Oleic acid albumin dextrose complex

A virulent strain of MAP (W09/710) previously isolated from a JD infected cow ^{56 (link)} was used to challenge the mice. MAP was grown to mid-log phase in Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with 10% Oleic acid albumin dextrose complex (OADC), 0.5% glycerol (Sigma, St. Louis, MO), 0.05% Tween 20 (Sigma, St. Louis, MO), and 2 µg/mL mycobactin J (Allied Monitor, Inc., Fayette, MO) for 8 weeks. The MAP culture at passage 9 was harvested by centrifugation at 4000×g for 15 min and washed twice with PBS (10 mM, pH-7.4). The organisms were then diluted in 10 mL of PBS and de-clumped using a high-performance ultrasonic cleaning system. The concentration of the bacteria was adjusted to the required challenge dose.

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.

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 in vitro and in vivo experiments, bacteria in log phase were centrifuged (3000 rpm for 15 minutes) and resuspended in cRPMI without antibiotics for in vitro studies, or sterile PBS for in vivo vaccination studies (see below for additional details).