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).

基于胸腔血液的威尔士獾种群代谢分型

Scott Baumann J, Pizzey R, Beckmann M., Villarreal-Ramos B., King J., Hopkins B., Rooke D., Hewinson G., Mur L

方法：
本研究所用獾均为威尔士政府与动物与植物卫生署（Animal and Plant Health Agency, APHA）正在推进的全威尔士獾尸检监测研究（All Wales Badger Found Dead study, AWBFD研究）中，于路边发现的死亡个体。公众上报的獾遗体将被送至实验室，若遗体完整、无胀气、未出现严重蝇蛆病且未经过冷冻，则可开展尸检（post-mortem, PM）。遗体在尸检前的冷藏时长不超过4天。尸检包含外观检查环节：称量体重、测量体长、性别鉴定、基于牙齿磨损程度进行大致年龄评估，以及雌性个体的泌乳状态检查。研究人员会对獾进行微芯片扫描，并通过剪取护毛或提取有色标记物，以追溯其过往的诱捕与疫苗接种史。同时记录所有外部损伤、咬伤、非法诱捕或套猎痕迹。
内部检查重点为识别大体病变，并采集组织样本用于分枝杆菌培养。对心包囊、肺脏、肝脏及肾脏开展详细检查，包括通过多处纵向切口进行内部探查。每一处淋巴结至少切开一次，并制备两份混合样本：第一份混合样本包含咽后淋巴结、支气管淋巴结、纵隔淋巴结及肝淋巴结；第二份混合样本取自任意咬伤部位，或任意疑似结核病的内部可见病变区域。所有被判定为牛结核病（bovine tuberculosis, bTB）阳性的獾，其判定仅基于培养检测结果。本研究仅使用培养检测阴性的獾血液样本。
所有样本均以盲法开展代谢组学处理。每份样本取200 μL，加入1520 μL体积比为4:1的甲醇-氯仿（高效液相色谱级，HPLC grade）混合液，以及50 mg经丙酮洗涤的玻璃微珠（粒径<160 μm，Sigma公司，英国）。将样本涡旋混匀后，于4℃下振荡15分钟，随后静置沉降（-80℃，20分钟），再以1800×g的相对离心力离心10分钟。取100 μL上清液转移至带玻璃内衬的样品瓶中，随机化样本进样顺序以降低批次效应，随后按照Baptista等（2018）的方法，在Exactive™ Orbitrap质谱仪（赛默飞世尔科技，Thermo Scientific）上进行检测。