Identification of Factors that Promote the Growth of Dormant Mycobacterium avium subsp. paratuberculosis

Johne\u27s disease, which is caused by the acid-fast bacterium Mycobacterium avium subspecies paratuberculosis (Mpt), is a chronic, inflammatory intestinal disease that primarily affects ruminants. This disease has a significant effect on the economics of dairy farming. Mpt has a remarkable ability to survive in host tissues for 2-6 years without producing any signs of infection. However, the reliability of diagnostic techniques is limited only to those animals with clinical disease. This may be due to the entry of the organism into a dormant state, which has been reported for other mycobacteria. Reversion to the actively growing state (resuscitation) would improve the sensitivity of diagnostic culture; however, the substances effecting resuscitation have not been defined. Previous work in our laboratory showed that conditioned medium obtained from starvation-dormant Mpt (SDCM) enhanced the resuscitation of dormant Mpt. Research by others demonstrated that resuscitation of other dormant mycobacterial pathogens was enhanced by resuscitation promoting factors (Rpf), a class of 17-19 kDa proteins that are secreted by these organisms. We therefore hypothesized that one or more Rpf was responsible for the resuscitative activity in SDCM. In this study, we sought to identify and characterize fractions of SDCM that contain resuscitative activity. SDCM was fractionated into 6 molecular weight classes, and each fraction was tested for the ability to promote the resuscitation of anaerobically dormant Mpt responder cells. Most (if not all) of this activity was confined to a fraction containing molecules with a molecular weight of 5 kDa or less (F5). To characterize F5, this fraction was subjected to heat, protease treatment, and combinations of the two. The resuscitative activity of F5 was stable at high temperatures (65oC and 80oC), resistant to trypsin and thermolysin, and was not impaired by a combination of heat and protease treatment. Growth factors in F5 were concluded to be non-protein in nature, thereby refuting our hypothesis. F5 prepared from 5-, 6, and 8-month-old cultures were analyzed by reversed-phase high performance liquid chromatography to compare peptide and acyl homoserine lactone (AHL) components in F5. AHL peaks steadily accumulated with time, whereas peptide analysis identified hydrophobic peaks that generally diminished over time, and hydrophilic peaks that increased with time. Further characterization and identification of this Mpt-resuscitative growth factor will be necessary in order to test its ability to enhance the recovery of dormant Mpt in clinical specimens