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).