Identification and development of novel antimicrobial peptides as alternatives to antibiotic growth promoters in poultry
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Poultry are vital to food security, with 60 billion chickens reared worldwide per annum and demand fast accelerating. For many years antibiotic growth promoters have been used to promote energy retention from the diet and control intestinal bacterial growth. Antibiotic use for prophylaxis or growth-promotion in farmed animals is prohibited under EU Directives due to human health concerns, but a pressing need exists to maintain the efficiency of animal production by finding alternatives. Antimicrobial peptides (AMPs), part of the innate immune system exist naturally in most species and could provide a vast array of potential therapeutics. Microbial resistance to AMPs is unlikely due to their relatively unspecific mode of action, their ability to target multiple sites within a cell and diverse immune-modulatory activities. The avian egg provides antimicrobial protection through many mechanisms including AMPs which are incorporated into the egg white by the hen. The ovodefensin family and ‘transiently expressed in neural precursors’ (TENP) have been identified as potential novel antimicrobials in egg white and therefore formed the basis of the peptide portfolio of this study. TENP was first identified as having a role in neurological development but has since been shown to be an important egg component constituting ~0.1-0.5% of the total protein. TENP is conserved across avian species being found in chicken, turkey, duck and zebra finch. Its homology with the bacterial permeability-increasing family of innate immune genes suggests it may contribute to antimicrobial function in the egg. This study confirmed that expression of TENP is confined to the albumen forming region of the oviduct in adult hens and is under gonadal steroid control, typical of an oviduct and egg specific gene. The ovodefensin family are β defensin related antimicrobial peptides thought to be restricted to the albumen producing region of the avian oviduct. This study identified twenty five novel ovodefensin members through genome analysis, expanding the ovodefensin family to include reptiles for the first time. Phylogenetic analysis showed a unique example of the evolution of a cysteine spacing motif alongside traditional sequence evolution. The expression of eight ovodefensins was shown to be oviduct specific supporting the hypothesis that ovodefensins evolved to protect the egg. Antimicrobial activity for three ovodefensins from chicken and duck was investigated against gram negative organisms E. coli and Salmonella including pathogenic strains as well as a gram positive organism, S. aureus, for the first time. The spectrum of activity varied greatly between peptides suggesting a link between structure and function. Inclusion of recombinant ovodefensin peptides in the feed of chickens showed beneficial effects on the gut microbiome, metabolite profile and most crucially an increase in mean body weight. This demonstrates the potential of antimicrobial peptides as alternatives to antibiotic growth promoters in poultry.