Optimisation of culture of Mycobacterium avium subspecies paratuberculosis from blood samples.
Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease or paratuberculosis, a chronic enteritis of ruminants, and has been suggested to play a role in Crohn's disease in humans. While disease expression is primarily in the gastrointestinal tract, isolation of MAP or MAP DNA at distant sites indicates that disseminated infections also occur. This is the first study to compare, optimise and critically evaluate different methods of concentrating MAP from spiked blood samples to enable its culture. Whole blood, erythrocytes, but not plasma, delayed the growth of MAP in BACTEC 12B medium. Culture of MAP from buffy coats (concentrated leukocytes) after lysis of erythrocytes and washing of the cells was an effective preparatory method. Several antibiotics were evaluated to reduce contamination of the slow growing MAP cultures with microbes which were derived from the skin during venipuncture, but were detrimental to the growth of MAP. However, decontamination of erythrocyte-lysed washed buffy coat samples in 0.75% hexadecylpyridinium chloride (HPC) for 72 h prior to inoculation of culture media did not inhibit the growth of MAP. The prepared samples can be stored at -80 degrees C prior to batch culture. MAP was isolated from the blood of 2 of 23 sheep 20 months after experimental inoculation. The optimised method has an analytical sensitivity of at least 10(1) MAP per ml of spiked whole blood and will enable trials to determine the incidence, duration and magnitude of mycobacteraemia in infected animals and humans.