Passive maternal exposure to environmental microbes selectively modulates the innate defences of chicken egg white by increasing some of its antibacterial activities

Abstract

Background: Egg defence against bacterial contamination relies on immunoglobulins (IgY) concentrated in the yolk and antimicrobial peptides/proteins predominantly localized in the egg white (EW). Hens contaminated with pathogenic microorganisms export specific IgYs to the egg (adaptative immunity). No evidence of such regulation has been reported for the antimicrobial peptides/proteins (innate immunity) which are preventively secreted by the hen oviduct and are active against a large range of microbes. We investigated whether the egg innate defences can be stimulated by the environmental microbial contamination by comparing the antimicrobial activity of EW of hens raised in three extreme breeding conditions: Germ-free (GF), Specific Pathogen Free (SPF) and Conventional (C) hens.

Results: The difference in the immunological status of GF, SPF and C hens was confirmed by the high stimulation of IL-1β, IL-8 and TLR4 genes in the intestine of C and SPF groups. EW from C and SPF groups demonstrated higher inhibitory effect against Staphylococcus aureus (13 to 18%) and against Streptococcus uberis (31 to 35%) as compared to GF but showed similar activity against Salmonella Enteritidis, Salmonella Gallinarum, Escherichia coli and Listeria monocytogenes. To further investigate these results, we explored putative changes amongst the three main mechanisms of egg antimicrobial defence: the sequestration of bacterial nutrients, the inactivation of exogenous proteases and the direct lytic action on microorganisms. Lysozyme activity, chymotrypsin-, trypsin- and papain-inhibiting potential of EW and the expression of numerous antimicrobial genes were not stimulated suggesting that these are not responsible for the change in anti-S. aureus and anti-S. uberis activity. Moreover, whereas the expression levels of IL-1β, IL-8 and TLR4 genes were modified by the breeding conditions in the intestine of C and SPF groups they were not modified in the magnum where egg white is formed.

Conclusions: Altogether, these data revealed that the degree of environmental microbial exposure of the hen moderately stimulated the egg innate defence, by reinforcing some specific antimicrobial activities to protect the embryo and to insure hygienic quality of table eggs.

Figure 1

Gene expression levels in the jejunum and the caecum of GF, SPF and C groups. In the jejunum, the gene expression levels of IL-1β and IL-8 (A and B respectively) were higher in C and SPF as compared to GF. In the cæcum, IL-1β and IL-8 were overexpressed in C group as compared to SPF and GF. IL-8 and TLR4 mRNA level were also higher respectively in SPF and C groups compared to GF. (n = 8; mean ± standard deviation;*p < 0.05; **p < 0.01; ***p < 0.001). IL-1β and IL-8 data (A, B, D, E) were analysed using the Kruskal-Wallis test followed by the Mann–Whitney test; TLR4 data (C, F) were analysed using one-way ANOVA followed by the Bonferroni-Dunn test.

Figure 2

Growth of several bacterial strains in presence of GF, SPF and GF egg whites. The growth inhibition of S. aureus (A), S. uberis (B) was significantly higher in C and SPF hens as compared to GF (p < 0.001) while no differences were recorded among these three groups regarding the growth of L. monocytogenes (C), S. Enteritidis (D), S. Gallinarum (E) and E. coli (F). Germ free (GF), Specific Pathogen Free (SPF) and conventional (C) hens (n = 10, mean ± standard deviation).

Figure 3

Genes expression levels in the magnum of GF, SPF and C groups. Gene expression levels of lysozyme (A), AvBD 10 (B), AvBD 11 (C), AvBD 12 (D), gallin (E), ovotransferrin (F), avidin (G), ovoinhibitor (H), cystatin (I), ovomucoid (J), IL1-β (K), IL8 (L) and TLR4 (M) in the magnum as assessed by RT-qPCR showed no difference among the three experimental groups GF, SPF and C (n = 8; mean ± standard deviation, * p < 0.05). Data in A, D, G, H, I, K, L and M were analysed using one-way ANOVA followed by the Bonferroni-Dunn test; data in B, C, E, F and J were analysed using the Kruskal-Wallis test followed by the Mann–Whitney test.