Stereologic sampling and quantitation of Mycobacterium tuberculosis (M.tb.) lesions in rhesus monkey lungs

Abstract

TB is a global infectious killer. Detection systems for TB are not efficient, TB drugs are over 40 years old, and the BCG vaccine dates back over 80 years; hence, there is an urgent need to develop methods to find faster, easier and more effective diagnostics, drugs and vaccines to control TB. Research in nonhuman primate models addresses these needs. Six healthy macaques were intratracheally inoculated with 500 CFU of the clinical M.tb. Erdman strain and followed for disease progression. M.tb. infection was confirmed in all 6 monkeys. At 3 months after inoculation, 2 animals developed significant TB symptoms and were euthanized and necropsied. The remaining 4 animals were necropsied at 6 months. Lungs slices (5 mm thick), overlaid with a Plexiglas template with 5 mm holes, were used to cut cores for the volume of TB lesions and bacterial burden. Lung cores were fixed, embedded, sectioned and stained with H and E. We estimated the volume of TB lesion by stereologic point counting, and homogenates of lung cores were serially diluted and plated to measure CFU. The volume TB lesions and the number of M.tb. CFU were estimated from lung core samples. Our results show that a Plexiglas overlay and circular knives enables a stratified random sampling of TB infected lung tissue in a BSL‐3 environment that produces accurate and efficient estimation of the amount of TB lesions and the number of TB bacteria.NCRR RR000169 and Aeras Foundation