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

在欧洲，库蠓叮咬蠓是反刍动物虫媒病毒最重要的生物载体，在那里它们作为蓝舌病和施马伦贝格病毒以及潜在的其他新兴威胁的载体。本研究旨在检验以下与减少库蠓传播虫媒病毒影响相关的假设。1.动物房中库蠓成虫的存在对虫媒病毒的越冬和传播起着重要作用，但可以通过防病媒住宿部分解决。2.免疫反应过度暴露的载体唾液蛋白可以利用，以减少生存的吸血库蠓与未来的疫苗设计的潜力。学生将检查库蠓种群在英国的牛持有已经建立了由Pirbright研究所（TPI：学术带头人）和APS生物控制（工业合作伙伴）。将使用标准化的吸力诱捕器和真空调查研究动物舍内成年库蠓的冬季存活率。直接采样的结果将与TPI已经进行的基于外部光吸捕集器采样的季节性丰度模型进行比较。为了提高我们对动物舍内生物安全的理解，然后将在实验室中使用行为分析来检查紫外线陷阱和空气幕的功效，以补充TPI已经完成的杀虫剂浸渍屏障的测试。在接下来的两个季节里，在库蠓传播虫媒病毒的高峰期，将在选定的地点进行努力，通过实施优化的程序和通过库蠓与外部收集的丰度评估的生物遏制程度，将库蠓排除在住房之外。该学生奖学金还将研究基于宿主对不自然的高剂量节肢动物唾液的反应的疫苗开发。与Moredun研究所的Alasdair Nisbet博士合作的初步数据表明，用库蠓提取物接种绵羊会产生血清抗体水平升高。与未接种疫苗的对照组相比，饲喂接种后绵羊的血液会降低库蠓的存活率。最初将对自然暴露的牛和羊的血清进行抗库蠓抗体水平筛查，评估暴露的季节性和库蠓种属之间唾液蛋白交叉识别的可能程度。采样将比较主机在已知的地区占主导地位的特定库蠓动物群和唾液蛋白纯化从殖民地饲养线同源现场收集的物种提取物。将通过蛋白质印迹试验和蛋白质组学鉴定样品中保守的免疫原性库蠓唾液蛋白。用纯化库蠓唾液蛋白对绵羊进行疫苗接种试验，然后分析免疫应答（例如血清抗体的滴度和同种型以及细胞因子的潜在存在）。将使用增殖试验和体外暴露于唾液蛋白（组合和分级）的接种绵羊外周血细胞的细胞因子产生来检查细胞介导的免疫应答。然后对接种和对照绵羊上吸血库蠓的实验室存活率和繁殖率进行统计学评估。学生实习将持续三个月，旨在设计收集C.impunctatus唾液的技术，利用Alison Blackwell博士（APS Biocontrol）对该物种的专业知识。这将为项目中的所有合作伙伴提供重要的资源，并且由于合作伙伴之间的地理距离，APS总部设在苏格兰。此外，学生还将与APS团队合作，估计英国遏制设施的商业潜力，这是2007年蓝舌病病毒入侵期间实施行动限制时讨论的一个主要领域。

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.