*4.5. Mice Infection*

Specified pathogen-free female C57BL/6 mice (*n* = 51) were obtained from Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS (Moscow, Russia). All mice were used at 7–8 weeks of age and 20–22 g in weight. Randomization was used to allocate three experimental groups: control, *M. tuberculosis* H37Rv, and *M. tuberculosis* Rostov.

*M. tuberculosis* strains were grown to mid-logarithmic phase (OD600 = 1.0), cells were collected by centrifugation, and washed with PBS containing 0.05% Tween-80. Mice were intravenously injected into the lateral tail vein with 5 × 10<sup>6</sup> CFU/mice (in 0.1 mL of 0.9% NaCl) of the H37Rv strain and of the Rostov strain. All animals were weighed each day after infection. Animals were observed for 90 days; the physical appearance and behavior of animals were estimated; the daily animal weight loss and mortality were calculated.

On the 30th day after *M. tuberculosis* injection, six mice were euthanized by CO2 gas in each experimental group. Lungs and livers tissues were examined for mycobacterial load and pathology. The *M. tuberculosis* bacillary burden in lungs and livers was counted by homogenates plating onto Middlebrook 7H11 agar. Some samples of lungs and livers were fixed in 10% formalin (BioChem-NN, Nizhny Novgorod, Russia), graded concentrations of ethanol and butanol were used for dehydration, embedded in para ffin, and serial sections (5 μm width) were prepared with the Ultracut microtome (Reichert-Jung, Bensheim, Germany). Sections were depara ffinated and stained with hematoxylin and eosin. All slides were examined with a Nikon Eclipse 80i microscope and a Nikon DS-U2digital camera (Nikon, Tokyo, Japan).
