Metabotyping the Welsh population of badgers based on thoracic blood

Metabotyping the Welsh population of badgers based on thoracic blood Scott Baumann J, Pizzey R, Beckmann M., Villarreal-Ramos B., King J., Hopkins B., Rooke D., Hewinson G., Mur L Methods: The badgers involved in this study where collected dead on the side of the road as part of an ongoing surveillance study by Welsh Government and the Animal Plant Health Agency (APHA), the All Wales Badger Found Dead study (AWBFD study). Badgers reported by members of the public are collected and brought to a laboratory where they are deemed appropriate for post-mortem (PM) if they are intact, not distended with gas, with no severe myiasis and have not been frozen. Carcasses spend no more than four days in cold storage before PM. PM involves an external examination; including, weighing, measuring, sexing, approximate aging based on dental wear, and checking for lactation if female. Badgers were scanned for microchips as well as clipping of guard hairs or any colour marker to indicate historical trapping and vaccination. Any signs of external injury, bite wounds, illegal trapping or snaring are also noted. Internal examination focussed on identification of any gross lesions and sampling of tissues for mycobacterial culture. Detailed examination is made of the pericardial sac, lungs, liver and kidneys including internally by making several, longitudinal incisions across each. Each lymph node is incised at least once and two pools of samples material are created. Pool one contains retropharyngeal, bronchial lymph nodes, mediastinal and hepatic lymph nodes. Pool two contains a section of any bite wound or any internal visible lesions suggestive of tuberculosis. All badgers designated as bTB positive were done so based solely on the results of culture. For this study only culture negative badger blood samples were used. All samples were processed for metabolomics in a blinded manner. 200 L of each sample were added to 1520 L of 4:1 (v/v) mix of MeOH:chloroform (HPLC grade) and 50 mg of acetone-washed glass beads (< 160 g, Sigma, UK). These were vortexed, shaken (15 min at 4 °C), left to settle (-80 °C for 20 min), then centrifuged (1800 × g for 10 min). 100 L of the supernatant was transferred to a glass vial with glass insert, sample injection order was randomised to reduce batch effects and run on an ExactiveTM Orbitrap Mass Spectrometer (Thermo Scientific) as described in Baptista et al. (2018).基于胸腔血液对威尔士獾种群进行代谢分型 Scott Baumann J, Pizzey R, Beckmann M., Villarreal-Ramos B., King J., Hopkins B., Rooke D., Hewinson G., Mur L 方法：本研究涉及的獾均为威尔士政府与动植物卫生检疫局（Animal and Plant Health Agency, APHA）开展的持续监测项目——全威尔士死亡獾监测研究（All Wales Badger Found Dead study, AWBFD研究）中，于路边收集的死亡个体。公众上报的獾遗体将被送至实验室，若遗体完整、无气体膨胀、无严重蝇蛆病且未经过冷冻，则符合尸检（post-mortem, PM）标准。尸检前，遗体在冷藏环境中的存放时长不超过4天。 尸检流程包含外部检查：包括称重、体长测量、性别鉴定、基于牙齿磨损程度的大致年龄评估，以及雌性个体泌乳情况检查。同时对獾进行微芯片扫描，并剪下护毛或识别其身上的颜色标记，以追溯其过往的诱捕与疫苗接种史。此外，研究人员还会记录所有外部损伤、咬伤伤口、非法诱捕或套索捕猎的迹象。 内部检查重点为识别大体病理病变，并采集组织样本用于分枝杆菌培养。研究人员将详细检查心包囊、肺脏、肝脏与肾脏，具体操作是对每个器官做多处纵向切口。每一处淋巴结至少切开一次，并制备两份样本混合池：混合池1包含咽后淋巴结、支气管淋巴结、纵隔淋巴结及肝淋巴结；混合池2包含任意咬伤伤口的组织，或任何疑似结核病的内部可见病变组织。 所有被判定为牛结核病（bTB）阳性的獾，仅依据培养检测结果确定。本研究仅使用培养结果为阴性的獾血液样本。所有样本均以盲法开展代谢组学处理。 取每份样本200 μL，加入1520 μL体积比为4:1（v/v）的甲醇:氯仿（高效液相色谱级）混合液，以及50 mg丙酮洗涤玻璃微珠（粒径<160 μm，英国Sigma公司）。将样本进行涡旋振荡、摇荡处理（4 ℃下持续15分钟），随后静置沉降（-80 ℃下20分钟），再以1800 × g的离心力离心10分钟。取100 μL上清液转移至带玻璃内插管的玻璃进样瓶中，随机化样本进样顺序以降低批次效应，并按照Baptista等人（2018）所述的方法，在Exactive™轨道阱质谱仪（赛默飞世尔科技）上完成检测。