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

急性时相反应是机体抵抗创伤、炎症和感染的先天防御系统的核心，由一系列的全身反应组成，包括肝脏产生急性时相蛋白(APP)和分泌到循环中。人们对APP在鸡中的反应动态知之甚少，APP在生产群中的流行程度，基线或刺激APP表达的遗传力，或监测鸡肉APP作为禽类健康工具或用于抗病育种的行业价值。这个项目将为弥合我们知识上的这些差距提供基础。在家禽中与APP相关的研究有限，在鸡或火鸡中，血清淀粉样蛋白A(SAA)、卵转铁蛋白(OTFR)、血凝蛋白(Hx)、铜蓝蛋白(CP)和α-1酸性糖蛋白(AGP)被证明是主要/中度APP。鸡血中的结合珠蛋白(HP)或其等价物PIT 54也已被鉴定为APP，而白蛋白则是阴性APP，因为其浓度在APP响应期间会下降。一项初步研究表明，由于细菌学病理学的原因，被扑杀的肉鸡血清蛋白的变化。补体C3(CC3)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、表征和量化鸡急性时相蛋白的病理生理反应；(2)评估APP血清浓度与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，来自细菌学病理的肉鸡的混合血清将为已知APP的蛋白质组分析提供证实的样本，并将通过与健康鸡的血清蛋白质组比较来鉴定潜在的新APP。在格拉斯哥实验室建立的用于从其他物种中纯化APP的方法将被适应于纯化鸡肉APP和提高抗体以允许免疫分析开发。在学员期间开发的免疫分析将通过与个别肉鸡的病理检查进行比较来验证，这些肉鸡有细菌病理和其他急性阶段炎症或创伤原因的诱导条件。在其他物种中已有报道的APP的非肝脏合成，将通过使用已识别的APP的引物进行定量PCR来表征。为了实现第二个目标，将由Aviagen(实物捐助)收集大约6周龄的生长肉鸡的血清样本。我们将研究两种截然不同的鸡遗传品系。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)更敏感。这些鸡将在两个不同的环境中饲养。第一批人口将处于高健康状况的环境中，并将临时提供饲料。第二个种群将在Aviagen的挑战环境中饲养，该环境已被开发为模仿英国肉鸡生产设施的较低四分之一。在这里，鸟类暴露在较低的营养标准下，面临更高水平的环境疾病挑战，并暴露在更具毒性的商业疫苗中。每组将从100只鸡身上采集血清样本。功率计算表明，当n=100时，对于CP，有99%的概率检测到在试点研究中发现的中位数之间的差异3%，对于CC3有10%的差异。在这个范围内，可以检测到适合作为疾病生物标志物的APP的差异。这些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历史。将对每种环境中的一组鸟类进行尸检。

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.