in the subcutaneous tissue of the abdominal wall.

Conclusions: Inoculation of E. coli caused cellulitis and prompted a rapid activation/redistribution of circulating monocytes followed by antibody production. The responses were most pronounced in chickens inoculated with E. coli strain ECB11, presumably because of a higher virulence.

Keywords: Post-mortem findings; APEC; Cellulitis; Chicken mannose receptor MRC1L-B; Clinical signs; IgY; Immune response.

Fig. 1

Body weight A and daily weight gain B, C for control chickens (filled triangles, 10 ≥ n ≤ 15), and chickens inoculated subcutaneously with E. coli strain ECA18 (group A; filled squares, 10 ≥ n ≤ 15) or strain ECB11 (group B; filled circles, 8 ≥ n ≤ 15) on experimental day 0 (dotted line). Chickens were weighed on the indicated experimental days and daily weight gains were calculated for time periods between weight recordings. Body weight on day 14 PI was only measured for 10 of 15 chickens in group A, 8 of 13 in group B and 10 of 15 in the control group. Results are shown as group mean values ± 95% CI, where non-overlapping CI indicate statistically significant differences, and as individual values for chickens no 35, 36, 38, 41 and 43, respectively, from group B that had bacteraemia on one or more sampling occasions post-inoculation

Fig. 2

Chickens with subcutaneous lesion on the right side of the abdomen two weeks after inoculation with E. coli strain ECB11. A 40 × 10 mm incised lesion of plaque-like material enclosed in subcutaneous tissue; B 30 × 40 mm lesion of plaque-like material partly embedded in the abdominal wall and extending to the distal part of the pectoral muscle

Fig. 3

Quantification of E. coli by direct culture of blood collected on the indicated days after inoculation from chickens inoculated subcutaneously with E. coli on day 0. Results are shown as individual values for bacteraemic chickens in group B inoculated with strain ECB11 (n = 5)

Fig. 4

Numbers of heterophils A, B monocytes C, D and lymphocytes E, F in blood collected on the indicated days from control chickens (filled triangles, 7 ≥ n ≤ 15) and chickens inoculated subcutaneously with E. coli strain ECA18 (group A; filled squares, 7 ≥ n ≤ 15) or strain ECB11 (group B; filled circles, 6 ≥ n ≤ 15) on experimental day 0 (dotted line). Results are shown as group mean values ± 95% CI, where non-overlapping CI indicate statistically significant differences, and as individual values for chickens no 35, 36, 38, 41 and 43, respectively, from group B that had bacteraemia on one or more sampling occasions post-inoculation. For details, see the materials and methods section

Fig. 5

Expression of MRC1L-B (A, B) on monocytes in blood collected on the indicated days from control chickens (filled triangles, 7 ≥ n ≤ 15) and chickens inoculated subcutaneously with E. coli strain ECA18 (group A; filled squares, 7 ≥ n ≤ 15) or strain ECB11 (group B; filled circles, 6 ≥ n ≤ 15) on experimental day 0 (dotted line). Results are shown as group mean values ± 95% CI, where non-overlapping CI indicate statistically significant differences, and as individual values for chickens no 35, 36, 38, 41 and 43, respectively, from group B that had bacteraemia on one or more sampling occasions post-inoculation

Fig. 6

Adhesion of E. coli to different leukocyte populations: (a) heterophils, (b) monocytes and (c) thrombocytes in whole blood cultures established with blood collected on the indicated days from control chickens (7 ≥ n ≤ 15) and chickens inoculated subcutaneously with E. coli strain ECA18 (group A, 7 ≥ n ≤ 15) or strain ECB11 (group B, 6 ≥ n ≤ 15) on experimental day 0 (dotted line). Cultures were incubated for 15 min with either strain ECA18 (A) or strain ECB11 (B), for details see materials and methods. Results are shown as group mean values ± 95% CI, where non-overlapping CI indicate statistically significant differences

Fig. 7

IgY titers to a sonicated antigen preparation of ECA18 A or ECB11 B in sera collected on the indicated experimental days from control chickens (C; triangles, 14 ≥ n ≤ 15) and chickens inoculated subcutaneously with E. coli strain ECA18 (group A; squares, n = 15) or strain ECB11 (group B; circles, 13 ≥ n ≤ 15) on experimental day 0. Results are shown as individual values for all chickens with titers > 1, with filled symbols for chickens from group B that had bacteraemia on one or more sampling occasions post-inoculation