Cute-Egg, improvement of eggshell cuticle quality to reduce vertical transmission of zoonotic and pathogenic organisms

The poultry industry relies on artificial incubation of eggs to prevent the transfer of micro-organisms from one generation to the next. Despite this the vertical transmission from broiler and layer breeders to production flocks has been identified by the European food safety agency as the most likely route of transfer of antibiotic resistant e-coli and salmonella. There is also the opportunity for transmission of micro-organisms to occur during the collection and transport of eggs. Irrespective of the route or site of transfer, the entry of micro-organisms harmful to the egg contents is undesirable for food safety, animal and human health.

The cuticle is a protein layer which covers the surface of the egg and fills the gas exchange pores in the shell through which the embryo 'breathes'. The cuticle forms the first line of defence to the penetration of bacteria derived both from transmission in the cloaca (where the egg and faeces exit the chicken) and transmission from the environment e.g. contact with collecting belts or handling equipment.

In preliminary studies we have been able for the first time to quantify the cuticle deposition on eggs using a relatively cumbersome two-stage process and estimate the importance of genetics for this trait (heritability, 0.27). This suggested that the trait has good potential for genetic selection to improve it. We also have evidence that the natural variation in cuticle deposition is responsible for differences in the ability of bacteria to penetrate the egg. This demonstrated that eggs with good cuticle deposition were never penetrated by E. coli whereas eggs with poor cuticle quality were frequently penetrated. This means that selection for better cuticle deposition will reduce micro-organism contamination of eggs.

In this proposal we will address the physiochemical, physiological and genetic parameters that characterise the cuticle and use this information to develop a simple one step measurement tool for cuticle assessment using the natural fluorescence of the cuticle. Both the amount and the degree to which the proteins are modified by the addition of sugar residues (glycosylation) will be quantified. In the first instance this will be achieved using a matrix of wavelengths of light both shone on and emitted from the egg surface. This will allow the wavelengths to be selected which can best distinguish the cuticle from potential interfering substances such as feather debri or pigment. A simple to use measurement tool will be built using this information. The amount of modification of the cuticle proteins by sugar residues will be assessed by how they change the rate at which the emission of fluorescent light occurs.

Both measurements will be used to estimate reliably the contribution of genetics to variation in cuticle coverage in meat and egg laying strains of poultry. We will also determine genetic correlations with important production parameters to ensure there are no major negative consequences that might occur when the cuticle is selected for. As well as the contribution of genetics to variation in the trait, we will measure the contribution of environmental influences such as stress, age and how the hormones involved in the expulsion of the egg control cuticle deposition.

In the project we will also characterise how the cuticle and its degree of modification by sugars acts both as a physical and a chemical barrier to bacteria and extend our understanding of the genes involved with cuticle formation and their expression.

Overall this will provide the information required to allow accurate genetic selection for improved cuticle coverage to reduce the risk of pathogens entering the egg. We will also gain fundamental knowledge about the biological mechanisms which give rise to the cuticle and its role as a physical and chemical barrier to microbial penetration.

The cuticle is a glycosylated protein layer which covers the external surface of the egg and fills the gas exchange pores through which the embryo 'breathes'. The cuticle is critical to minimising the risk of vertical and horizontal transmission of pathogenic or zoonotic organisms to the egg contents which is un-desirable for food safety, animal and human health. This project will use physiochemical, physiological and genetic methods to provide the information and tools necessary to make precision genetic selection for improved cuticle quality in both meat and egg laying hens.
The objective of the work is;
1) to use excitation-emission-matrix spectroscopy to determine the parameters for discrimination of the cuticle from background fluorescence and to design and build an instrument that can rapidly and accurately measure cuticle coverage,
2) to apply this direct measurement to estimate reliable heritabilities for cuticle coverage in meat and egg laying strains of poultry and to determine genetic correlations with important production parameters to ensure there are no major negative consequences,
3) to test the hypothesis that (i) moderate levels of stress, (ii) events determining the timing of oviposition and (iii) bird age influences the level of cuticle coverage and to estimate the contribution of these environmental factors to cuticle variation,
4) to develop a method to measure the degree of glycosylation of the cuticle using time resolved fluorescence,
5) to test the hypothesis that the cuticle and its degree of glycosylation acts both as a physical and a chemical barrier to bacteria using penetration and recovery experiments with a range of GFP bacteria,
6) Using animals killed during the transition from shell to cuticle formation and oviposition we will profile the expression of genes to increase our understanding of the control of cuticle formation. We will also specifically measure genes we know from proteomic studies that encode cuticle proteins.

The principle benefit from this research will be a reduction in the transmission of potential pathogens from parent to offspring via the egg of poultry. In particular organisms of enteric origin such as E Coli, Enterococcus faecalis, Yersinia enterocolitica, Clostridium Perfringens all of which reduce growth, health and welfare and are known zoonotics.

This is of benefit to the poultry industry where birds that lay eggs with good cuticles will result in savings to farmers all the way down the production chain with reduced losses and better protection from disease. For E. Coli infection alone it is estimated that the cost for a typical farm is in the region of £360,000 per annum (World Poultry Vol. 25 No. 5, 2009). With continual pressure to remove antibiotics any system that improves natural antimicrobial defence is important.

The proposal also addresses the concerns of governmental agencies such as the European Food Safety Authority which has identified vertical transmission of antibiotic resistant strains of bacteria in poultry as a major source of flock infection and an impediment to creating clean flocks.

The development of a simple tool and its application to breeding poultry will therefore ultimately reduce the risk to consumers and improve the safety and sustainability of poultry production by utilizing the natural antimicrobial defenses of the egg.

The development of methods to measure the cuticle will be of interest to a range of academics involved in the poultry industry and in wild birds and may provide commercial opportunities.
The novel techniques for measuring glycosylation are likely to have interest across biological disciplines and would have wide application beyond that envisaged in the proposal.