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

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description Establishment of a number of methods to potentially measure cuticle coverage on eggs in a high throughput manner. These have been tested on a number of lines of hen. One method is emerging as the most practical. We have also established basic facts about the deposition of the cuticle which we can manipulate to be absent or present by endocrine tools.
Genetic parameters so far are extremely promising in particular in all the lines examined including meat type hens. In the brown egg laying hen cuticle deposition at the beginning of lay is predictive of the hens deposition of cuticle nearer the end of lay. This is important as it means only one measurement is required.
Most gratifyingly in all the lines tested the better the cuticle the less bacteria penetrate the egg. This strongly supports the hypothesis that selecting hens for better cuticle will minimise the chances of bacterial contamination of the egg contents.

More specifically in relation to knowledge on the nature of the cuticle;
The cuticle is a unique oviduct secretion that protects avian eggs from bacterial penetration through gas exchange pores. Despite its importance, experimental evidence is lacking for assertions on where, when and what is responsible for its deposition. By using knowledge about the ovulatory cycle and its relationship to oviposition we have been able to manipulate cuticle deposition to get evidence on these key points. Cuticle deposition was measured using staining and spectrophotometry. Experimental evidence supports the shell gland pouch (uterus) of the oviduct as the location of cuticle deposition and not the vagina and this occurs within the final hour before oviposition. Oviposition induced by arginine vasotocin or prostaglandin, the penultimate and ultimate factors for the induction of oviposition produces an egg with no cuticle, therefore these factors are not responsible for cuticle secretion. Conversely oviposition induced by GnRH, which mimics the normal events of ovulation and oviposition, results in a normal cuticle. There is no evidence that cuticle deposition differs at the end of a clutch and therefore there is no evidence that the ovulatory surge of progesterone effects cuticle deposition. Overall the results demonstrate that the cuticle is a specific secretion and is not contiguous with the organic matrix. It was demonstrated that a stressor reduced cuticle deposition and whilst the cuticle probably contains some of the pigment that give eggs their colour there is no absolute inter-dependence of pigment and cuticle. Defining the basic facts surrounding cuticle deposition will aid efforts to reduce contamination of laid eggs.
Exploitation Route Lessons learned from use of the prototypes will be incorporated in further versions to improve measurement and use. A prototype commercial device has been built and tested using that knowledge. It is hoped this practical tool will be used to further validate the conclusions and help breeders to improve biosecurity. Current indications are that breeders are going to invest in the technology and try it on a larger scale to validate its usefulness.

The observations on the cuticle deposition indicate that an oviposition produced by AVT does not result in a cuticle being formed . This demonstrates that the cuticle is a specific layer which is added just prior to oviposition and it is not simply contiguous with the shell matrix. We have ruled out a number of factors in the normal process of inducing an ovulation/oviposition that do not stimulate secretion of the cuticle but we have not yet identified what does prompt its deposition. However the methods developed do give us powerful tools to dissect this in the future.
The genetic parameters suggest that for all lines of hen tested the method can be adopted to improve cuticle and reduce contamination. There seems to be no major negative correlations. So the method has, we believe, universal application.
Sectors Agriculture, Food and Drink

URL https://academic.oup.com/biolreprod/article/97/1/39/3920755
 
Description Measurements of estimate genetic parameters have been made for 2 layer and one broiler line. The results indicated that we can select for cuticle in all lines and that one measurement in the life of a hen was adequate to estimate its genetic potential. We now have a clear idea of how we need to implement a quantitative measurement of the cuticle for practical use. We have produced a machine with high throughput which should facilitate the ultimate implementation in breeding programmes. We are continuing to work with the companies to overcome any obstacles to implementation. However all signs are that a major effort is going to be made to incorporate this measure, so we should succeed in our efforts to reduce vertical transmission of micro-organisms between generations. The other major output has been a greater understanding and clarification of cuticle deposition physiology. Specifically the timing of the deposition, its susceptibility to environmental factors, the reliance on hormonal signals and the precise location of deposition, most of these points were not proven in the literature before we started. This will help the research community to design further experiments to characterise the nature and the mechanisms of cuticle deposition.
First Year Of Impact 2015
Sector Agriculture, Food and Drink
Impact Types Societal,Economic

 
Title High speed integrated spectrophotometer and analysis tool, eCutimeter 
Description Custom designed system for the rapid assessment of cuticle deposition using spectrophotometry. The final 'production' prototype was capable of rapid measurement across a range of wavelengths but was designed principally for high throughput reflectance measurement of eggs which would be used for cuticle measurement by staining. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact The machine is in trial at breeding organisations with a view to assessing its incorporation into breeding practice. 
 
Title prototype egg cuticle measurement device 
Description Device to hold rotate and measure surface fluorescence of an egg 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Ability to conduct measurements for genetic evaluation . 
 
Description Improving egg quality; win - win traits 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presentation to breeders and geneticists from main breeding companies . X European Symposium on Poultry Genetics
Year(s) Of Engagement Activity 2017
URL http://www.wpsa.com/index.php/publications/wpsa-proceedings/2017/x-european-symposium-on-poultry-gen...
 
Description AUSTRALIAN POULTRY SCIENCE SYMPOSIUM 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Review of the challenges for the laying hen industry in improving sustainability. Published on line with international reach, reviewed

INCREASING PERSISTENCY IN LAY AND STABILIZING EGG QUALITY IN LONGER LAYING CYCLES - WHAT ARE THE CHALLENGES?
Year(s) Of Engagement Activity 2015
URL http://sydney.edu.au/vetscience/apss/documents/2015/APSS%202015%20Proceedings%20Final.pdf
 
Description Activity for Farm Sunday 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Produced a kit and advertising material to educate about eggs, science and its application. We hope to use this in interaction with schools and other engagement activities. Was distributed to farms we don't know the feedback although when run at our open days it was very favourable.

http://www.roslin.ed.ac.uk/CuteEgg/
Year(s) Of Engagement Activity 2016
URL https://farmsunday.org/open-my-farm/science-and-technology
 
Description BUAS Schools Countryside Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Primary 5 children and teachers from the entire Scottish Borders region. Ran activities around the cuticle and its importance.
Year(s) Of Engagement Activity 2017
URL http://www.buas.org/calendar/buas_schools_countryside_day_23_may_17.html
 
Description CUTE EGG: PROVIDING A SAFE SUPPLY OF EGGS TO HATCH THE NEXT GENERATION OF CHICKENS 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Article on Cute egg featured in LOHMANN TIERZUCHT "Poultry News". Reaches huge number of farmers across the world
Year(s) Of Engagement Activity 2017
URL http://www.ltz.de/de-wAssets/docs/poultry-news/2017/Poultry-News-02-2017.pdf
 
Description CUTICLE DEPOSITION DOES NOT AFFECT WATER VAPOUR CONDUCTANCE AND CAN BE MEASURED IN HATCHING EGGS WITHOUT COMPROMISING EMBRYO DEVELOPMENT 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presentation to specialists in egg science, breeders and state veterinarians, an industry Science meeting. XVIIth European Symposium on the Quality of Eggs and Egg Products, XXIIIth European Symposium on the Quality of Poultry Meat. Increases awareness across Europe of applications in poultry science.
Year(s) Of Engagement Activity 2017
URL http://www.wpsa.com/index.php/publications/wpsa-proceedings/2017/xxiii-european-symposium-on-the-qua...
 
Description CUTICLE DEPOSITION ENHANCES THE EGGS NATURAL DEFENCE AND IS A TRAIT WHICH DOES NOT TEND TO DECREASE WITH BIRD AGE 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presentation to specialists in egg science, breeders and state veterinarians, an industry Science meeting. XVIIth European Symposium on the Quality of Eggs and Egg Products, XXIIIth European Symposium on the Quality of Poultry Meat. Increases awareness across Europe of applications in poultry science.
Year(s) Of Engagement Activity 2017
URL http://www.wpsa.com/index.php/publications/wpsa-proceedings/2017/xxiii-european-symposium-on-the-qua...
 
Description CUTICLE QUALITY GENETICS IN LAYERS AND BROILERS 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presentation to specialists in egg science, breeders and state veterinarians, an industry Science meeting. XVIIth European Symposium on the Quality of Eggs and Egg Products, XXIIIth European Symposium on the Quality of Poultry Meat. Increases awareness across Europe of applications in poultry science.
Year(s) Of Engagement Activity 2017
URL http://www.wpsa.com/index.php/publications/wpsa-proceedings/2017/xxiii-european-symposium-on-the-qua...
 
Description OPEN day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact To introduce our work on the sustainability of poultry and egg production to a wider audience. We had a number of activities which introduced the role of the cuticle in preventing bacterial penetration of the egg. For many people this was a revelation. It allowed an opening to discussions on modern farming and to challenge many misconceptions. There was also a lot of hands on opportunities. The event was so successful that we have developed it into a grant for building kits for using at farm visits.
Year(s) Of Engagement Activity 2016
URL http://www.bbsrc.ac.uk/news/people-skills-training/2016/160107-pr-farm-science-at-open-farm-sunday/
 
Description Poster presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Poster presentation at an RSC local Photophysics meeting, Newcastle

Interest in novel application
Year(s) Of Engagement Activity 2015
 
Description Presentation at World Poultry Congress (Beijing) Cuticle 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Largest Poultry science conference >4000 people. 50% industry Interest engendered from breeding companies.
Year(s) Of Engagement Activity 2016
URL http://www.wpc2016.cn/uploadfiles/2016-9/The%20Proceedings%20of%20WPC2016-Abstracts-20160920.pdf
 
Description Presentation to the European Egg Quality meeting. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact XXII European Symposium on the Quality of Poultry Meat and the XVI European Symposium on the Quality of Eggs and Egg Products Eggmeat2015, Nantes (France), May, 10 to 13, 2015

Science/industry meeting on quality of eggs. Good links to encourage uptake.
Year(s) Of Engagement Activity 2015
URL http://www.eggmeat2015.com/home/index.php/the-program/tuesday-12-may-program
 
Description Science festivals 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact As a result of a Beltane grant and workshop an activity was built up to engage the public including children in the research. Specifically to understand the role of the cuticle in keeping bacteria out of the egg (in a fun way!).
This was used at a number of different science festival events

Developed an interactive tool to explain the research aim which can be used in the future.
Year(s) Of Engagement Activity 2014
URL http://scoffingscottishscience.com/about/