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

急性期反应是先天宿主防御创伤、炎症和感染的核心，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。人们对鸡中APP反应的动态、APP在生产禽群中增加的普遍程度、基线或受刺激APP表达的遗传力以及监控鸡中APP作为禽类健康或抗病育种工具的价值知之甚少。这个项目将为缩小我们知识上的这些差距提供基础。有关家禽APP的有限数量的研究已被报道，在鸡或火鸡的血清淀粉样蛋白A （SAA）、卵黄转铁蛋白（oTFR）、血凝素（Hx）、铜蓝蛋白（Cp）和α -1酸性糖蛋白（AGP）中已被证明是阳性的主要/中度APP。鸡血液中的Haptoglobin （Hp）或其等同物PIT 54也被确定为APP，而白蛋白是阴性APP，因为其浓度在APP反应期间下降。一项初步研究表明，由于细菌学病理而被扑杀的肉鸡血清蛋白发生了变化。补体C3 （CC3）为鸡的中等APP， Cp为鸡的中等APP。本研究的目的是：(1)识别、表征和量化鸡急性期蛋白的病理生理反应；(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，汇集了具有细菌学病理学的肉鸡的血清，将为已知APP的蛋白质组学分析提供样本，并将通过与健康鸡的血清蛋白质组学比较，确定潜在的新型APP。格拉斯哥实验室建立的用于从其他物种中纯化APP的方法将适用于鸡APP的纯化和培养的抗体，以便进行免疫测定。在学习期间开发的免疫分析将通过与单个肉仔鸡的细菌病理和其他急性期炎症或创伤原因诱导条件的病理调查进行比较来验证。APP的非肝合成，已在其他物种中报道，将通过使用鉴定APP的引物进行定量PCR表征。为了实现第二个目标，将由Aviagen（实物贡献）从大约6周龄的生长肉鸡中收集血清样本。将研究两种截然不同的鸡基因系。一个品系（品系A）在遗传上相对抵抗感染，另一个品系（品系B）更容易受到感染。这些鸡将在两种不同的环境中饲养。第一批人口将处于高健康状况的环境中，饲料将免费供应。第二个种群将在Aviagen的挑战环境中饲养，该环境已被开发成模仿英国肉鸡生产设施的较低四分之一。在这里，禽类暴露于较低的营养标准，更大程度的环境疾病挑战，并暴露于毒性更强的商业疫苗。每组100只鸡采集血清样本。幂次计算表明，当n = 100时，有99%的概率检测到初步研究中发现的Cp中位数差异的3%和CC3中位数差异的10%。在这个范围内可以检测到适合作为疾病生物标志物的APP差异。鸡的健康状况将由圣大卫兽医诊所的巴里·索普医生核实，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.