Antimicrobial activity of the Anseriform outer eggshell and cuticle

The avian eggshell is a complex, multifunctional biomineral composed of a calcium carbonate mineral phase and an organic phase of lipids and proteins. The outermost layer of the eggshell, the eggshell cuticle, is an organic layer of variable thickness composed of polysaccharides, hydroxyapatite crystals, lipids and glycoprotein. In addition to regulating gas exchanges, the eggshell cuticle may contain antimicrobial elements. In this study, we investigated the antimicrobial activity of eggshell cuticle and outer eggshell protein extracts from four Anseriform species: wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis) and mute swan (Cygnus olor). Cuticle and outer eggshell protein was extracted by urea or HCl treatment of eggs. C-type lysozyme, ovotransferrin and an ovocalyxin-32-like protein were detected in all extracts. Cuticle and outer eggshell protein extracts inhibited the growth of Staphylococcus aureus, Escherichia coli D31, Pseudomonas aeruginosa and Bacillus subtilis. The presence of active antimicrobial proteins within the avian cuticle and outer eggshell suggests a role in antimicrobial defense. Protein extracts from the cavity nesting hooded merganser were especially potent. The unique environmental pressures exerted on cavity-nesting species may have led to the evolution of potent antimicrobial defenses.