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Banning the use of antibiotic growth promotors in animal feed has forced poultry breeding companies to select birds with improved disease resistance. One area of focus is the chicken innate immune system, which includes a family of avian β-defensins (AvBDs) that are synthesised by epithelia in response to microbial challenge. The aim of this thesis was to investigate the in vivo gene expression, antimicrobial activity (AMA) and mechanisms of action of AvBDs 6 and 9. Endpoint and qPCR were used to investigate AvBD6 and 9 gene expression in an array of epithelial tissues taken from day 7 and 35 broiler chickens reared in low and high hygiene conditions. The expression profiles of pro (interleukin-6: IL-6) and anti (Transforming Growth Factor β4:TGFβ4) inflammatory cytokine genes, and the chicken galectin-3 gene (CG3) were also examined. To explore AMA recombinant (r)AvBD peptides and their variants, including rAvBD9 3CA (lacking 3 of the 6 conserved cysteine (C) amino acids), rAvBD9 6CAG (lacking all the conserved C amino acids) and rAvBD9 W38G were synthesised, and their AMAs against E. coli and E. faecalis tested in vitro using time-kill assays. The structural properties and membrane interactions of the rAvBDs and custom synthesized linear (s) AvBD6 and 9 peptides were also investigated using circular dichroism (CD), liposome entrapped calcein leakage assays and peptide modelling. The in vivo gene expression analyses revealed that AvBD6 and 9, IL-6, TGFβ4 and CG3 were expressed in all the broiler chicken tissues examined. However, the data were compromised by the small bird numbers, and the variability in the tissue expression data between individual birds within a group resulted in no statistically significant trends associated with rearing environment being detected. The AMA data showed that the rAvBD6 and 9 peptides were antimicrobial against both Gram negative and positive microbes with rAvBD6 > rAvBD9 (using 100 μg/ml peptide, 76.4% (23.6±2.3 % survival) of E. coli were killed by rAvBD6 compared to 64.3% (35.7±7.2% survival) for rAvBD9). These data were potentially related to the physical properties of the peptide with AvBD6 being more cationic (+6.8) and hydrophobic than AvBD9 (+3.8), although modelling data also suggested that AvBD6 contained a hook-like foramen structure. The AMAs of the rAvBD9 and AvBD9 3CA peptides were not significantly different. Using 50 μg/ml peptide 49% (51±12% survival) and 60% (40±8% survival) of E. coli were killed compared to 53% (47±20% survival) and 69% (31±13% survival) killing for E. faecalis. Nu-PAGE data suggested the AMA potency of AvBD9 3CA was associated with dimer formation. At 50 μg/ml neither the rAvBD9 6CAG nor the synthetic linear AvBD9 peptides were active against the E.coli isolate. However, in the presence of proteinase inhibitor (Roche-1:1000 dilution), 45% E.coli BL21 killing (55±1% survival) was observed, which strongly supported the roles of the di-sulphide bonds in protecting the AvBD peptides against proteolysis. Substituting the C-terminal tryptophan (W) of rAvBD9 for a glycine (G) also resulted in a loss of AMA against bacteria (at 50 μg/ml 0% killing of E. coli and 15% (85.5 ±20.5% survival) killing of E. faecalis was detected). These data indicated that the C terminal W amino acid is also important for AvBD9 AMA. In the presence of SDS micelles mimicking the bacterial membrane synthetic AvBD1, 6 and 9 peptides showed increased α-helicity. Membrane leakage experiments using calcein-entrapped liposomes and synthetic peptides (1.5 μg/ml) showed sAvBD6 induced more leakage at 4 minutes than sAvBD9 (60.3±6.3% (n=4) versus 11.5% (n=1)). Although compromised by the lack of replicates these data suggested that AvBD9 may not function through membrane disruption suggesting other mechanisms including inhibition of nucleic acid synthesis and/or cell division. Membrane leakage experiments using sAvBD1 peptides modelling an AvBD1 SNP found in poultry showed NYH >SSY> NYY. Overall these data show AvBD6 and 9 are expressed in bird tissues and have AMA against gram negative and positive bacteria. The studies supported different mechanisms of action of the two defensins with AvBD6 causing membrane damage compared to AvBD9, which probably functions through disrupting intracellular systems. These data suggest that the AvBD peptides work in synergy in defending the epithelia and warn against poultry geneticists selecting individual AvBD genes for breeding purposes.

The E.U. ban on the use of anti-microbial growth promoters in poultry feed, introduced to counter global problems of bacterial antibiotic resistance, has increased the risk of enteric disease in commercially reared broiler chickens. Development of strategies to prevent such diseases requires further knowledge and understanding of avian gut defences and particularly the innate immune defences. In collaboration with Aviagen Ltd., the objective of this study was to investigate, through two farm trials, the effects of bacterial exposure on host avian β-defensin (AvBD) expression profiles and gut health of two commercial broiler lines (X and Y). Furthermore, two host defense peptides, avian beta defensin 1 (AvBD1) and 10 (AvBD10) were analysed in vitro for their anti-microbial efficacies. In Trial 1, Lines X and Y, differing in their gut health, were exposed to one of three bacterial challenges on the day of hatch, namely a combination of Bacteroides dorei and Barnesiella viscericola (B/BV), Lactobacillus johnsonii (LJ) or a mixture of the two challenges (B/BV + LJ). At days 4, 7, 14, 21, 28 and 35, birds were scored for gut health using an industry approved system and digesta were sampled and analysed for microbiotae (pyrosequencing). The data revealed that, relative to control and LJ challenged birds, the B/BV challenge was associated with gut health deterioration. Furthermore, relative to Line X, there was a trend for the gut health of Line Y birds to be superior for all challenged groups. Although microbiome analyses did not reveal any clear differences between Lines X and Y, the data did suggest that birds with better gut health outcomes were associated with higher ileal Lactobacillus spp. levels at Day 4 and higher caecal levels of Bacteroides spp. at Day 21. Despite less optimal gut health, Line X is important to the Aviagen Ltd. breeding programme. To understand the roles, if any, of the AvBDs in bird gut health, a second trial was performed in which gut AvBD1 and 10 gene expression were assessed in Line X birds following B/BV challenge. Relative to control birds, the B/BV challenge suppressed gene expression of AvBD1 in the duodenum/jejunum (P < 0.05) and AvBD10 in the duodenum/caecum (P < 0.05) and AvBD1 down-regulation was confirmed at the cellular level by data from an in vitro challenge model (P < 0.001). Interestingly, within the B/BV challenged group, birds with higher AvBD1 expression were associated with better gut health assessments. The AvBD1 gene contains single nucleotide polymorphisms (SNP) within the region encoding region the mature peptide. Three AvBD1 variants were synthesised that were typical to Line X (NYH), Y (SSY) and another commercial Line, Z (NYY), and were assessed, together with AvBD10, for in vitro anti-microbial activities (AMAs) against a variety of gut bacterial isolates. Despite Line X displaying the least optimal gut health, the ‘NYH’ variant exhibited the greatest potency against all bacterial species. The data for AvBD10 revealed that, although bacterial growth was inhibited, this peptide had lower AMA than AvBD1, indicative of additional physiological functions. An in vitro examination of wound healing capacity using a scratch assay was inconclusive. The in vivo data indicated that gut AvBD expression is susceptible to gene down-regulation by bacteria and that this, in turn, may have an adverse outcome on gut health. However, selectively breeding for birds able to maintain high AvBD expression presents a strategy to protect flocks against the threat of endemic gut health problems.

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