Antimicrobial peptides (AMPs) are essential components of innate immunity in a range of species fromDrosophila to humans and are generally thought to act by disrupting the membrane integrity of microbes. In order to discover novel AMPs in the chicken, we have implemented a bioinformatic approach that involves the clustering of more than 420,000 chicken expressed sequence tags (ESTs). Similarity searching of proteinspredicted to be encoded by these EST clusters-for homology to known AMPs has resulted in the in silico identification of full-length sequences for seven novel gallinacins (Gal-4 to Gal-10), a novel cathelicidin and a novel liver-expressed antimicrobial peptide 2 (LEAP-2) in the chicken. Differential gene expression of these novel genes has been demonstrated across a panel of chicken tissues. An evolutionary analysis of the gallinacin family has detected sites-primarily in the mature AMP-that are under positive selection in these molecules. The functional implications of these results are discussed.

Antimicrobial peptides are essential components of innate immunity and are generally thought to act by disrupting the membrane integrity of microbes. Here we report the discovery of two novel chicken beta-defensins, gallinacin (Gal)-11 and Gal-12, found by hidden Markov model profile searching of the chicken genome. We have sequenced the genes and elucidated the 3'UTR of Gal-11. Differential mRNA expression of these novel genes has been shown across a panel of chicken tissues. Gal-11 mRNA was highly expressed in the small intestine, the liver, the gall bladder and the spleen and also showed moderate expression in several other areas of the chicken anatomy, whilst Gal-12 mRNA was found only in the liver and the gall bladder. Antimicrobial activity of synthetic Gal-11 has been demonstrated against a range of bacteria and is predominantly active against the intestinal pathogens Salmonella typhimurium and Listeria monocytogenes.

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Oral administration of hen-egg yolk provides protection against specific pathogens. We examined the antibacterial activity of fractionated egg yolk against 2 pathogenic Streptococcus strains, using an in vitro assay. A water-soluble protein fraction (WSPF) of egg yolk consistently inhibited the growth of S. mutans by 25%. The WSPF treated with pancreatin demonstrated > 80% inhibition of bacterial growth. Growth of S. sanguis was completely inhibited. Gel filtration and ion exchange chromatography established that anti-Streptococcal activity resided with lipoproteins. Antibacterial activity was released by crude lipase or a combination of lipase and protease treatment of egg lipoproteins. Thus, hen-egg yolk lipoproteins are important molecules for lipid-mediated antimicrobial activity.

Hen egg yolk contains significant antibacterial activity. We show that this activity is associated with the release of free fatty acids. Chloroform-methanol extraction on egg yolk demonstrated the activity to be lipoprotein-bound before enzymatic digestion and associated with the lipid-soluble chloroform phase afterward. Acetone extraction yielded a fraction of egg yolk, which was 97% triglyceride and highly antibacterial to Streptococcus mutans when treated with pancreatin. The fatty acid profile of the extract reflected the proportions known to exist in egg yolk. Both oleic and linoleic acid were found to inhibit growth of S. mutans. These findings highlight a previously unreported potential of hen egg yolk in future healthcare applications.