Behavioural and immunological control methods for Culicoides biting midges in the UK

库蚊叮咬蚊子是欧洲反刍动物虫媒病毒最重要的生物媒介，在欧洲，它们是蓝舌病和施马伦堡病毒以及其他潜在威胁的媒介。这项研究测试了以下假设，这些假设与减少库蚊虫媒病毒的影响有关。1.成虫在动物舍内的存在对虫媒病毒的越冬和传播具有重要作用，但部分可通过防止媒介传播来解决。2.对载体唾液蛋白过度暴露的免疫学反应可被利用来降低嗜血库蚊的存活率，并具有未来疫苗设计的潜力。这名学生将研究皮尔布赖特研究所(TPI：学术领先)和APS BioControl(工业合作伙伴)已经建立的英国养牛场的库蚊种群。将使用标准化的吸力诱捕器和真空调查来调查成虫在动物舍内的越冬存活率。这次直接采样的结果将与TPI已经进行的基于外部光吸力陷阱采样的季节性丰度模型进行比较。为了更好地了解动物舍内的生物安全，随后将在实验室使用行为分析来检验基于紫外线的捕集器和空气幕的有效性，以补充已经在TPI完成的杀虫剂浸渍滤网的测试。在接下来的两个野外季节，在库蚊传播虫媒病毒的高峰期，将在选定的地点通过实施优化的程序和通过库蚊与外部收集的库蚊的丰度来评估生物遏制程度来努力将库蚊排除在房屋之外。学生还将根据宿主对非自然高剂量节肢动物唾液的反应来审查疫苗的开发。与莫雷登研究所的Alasdair Nisbet博士合作的初步数据表明，给绵羊接种库蚊提取物会产生更高水平的血清抗体。与未接种疫苗的对照组相比，以接种后绵羊的血液为食可降低库蚊的存活率。最初将对自然暴露的牛和羊的血清进行抗库蚊抗体水平的筛选，以评估暴露的季节性和库蚊物种之间可能出现的唾液蛋白交叉识别程度。抽样将比较已知以特定库蚊动物群和从群体饲养的品系中提纯的唾液蛋白为主的地区的宿主与现场收集的同源物种提取物。样本中保守的免疫原性库蚊唾液蛋白将通过蛋白质印迹分析和蛋白质组学进行鉴定。然后用纯化的库蚊唾液蛋白对绵羊进行免疫试验，分析免疫反应(例如血清抗体的滴度和同型(S)以及潜在的细胞因子的存在)。细胞介导的免疫反应将通过体外暴露于免疫绵羊的外周血细胞对唾液蛋白(合并和分离)的增殖分析和细胞因子产生来检测。然后，对接种疫苗的绵羊和对照绵羊血液喂养的库蚊的实验室存活率和繁殖率进行统计评估。学生安置将持续三个月，旨在利用艾莉森·布莱克韦尔博士(APS生防专家)对该物种的专业知识，设计出收集Imimpunctatus唾液的技术。这将为项目中的所有合作伙伴提供重要的资源，并通过合作伙伴之间的地理距离实现，APS的总部设在苏格兰。此外，该学生还将与APS团队合作，评估英国遏制设施的商业潜力，这是2007年实施行动限制时蓝舌病毒入侵期间的主要讨论领域。

Culicoides biting midges are the most important biological vectors of arboviruses of ruminants in Europe, where they act as vectors of bluetongue and Schmallenberg viruses and potentially other emerging threats. This studentship tests the following hypotheses which are relevant to reducing the impact of Culicoides-borne arboviruses. 1. The presence of adult Culicoides in animal housing has a major role in overwintering and spread of arboviruses, but can be addressed in part through vector-proof accommodation. 2. Immunological responses to over-exposure of vector salivary proteins can be harnessed to reduce the survival of bloodfeeding Culicoides with future potential for vaccine design. The student will examine Culicoides populations at cattle holdings in the UK already established by The Pirbright Institute (TPI: Academic lead) and APS Biocontrol (Industrial Partner). Winter survival of adult Culicoides within animal housing will be investigated using standardised suction trap and vacuum surveys. Results from this direct sampling will be compared with models of seasonal abundance based on outside light-suction trap sampling already conducted by TPI. To improve our understanding of biosecurity in animal housing, the efficacy of ultraviolet-based traps and air curtains will then be examined using behavioural assays in the laboratory to complement testing of insecticide impregnated screens already completed at TPI. In the subsequent two field seasons, at time of peak transmission of Culicoides-borne arboviruses,efforts will be made across selected sites to exclude Culicoides from housing through implementation of optimized procedures and the degree of bio-containment assessed by the abundance of Culicoides versus outside collections.The studentship will also examine development of vaccines based on responses of hosts to unnaturally high doses of arthropod saliva. Preliminary data in collaboration with Dr Alasdair Nisbet of the Moredun Institute demonstrated that inoculated of sheep with a Culicoides extract produce elevated levels of serum antibodies. Feeding on the blood of postvaccination sheep reduces survival in Culicoides when compared to unvaccinated controls. Serum from naturally exposed cattle and sheep will initially be screened for anti-Culicoides antibody levels, assessing the seasonality of exposure and the likely degree of saliva protein cross-recognition between Culicoides species. Sampling will compare hosts in areas known to be dominated by specific Culicoides fauna and saliva proteins purified from colony-reared lines with homologous field-collected species extractions. Conserved immunogenic Culicoides saliva proteins across samples will be identified by western blot assays and proteomics. A vaccination trial of sheep with purified Culicoides saliva proteins will then analyse immunological responses (e.g. titre and isotype(s) of serum antibodies and the potential presence of cytokines). Cell mediated immune responses will be examined using proliferation assays and cytokine production of in vitro exposed peripheral blood cells from vaccinated sheep to saliva proteins (combined and fractionated). Laboratory survival and fecundity rates of Culicoides blood-fed on vaccinated and control sheep will then be statistically assessed. The student placement will last three months and will aim to devise techniques for the collection of saliva from C.impunctatus, utilizing the expertise of Dr Alison Blackwell (APS Biocontrol) with this species. This will provide a significant resource for all collaborating partners in the project and is enabled by geographic distance between the partners with APS being based in Scotland. In addition, the student will also work with the APS team to estimate commercial potential for containment facilities in the UK which was a major area of discussion during the 2007 incursion of bluetongue virus when movement restrictions were implemented.