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

库蠓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贝格病毒以及潜在的其他新出现威胁的媒介。本研究测试了以下与减少库蠓传播的虫媒病毒影响有关的假设。1. 库蠓成虫在动物舍内的存在对虫媒病毒的越冬和传播起着重要作用，但可以通过防媒介住宿部分解决。2. 可以利用对媒介唾液蛋白过度暴露的免疫反应来降低吸血库蠓的存活率，并具有未来疫苗设计的潜力。该学生将研究英国皮尔布赖特研究所（TPI：学术领导）和APS生物防治（工业合作伙伴）已经建立的牛场中的库蚊种群。将采用标准化的吸盘和真空调查方法调查库蠓成虫在动物舍内的冬季存活情况。这种直接抽样的结果将与TPI已经进行的基于外部光吸陷阱抽样的季节性丰度模型进行比较。为了提高我们对动物住房生物安全的了解，我们将在实验室使用行为分析来检查基于紫外线的陷阱和空气幕的功效，以补充TPI已经完成的杀虫剂浸渍屏风的测试。在随后的两个野外季节，即库蠓携带的虫媒病毒传播高峰时，将在选定的地点努力通过实施优化程序，并通过库蠓丰度与外部采集的库蠓丰度评估生物遏制程度，将库蠓排除在住房之外。该学生还将研究基于宿主对非自然高剂量节肢动物唾液反应的疫苗开发。与莫尔顿研究所的Alasdair Nisbet博士合作的初步数据表明，给绵羊接种库蠓提取物可以提高血清抗体水平。与未接种疫苗的对照组相比，以接种疫苗后绵羊的血液为食可降低库蠓的存活率。最初将筛选自然暴露的牛羊血清中的库蠓抗体水平，评估暴露的季节性和库蠓物种之间唾液蛋白交叉识别的可能程度。采样将比较已知以特定库蠓动物群为主的地区的宿主，以及从菌落饲养系中纯化的唾液蛋白与现场收集的同源物种提取。保守的免疫原性库蠓唾液蛋白将通过western blot检测和蛋白质组学鉴定。然后用纯化库蠓唾液蛋白对绵羊进行疫苗接种试验，分析免疫反应（例如血清抗体的滴度和同型以及细胞因子的潜在存在）。细胞介导的免疫反应将通过增殖试验和体外暴露于唾液蛋白（联合和分离）的接种羊外周血细胞的细胞因子产生来检查。然后将统计地评估以接种疫苗和对照羊为食的库蠓的实验室存活率和繁殖率。学生实习将持续三个月，目的是利用Alison Blackwell博士（APS Biocontrol）对该物种的专业知识，设计出从impunctatus收集唾液的技术。这将为项目中的所有合作伙伴提供重要的资源，并且由于合作伙伴之间的地理距离，APS总部位于苏格兰。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年蓝舌病病毒入侵期间实施行动限制时讨论的主要领域。

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.