Biomarkers of the innate immune response to disease in chickens: acute phase proteins and resistance to disease

急性期反应是针对创伤、炎症和感染的先天宿主防御系统的核心，并且由广泛的全身反应组成，包括肝脏产生急性期蛋白（APP）并分泌到循环中。鸡中APP反应的动力学知之甚少，生产鸡群中APP增加的普遍性、基线或刺激的APP表达的遗传性或监测鸡中APP作为禽类健康或抗病育种工具的产业价值。该项目将为填补我们知识中的这些空白奠定基础。已经报道了与家禽中的APP相关的有限数量的研究，并且在鸡或火鸡中，血清淀粉样蛋白A（SAA）、卵转铁蛋白（oTFR）、血红素结合蛋白（Hx）铜蓝蛋白（Cp）和α-1酸性糖蛋白（AGP）已经显示为阳性的主要/中度APP。也被鉴定为APP，而白蛋白是阴性APP，因为其浓度在APP反应期间下降福尔斯。一项初步研究表明，由于细菌病理学原因，被淘汰的肉鸡血清蛋白质发生了变化。补体C3（CC 3）被鉴定为中度鸡APP，Cp被确认为该物种的APP。本研究的目的是（1）鉴定、鉴定和量化鸡急性期蛋白的病理生理学反应，（2）评估APP血清浓度与健康鸡生产相关的遗传、表型、营养和环境因素之间的关系。为了实现第一个目标，来自具有细菌病理学的肉鸡的合并血清将提供用于通过已知APP的蛋白质组学分析进行确认的样品，并且还将通过与健康鸡的血清蛋白质组进行比较来鉴定潜在的新型APP。在格拉斯哥实验室建立的用于从其他物种中纯化APP的方法将适用于纯化鸡APP和抗体，以允许免疫测定法的开发。在学生期间开发的免疫测定将通过与具有细菌病理学和其他炎症或创伤原因的急性期诱导条件的个体肉鸡的病理学研究进行比较来验证。APP的非肝脏合成（已在其他物种中报告）将通过定量PCR使用已鉴定APP的引物进行表征。为了实现第二个目的，将通过Aviagen（实物捐助）从约6周龄的生长肉鸡中采集血清样品。将研究两种明显不同的鸡遗传系。一个系（系A）在遗传上对感染具有相对抗性，一个系（系B）更易感。鸡将在两个不同的环境中饲养。第一批种群将处于高健康状态环境中，饲料将随意供应。第二个群体将在安伟捷的挑战环境中饲养，该环境已被开发为模拟英国肉鸡生产设施的下四分之一。在这里，鸟类暴露于较低的营养标准，更高水平的环境疾病挑战，并暴露于更具毒性的商业疫苗。将从每组100只鸡中采集血清样品。的把握度计算表明，n = 100时，有99%的概率检测到Cp初步研究中发现的中位数差异为3%，CC 3中发现的中位数差异为10%。适合作为疾病生物标志物的APP的差异在该范围内是可检测的。鸡的健康状况将由圣大卫兽医诊所的巴里索普博士核实，他将检查鸡群的记录和健康史。将对每种环境中的一部分鸟类进行尸检。

The acute phase response is central to the innate host defence system against trauma, inflammation and infection and is composed of a wide range of systemic reactions including the production by the liver of acute phase proteins (APP) and secretion into the circulation. Little is known of the dynamics of the APP response in chickens, the prevalence of increased APP in production flocks the heritability of baseline or stimulated APP expression or the value to the industry of monitoring APP in chicken either as a tool for bird health or for breeding for disease resistance. This project will provide the groundwork to close these gaps in our knowledge. A limited number of investigations relating to APP in poultry have been reported and in chickens or turkeys serum amyloid A (SAA), ovotransferrin (oTFR), hemopexin (Hx) ceruloplasmin (Cp) and alpha-1 acid glycoprotein (AGP) have been shown to be positive major/moderate APP. Haptoglobin (Hp) or its equivalent in chicken blood, PIT 54, has also been identified as an APP while albumin is a negative APP as its concentrations falls during the APP response. A pilot study has shown changes in serum proteins changes in broilers culled due to bacteriological pathology. Complement C3 (CC3) was identified as a moderate chicken APP and Cp was confirmed as an APP in this species. The objective of this studentship is (1) to identify, characterise and quantify the pathophysiological responses of the chicken acute phase proteins and (2) to assess the relationship between the APP serum concentrations and genetic, phenotypic, nutritional and environmental factors related to production of healthy chicken. To achieve the first objective pooled serum from broilers with bacteriological pathology will provide the sample for confirmation by proteomic analysis of known APP and will also identify potential novel APP by comparison to serum proteome of healthy chickens. Methods, established in the Glasgow laboratory for purification of APP from other species will be adapted for purification of the chicken APP and antibody raised to allow immunoassay development. Immunoassays developed during the studentship will be validated by comparison to pathological investigation of individual broilers with bacterial pathology and other acute phase inducing conditions of inflammatory or traumatic causation. Non-hepatic synthesis of APP, which has been reported in other species, will be characterised by quantitative PCR using primers for identified APP. To achieve the second objective, serum samples will be collected, by Aviagen (contribution in kind) from growing meat chickens of approximately 6 weeks of age. Two distinctly different genetic lines of chickens will be studied. One line (Line A) is genetically relatively resistant to infection and one (Line B) is more susceptible. The chickens will be reared in 2 different environments. The first population will be in a high health status environment and feed will be supplied ad libitum. The second population will be reared in Aviagen's challenge environment which has been developed to mimic the lower quartile of UK broiler production facilities. Here the birds are exposed to a lower standard of nutrition, a greater level of environment disease challenge and exposed to more virulent commercial vaccines. Serum samples will be collected from 100 chickens in each group. A power calculation of the indicated that with n = 100, there is a 99% probability of detecting 3% of the difference between medians found in the pilot study for Cp and 10% of the difference found for CC3. Differences in APP that are suitable as biomarkers of disease would be detectable within this range. The health status of the chickens will be verified by Dr Barry Thorp of the St David Vet Practice who will check flock records and health history of the flocks. Post mortem examination will be performed on a subset of the birds in each environment.