Properties, Genetics and Innate Immune Function of the Cuticle in Egg-Laying Species

Abstract

Cleidoic eggs possess very efficient and orchestrated systems to protect the embryo from external microbes until hatch. The cuticle is a proteinaceous layer on the shell surface in many bird and some reptile species. An intact cuticle forms a pore plug to occlude respiratory pores and is an effective physical and chemical barrier against microbial penetration. The interior of the egg is assumed to be normally sterile, while the outer eggshell cuticle hosts microbes. The diversity of the eggshell microbiome is derived from both maternal microbiota and those of the nesting environment. The surface characteristics of the egg, outer moisture layer and the presence of antimicrobial molecules composing the cuticle dictate constituents of the microbial communities on the eggshell surface. The avian cuticle affects eggshell wettability, water vapor conductance and regulates ultraviolet reflectance in various ground-nesting species; moreover, its composition, thickness and degree of coverage are dependent on species, hen age, and physiological stressors. Studies in domestic avian species have demonstrated that changes in the cuticle affect the food safety of eggs with respect to the risk of contamination by bacterial pathogens such as Salmonella and Escherichia coli. Moreover, preventing contamination of internal egg components is crucial to optimize hatching success in bird species. In chickens there is moderate heritability (38%) of cuticle deposition with a potential for genetic improvement. However, much less is known about other bird or reptile cuticles. This review synthesizes current knowledge of eggshell cuticle and provides insight into its evolution in the clade reptilia. The origin, composition and regulation of the eggshell microbiome and the potential function of the cuticle as the first barrier of egg defense are discussed in detail. We evaluate how changes in the cuticle affect the food safety of table eggs and vertical transmission of pathogens in the production chain with respect to the risk of contamination. Thus, this review provides insight into the physiological and microbiological characteristics of eggshell cuticle in relation to its protective function (innate immunity) in egg-laying birds and reptiles.

Keywords: bacterial pathogens; egg-laying birds; eggshell cuticle; evolution; food safety; genetics; microbiome.

Figure 1

Graphical abstract navigating/cataloging the contents of this review article (Original figure by GK).

Figure 2

Stylized depiction of the reproductive system of the hen, containing an incomplete egg in the uterus. Reprinted from Front Bioscience., Vol. 17, Issue 1, Hincke et al., The eggshell: structure, composition and mineralization, 1266-1280, 2012, with permission from Frontiers.

Figure 3

Scanning electron micrograph of cross-fractured eggshell showing different layers: cuticle, palisade layer, mammillary layer with associated inner and outer shell membranes and full-length respiratory pore with pore plug. (Original image by GK).

Figure 4

Eggshell ultrastructure in representative species of amniote vertebrates. The eggshell cuticle (arrows) is the most superficial layer covering the calcified or fibrous shell. (A) Common eider (Somateria mollissima); (B) Great ani (Crotophaga major); (C) red-footed tortoise (Chelonoidis carbonarius); (D) Mediterranean chameleon (Chamaeleo chamaeleon); (E) Madagascar day-gecko (Phelsuma madagascariensis); (F) Mountain pit viper (Ovophis monticola). Insets show detail of representative amorphous (am) and nanostructured (n; nanospheres) cuticles. Scale bars: (A, B, D) -100 μm; (C) - 200 μm; (E, F) - 50 μm. (Original figure by LDA).

Figure 5

Scanning electron micrograph (SEM) image of outer surface of chicken eggshell cuticle at 1000X showing patchy distribution with cracks and fissures. (Original figure by GK).

Figure 6

Proposed compositional gradient in the eggshell cuticle with predicted distribution of proteins, phosphoproteins, glycoproteins, and sulphated proteoglycans. Reprinted from Foods, Vol. 10, Issue 11, Kulshreshtha et al., Impact of different layer housing systems on eggshell cuticle quality and Salmonella adherence in table eggs, 2559, 2021, with permission from MPDI.

Figure 7

Characterization of the eggshell cuticle. (A) Eggs demonstrating a good degree of uniform staining with MST cuticle blue dye; (B) ATR-FTIR spectra of an eggshell surface showing the main IR bands from the cuticle and from the shell mineral. (Original figure by ARN).

Figure 8

Crystal violet stained cuticle protein in pores and plug visualized by stereomicroscopy. (A) Outer surface of white ungraded chicken eggshell (21 wk) showing pore surfaces at 10X. (B) Cross-fractured eggshell showing plug and pore lined by stained protein at 150X magnification. (C) Outer surface of eggshell showing magnified pore plug at 150X. Reprinted from Poultry Science, Vol. 97, Issue 4, Kulshreshtha et al., Cuticle and pore plug properties in the table egg, 1382-1390, 2018, with permission from Elsevier.

Figure 9

Confocal fluorescent image of Salmonella Typhimurium on the outer surface of white ungraded chicken eggshell cuticle. S. Typhimurium localized near cracks and fissures of cuticle on the outer surface of ungraded eggshell. Red fluorescence= cuticle protein; Green fluorescence= GFP expressing S. Typhimurium cells. (Original figure by GK).

Figure 10

Scanning electron microscopy (SEM) images of (A) Outer surface of chicken eggshell showing openings of pores at 1000x. (B) and (C) at higher magnification 5000X. (Original figure by GK).