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

家禽业依靠鸡蛋的人工孵化来防止微生物从一代转移到下一代。尽管如此，从肉鸡和蛋鸡饲养者到生产禽群的垂直传播已被欧洲食品安全局确定为最有可能的抗生素耐药性大肠杆菌和沙门氏菌转移途径。在收集和运输鸡蛋的过程中也有可能发生微生物的传播。无论转移的途径或地点如何，对鸡蛋内容物有害的微生物的进入对食品安全、动物和人类健康都是不可取的。角质层是覆盖在卵子表面的一层蛋白质，它填满了蛋壳上的气体交换孔，胚胎通过这些孔“呼吸”。角质层是防止细菌渗透的第一道防线，这些细菌来自泄殖腔（鸡蛋和粪便离开鸡的地方）的传播，以及来自环境的传播，例如接触收集带或处理设备。在初步研究中，我们首次能够使用一个相对繁琐的两阶段过程来量化鸡蛋上的角质层沉积，并估计遗传对这一性状的重要性（遗传率，0.27）。这表明该性状具有良好的遗传选择潜力。我们也有证据表明，角质层沉积的自然变化是细菌穿透卵子能力差异的原因。这表明表皮沉积良好的鸡蛋从未被大肠杆菌穿透，而表皮质量差的鸡蛋却经常被穿透。这意味着选择更好的角质层沉积将减少鸡蛋的微生物污染。在本提案中，我们将解决表征角质层的理化，生理和遗传参数，并利用这些信息开发一个简单的一步测量工具，用于角质层的自然荧光评估。通过添加糖残基（糖基化）对蛋白质进行修饰的量和程度将被量化。在第一个实例中，这将通过使用照射在鸡蛋表面和从鸡蛋表面发射的光波长矩阵来实现。这将允许波长的选择，可以最好地区分角质层和潜在的干扰物质，如羽毛碎片或色素。使用这些信息将构建一个简单易用的测量工具。糖残基对角质层蛋白的修饰量将通过它们如何改变荧光发射的速率来评估。这两项测量将用于可靠地估计遗传对家禽产肉和产蛋菌株角质层覆盖变异的贡献。我们还将确定与重要生产参数的遗传相关性，以确保在选择角质层时不会发生重大的负面后果。除了遗传对性状变异的贡献外，我们还将测量环境影响（如压力、年龄）的贡献，以及参与卵子排出的激素如何控制角质层沉积。在该项目中，我们还将描述角质层及其被糖修饰的程度如何作为细菌的物理和化学屏障，并扩展我们对角质层形成及其表达相关基因的理解。总的来说，这将提供所需的信息，以允许精确的遗传选择，以改善角质层覆盖，以减少病原体进入卵子的风险。我们还将获得关于产生角质层的生物机制及其作为微生物渗透的物理和化学屏障的作用的基本知识。

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.