Middlebrook 7h9 liquid media

M. tuberculosis H37Rv and HN878 were grown in Middlebrook 7H9 liquid media (Becton-Dickinson, Sparks, MD, USA) supplemented with 0.05% Tween 80, 0.2% glycerol and 10% oleic acid/albumin/dextrose/catalase (OADC) enrichment for 7–10 days and then sub cultured in Proskauer Beck (PB) medium, supplemented with 0.05% Tween 80 and 2% glycerol, to the mid-log phase. To determine the concentration of M. tuberculosis, 6 frozen aliquots were serial diluted and plated in Middlebrook 7H11 (Becton-Dickinson, Sparks, MD, USA) agar plates supplemented with 10% OADC and 0.5% glycerol. Viable bacteria were determined by CFU enumeration after 21–28 days of incubation at 37 °C.

The MCIs for AMK, CLR, EMB, MXF, NALC, Tween 80, ASA, IBP, PCM and DADS, alone and in combination, were determined in 96-well plates (Smartech Biosciences, Barcelona, Spain). In brief, 100 µL of Middlebrook 7H9 liquid media (Becton Dickinson, Sparks, MD, USA) were added to each well. Then, 100 µL of antibiotic were added to the first well and two-fold serial dilutions, ranging from 64 µg/mL to 0.5 µg/mL, were made. The same procedure and concentrations were used for the PAAs, except for Tween 80, which was tested in dilutions ranging from 12.5% to 0.09%. Finally, 100 µL of inoculum, at a concentration of 1.5 × 10⁵ CFU/mL, were added (1/1000 dilution of a 0.5 McFarland, using a nephelometer) (PhoenixSpec, Becton Dickinson). The positive control wells contained 100 µL of Middlebrook 7H9 and 100 µL of inoculum. The negative control wells were also included, by adding 200 µL of Middlebrook 7H9. The microplates were incubated at 37 °C for 7 days. After incubation, the plates were read using the Vizion System (Sensititre Vizion Digital MIC Viewing System, Thermo Fisher Scientific, Waltham, MA, USA). The MIC value was interpreted as the lowest antibiotic and/or PAA concentration inhibiting mycobacterial growth.

M. bovis BCG Danish strain was a gift from Dr. Ray Waters at the National Animal Disease Center, USDA. BCG was prepared using standard techniques in Middlebrook 7H9 liquid media (Becton Dickinson, Franklin Lakes, NJ) supplemented with 10% oleic acid-albumin-dextrose complex (OADC) plus 0.05% Tween 80 (Sigma, St. Louis, Missouri). For the first experiment, treatment groups consisted of non-vaccinated steers (n = 3) and animals receiving 1 × 10⁸ colony-forming units (CFU) of M. bovis BCG Danish strain (n = 11). For the second study, treatment groups consisted of non-vaccinated calves (n = 7) and animals receiving 1 × 10⁸ CFU of M. bovis BCG Danish strain (n = 7). For both studies, BCG inoculum was delivered to restrained calves by aerosol as described by Palmer et al.^{66 (link)}. Briefly, inoculum was nebulized into a mask (Trudell Medical International, London, ON, Canada) covering the nostrils and mouth, allowing regular breathing and delivery of the bacterial inoculum to the respiratory tract. Clinical signs, including cough, dyspnea, and loss of appetite were monitored daily throughout the study. No clinical signs or pathology associated with BCG immunization were observed.