The causative agent of Johne's disease is Mycobacterium avium subsp. paratuberculosis. This is a chronic, debilitating gastrointestinal disorder that affects ruminants and is responsible for significant economic loss. The specimen processing method that combines C(18)-carboxypropylbetaine (CB-18) treatment and lytic enzyme decontamination has been shown to improve the diagnosis of mycobacterioses. This processing method was applied to the isolation of M. avium subsp. paratuberculosis from ruminant tissue samples. The BACTEC 12B liquid culture system was used but was supplemented with 1% egg yolk emulsion, 4 microg of mycobactin J, and 0.5% pyruvate (12B/EMP) for use in conjunction with this method. The final concentration of antibiotics used was 10 microg of vancomycin, 30 microg of amphotericin B, and 20 microg of nalidixic acid (VAN) per ml. A 7H10-based solid medium was also used that included mycobactin J, pyruvate, and VAN but excluded the egg yolk emulsion (7H10/MPV). Several M. avium subsp. paratuberculosis isolates were examined during the evaluation of this processing method. It was observed that treatment with lytic enzymes stimulated the growth of M. avium subsp. paratuberculosis; however, the growth of one isolate was markedly inhibited due to the presence of vancomycin. Subsequently, the vancomycin concentration in the VAN formulation was reduced to 2 microg/ml. A blinded panel of 60 previously characterized tissue samples from bovine and bison were then processed and analyzed by smear and culture. Historically, 31 and 37 specimens were classified as positive by histology and culture, respectively. The overall sensitivity and specificity of smear relative to culture following CB-18 processing were 97.6 and 89.5%, respectively. The 12B/EMP/VAN liquid culture system recovered M. avium subsp. paratuberculosis from 39 specimens, whereas 7H10/MPV and Herrold's egg yolk media recovered M. avium subsp. paratuberculosis from 26 and 16 specimens, respectively. The average times to positive were 7.4 +/- 8.3, 29.9 +/- 2.6, and 24 +/- 0 days, respectively. The contamination rates were 4.8, 22.6, and 20.0%, respectively.