Stereological analysis of bacterial load and lung lesions in nonhuman primates (rhesus macaques) experimentally infected with Mycobacterium tuberculosis

Abstract

Infection with Mycobacterium tuberculosis primarily produces a multifocal distribution of pulmonary granulomas in which the pathogen resides. Accordingly, quantitative assessment of the bacterial load and pathology is a substantial challenge in tuberculosis. Such assessments are critical for studies of the pathogenesis and for the development of vaccines and drugs in animal models of experimental M. tuberculosis infection. Stereology enables unbiased quantitation of three-dimensional objects from two-dimensional sections and thus is suited to quantify histological lesions. We have developed a protocol for stereological analysis of the lung in rhesus macaques inoculated with a pathogenic clinical strain of M. tuberculosis (Erdman strain). These animals exhibit a pattern of infection and tuberculosis similar to that of naturally infected humans. Conditions were optimized for collecting lung samples in a nonbiased, random manner. Bacterial load in these samples was assessed by a standard plating assay, and granulomas were graded and enumerated microscopically. Stereological analysis provided quantitative data that supported a significant correlation between bacterial load and lung granulomas. Thus this stereological approach enables a quantitative, statistically valid analysis of the impact of M. tuberculosis infection in the lung and will serve as an essential tool for objectively comparing the efficacy of drugs and vaccines.

Idiopathic Microscopic Colitis of Rhesus Macaques: Quantitative Assessment of Colonic Mucosa

“…For parenchymal organs such as lung, liver, and kidney, it involves cutting the organ into slabs of equal thickness and determining the cumulative area by point counting the organ surface and multiplying by the average slab thickness. (Luciw et al, 2011) It is a very efficient estimator of volume. (Hyde et al, 1992) Performing a Cavalieri estimate on hollow organs such as the gastrointestinal tract does not require sectioning the organ into slabs.…”

“…Then starting with the random number, samples were collected at a set sampling interval throughout the tissue. (Gundersen and Jensen, 1987; Braendgaard et al, 1990; Wreford, 1995; Luciw et al, 2011) SURS ensures that every point in the tissue has an equal chance of being selected for analysis. To start the sampling, the whole colon was collected at the necropsy and cut into three sections (Figure 1A).…”

Modeling Tuberculosis in Nonhuman Primates

“…Last, determining bacterial load in monkeys is technically challenging. Some researchers homogenize whole lungs for plating (Langermans et al 2001;Verreck et al 2009), some use a stereologic approach to guide the selection of random tissue sections for plating (Luciw et al 2011), and some use a combination of lesion collection and random sampling (Lin et al 2009). Many researchers report bacterial burden as CFU/gram of tissue (Langermans et al 2001;Reed et al 2009;Verreck et al 2009) or CFU/ lung (Kita et al 2011).…”

Monkey Models of Tuberculosis: Lessons Learned

“…A series of subsequent investigations were dedicated to macaque research to assess novel TB vaccines and drugs, as well as to gain an understanding of the pathogenesis of M. tuberculosis infection and reactivation. Figures 2 and 3 abridge the RM (13,(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) and CM (10,15,25,(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59) models of experimental M. tuberculosis infection after the Golden Age, from 2001 to 2014.…”