Necropsy and Tuberculosis Lesion Analysis

Necropsy was performed as previously described [17 (link),24 (link),25 (link),29 (link)]. Briefly, an ¹⁸F-FDG PET-CT scan was performed on every animal 1–3 days prior to necropsy to measure disease progression and identify individual granulomas as described [29 (link)]. At necropsy, monkeys were maximally bled and humanely sacrificed using pentobarbital and phenytoin (Beuthanasia; Schering-Plough, Kenilworth, NJ). Individual lesions previously identified by PET-CT and those that were not seen on imaging from lung and mediastinal lymph nodes were obtained for histological analysis, bacterial burden, and immunological studies [29 (link)]. A veterinary pathologist described gross pathologic findings. To quantify gross pathologic disease (disease burden), a necropsy score was developed in which points were given for TB disease: number, size, and pattern of granulomas distributed in each lung lobe and mediastinal lymph node and in other organs each lung lobe, lymph node, and visceral organ were included and enumerated, and an overall score was determined as previously described [25 (link)]. The size of each granuloma was measured at necropsy and by pre necropsy scan [69 ]. Representative sections of each tissue were homogenized into single-cell suspensions for immunologic studies, flow cytometric analysis, and bacterial burden, as previously described [17 (link),24 (link),26 (link),65 (link)].