The effects of genetics, age and rearing environment on AvBD gene expression and gut anti-microbial activities in three chicken lines

Abstract

The defensins have been shown to be an important component of the innate immune system across many species from plants to man. To date a total of 14 beta defensins have been identified within the chicken genome and anti-microbial activity of an array of these peptides against pathogens has been demonstrated. The innate immune system has been shown to be important in chickens during the first week of life when exposure to pathogens in the environment occurs and the adaptive immune system is not fully developed. The research presented in this thesis attempts to investigate the effects of bird age (aged 0, 7, 14 and 35 days), genetics and rearing environment on components of the innate immune system. A farm trial was performed using three lines of Aviagen birds (lines X, Y and Z) and two distinct rearing environments (low and high hygiene farms). To determine which of the 14 avian beta defensins (AvBD) to investigate, a panel of potential single nucleotide polymorphisms (SNPs) within the AvBD locus was submitted as part of an Aviagen Ltd genomics initiative. Frequencies of the polymorphisms across the three lines of birds were determined. From these data, gene expression of AvBDs1, 4 and 10 were fully investigated using end-point PCR and then quantitative real-time PCR (qRT-PCR). A pronounced finding from the qRT-PCR was the marked intra and inter-group variation in gene expression levels, which lead to few statistical significant differences. All three genes were shown to be expressed across a panel of ten tissues analysed, but distinct patterns were also seen. Significantly AvBD1 gene expression in the duodenum indicated that of the 7 day old birds the line X birds reared in the low hygiene environment had the highest level of gene expression. In relation to AvBD4 gene expression, the highest level was observed in the spleen of the 0 day old birds, but overall environment did not appear to affect AvBD4 gene expression of the tissues examined. High levels of AvBD10 gene expression were observed in bird kidney and testicle tissue, but again environment in the case of the former tissue did not appear to statistically affect gene expression levels, the YH 7 day old bird testicle samples did have statistically significant higher expression levels compared to the other groups. The SNP analysis revealed three non-synonymous polymorphisms within the AvBD1 mature peptide locus. The three lines of birds had quite different patterns of these polymorphisms and so three different forms of the peptide along with a single form of the AvBD10 peptide were synthesised using a bacterial hyper-expression system. Peptide levels were quantified using an ELISA and subsequently tested in a bacterial time-kill assay using Salmonella enterica serovar Typhimurium phoP, Staphylococcus aureus and two strains of Enterococcus faecalis. All recombinant peptides showed anti-microbial activity against the bacteria tested. The exception was a clinical isolate of E. faecalis which showed resistance to the killing activities of recombinant AvBD10 peptide. Duodenal gut protein extracts were also tested using the same bacterial assay and marked differences in the anti-microbial activities of these samples were seen. The samples taken from the day 0 birds were found to have significant anti-microbial activity compared to those of the older birds. LC/MS identified differences in the proteomes of the respective gut extracts. These data support that bird genetics, age and the environment have an effect on AvBD gene expression and gut anti-microbial activity. These differences are not uniform for all genes and groups of birds, but clear patterns were observed.