Aerosol vaccination of chicken pullets with irradiated avian pathogenic Escherichia coli induces a local immunostimulatory effect

Abstract

The present study investigated the expression of cytokines and cellular changes in chickens following vaccination with irradiated avian pathogenic Escherichia coli (APEC) and/or challenge. Four groups of 11-week-old pullets, each consisting of 16 birds were kept separately in isolators before they were sham inoculated (N), challenged only (C), vaccinated (V) or vaccinated and challenged (V+C). Vaccination was performed using irradiated APEC applied via aerosol. For challenge, the homologous strain was administered intratracheally. Birds were sacrificed on 3, 7, 14 and 21 days post challenge (dpc) to examine lesions, organ to body weight ratios and bacterial colonization. Lung and spleen were sampled for investigating gene expression of cytokines mediating inflammation by RT-qPCR and changes in the phenotype of subsets of mononuclear cells by flow cytometry. After re-stimulation of immune cells by co-cultivation with the pathogen, APEC-specific IFN-γ producing cells were determined. Challenged only birds showed more severe pathological and histopathological lesions, a higher probability of bacterial re-isolation and higher organ to body weight ratios compared to vaccinated and challenged birds. In the lung, an upregulation of IL-1β and IL-6 following vaccination and/or challenge at 3 dpc was observed, whereas in the spleen IL-1β was elevated. Changes were observed in macrophages and TCR-γδ⁺ cells within 7 dpc in spleen and lung of challenged birds. Furthermore, an increase of CD4⁺ cells in spleen and a rise of Bu-1⁺ cells in lung were present in vaccinated and challenged birds at 3 dpc. APEC re-stimulated lung and spleen mononuclear cells from only challenged pullets showed a significant increase of IFN-γ⁺CD8α⁺ and IFN-γ⁺TCR-γδ⁺ cells. Vaccinated and challenged chickens responded with a significant increase of IFN-γ⁺CD8α⁺ T cells in the lung and IFN-γ⁺TCR-γδ⁺ cells in the spleen. Re-stimulation of lung mononuclear cells from vaccinated birds resulted in a significant increase of both IFN-γ⁺CD8α⁺ and IFN-γ⁺TCR-γδ⁺ cells. In conclusion, vaccination with irradiated APEC caused enhanced pro-inflammatory response as well as the production of APEC-specific IFN-γ-producing γδ and CD8α T cells, which underlines the immunostimulatory effect of the vaccine in the lung. Hence, our study provides insights into the underlying immune mechanisms that account for the defense against APEC.

Keywords: APEC; T cells; chicken; colibacillosis; cytokine; immune response; interferon gamma.

Figure 1

Schematic representation of the experimental setup. Pullets (n = 16 per group) from the negative control (N), challenged (C), vaccinated (V), vaccinated and challenged (V+C) groups were kept in separate isolators from the first day of the experiment. Two birds of group C, one bird of group V+C and one bird in group V died or had to be euthanized before predetermined sampling time points. The vaccination was performed using an irradiated APEC strain applied via aerosol. For challenge, the virulent, homologous strain was administered intratracheally.

Figure 2

Number of birds with lesion in lung, spleen, liver, air sac, heart, and peritoneum following vaccination and/or challenge of birds. The column bar indicates the number of birds showing lesions in the respective organ at different time points. Chickens of the remaining groups (only vaccinated birds and negative control birds) did not show lesions related to APEC inoculation.

Figure 3

Organ to body weight (BW) ratios following vaccination and/or challenge of birds. Comparison of lung-BW ratio and spleen-BW ratio for each experimental group (negative control group (N), challenged group (C), vaccinated group (V), vaccinated and challenged group (V+C)) after challenge. Statistical differences were evaluated using one-way ANOVA in R. Significance was declared at an alpha cut off of 5%. Pairwise comparisons between groups were corrected for multiple testing via the Bonferroni-Holm method. Asterisks indicate a significant difference (P ≤ 0.05).

Figure 4

Predicted probability of bacterial isolation in lung and spleen following vaccination and/or challenge of birds. Comparison of probability of bacterial isolation between experimental groups (negative control group (N), challenged group (C), vaccinated group (V), vaccinated and challenged group (V+C)) in lung and spleen. Statistical differences were evaluated analyzed using a binary logistic regression model in R. Significance was declared at an alpha cut off of 5%. Pairwise comparisons between groups were corrected for multiple testing via the Bonferroni-Holm method. Asterisks indicate a significant difference (P ≤ 0.05).

Figure 5

Histopathology of lung and spleen from different groups. Histopathological observations in tissues from a negative control ((A, B) bird identification: 1), challenged only ((C, D) bird identification: 19), vaccinated ((E, F) bird identification: 47) and vaccinated and challenged ((G, H) bird identification: 51) of representative birds are shown. Organ samples in (C, D) show multifocal areas of necrosis and severe infiltrations of leukocytes. Tissue sections in C and H indicate mainly an increased presence of inflammatory cells. Organ samples of the negative control and vaccinated birds do not show histopathological lesions.

Figure 6

Immunohistochemical detection of E. coli in representative positive tissue samples. Presence of E. coli (arrow heads) in the lung (A) and spleen (B) of an APEC-challenged bird (bird identification: 19).

Figure 7

Cytokines mRNA expression levels measured by RT-qPCR. Expression pattern of IL-1β, IL-6, IL-10 and IFN-γ in lung and spleen are shown as fold changes of mRNA expression levels at 3, 7, 14 and 21 dpc in groups C (red, n = 14) V (green, n = 15) V+C (purple n = 15). Statistical differences were calculated in comparison to the negative control group (n = 16). Error bars represent the standard error of ΔCq values. Statistical differences were evaluated using linear mixed model in R. Significance was declared at an alpha cut off of 5%. Pairwise comparisons between groups were corrected for multiple testing via the Bonferroni-Holm method. Significant changes are indicated as * (P ≤ 0.05).

Figure 8

Alterations of CD4⁺, CD8α⁺, TCR-γδ⁺, Bu-1⁺ and Kul-01⁺ cell subpopulations following vaccination and/or challenge analysed by flow cytometry in lung and spleen. The percentage of CD45⁺CD4⁺, CD45⁺CD8α⁺ TCR-γδ^-, CD45⁺TCR-γδ⁺, CD45⁺CD4^-CD8α^-Bu-1⁺ and CD45⁺KUL-01⁺ cells were determined in groups N (blue) C (red), V (green) V+C (purple). Statistical differences were evaluated using linear mixed model in R. Significance was declared at an alpha cut off of 5%. Pairwise comparisons between groups were corrected for multiple testing via the Bonferroni-Holm method. Asterisks indicate a significant difference in compare to the control group (P ≤ 0.05).

Figure 9

Frequencies of IFN-γ-producing cells subpopulations isolated from lung and spleen of birds following re-stimulation with irradiated APEC. The plots represent the mean frequencies with standard error of IFN-γ-producing CD45⁺CD4⁺, CD45⁺CD8α⁺ TCR-γδ⁻ and CD45⁺TCR-γδ⁺ T cells isolated from lung and spleen following re-stimulation with irradiated APEC. Neither for total Kul-01⁺ nor Bu-1⁺ cells, hardly any IFN-γ producing cells were identified. Statistical differences were evaluated using one-way ANOVA in R. Significance was declared at an alpha cut off of 5%. Pairwise comparisons between groups were corrected for multiple testing via the Bonferroni-Holm method. Asterisks indicate a significant difference of the C (red) V (green) V+C (purple) in compare to the N (blue) group (P ≤ 0.05). Different symbols represent respective time points: circles: 3 dpc, rectangles: 7 dpc, triangles: 14 dpc and rhombuses: 21 dpc.