Comparative Analysis of Low Molecular Weight Fraction of Conditioned Media Derived from Starvation-Dormant and Anaerobically-Dormant Mycobacterium Avium Subsp. Paratuberculosis

Johne\u27s disease (JD) caused by Mycobacterium avium subsp. paratuberculosis (MAP) is a contagious chronic enteritis affecting all ruminants and causing economic loss of at least $250 million annually in the US dairy industry. One of the major limitations in the diagnosis of JD is the low sensitivity of fecal culture in animals with no clinical signs of the disease, but who are actively shedding MAP. Persistence of MAP in the host with no apparent signs of infection is attributed to its ability to enter a dormant state wherein the bacterium is viable, but is not able to replicate until conditions become favorable. In vitro studies have shown that addition of culture media from starvation-dormant cells (SDCM) to growth media enhances the recovery of dormant cells. Resuscitative activity has been noted in the fraction of SDCM containing molecules equal to or smaller than 5kDa in size; the ability of this fraction to resuscitate was resistant to heat and enzyme treatment. In this study we sought to further investigate the composition of low molecular weight fraction of SDCM in order to identify the nature of the molecules with resuscitative effect. Starvation-dormant MAP cells were grouped into three distinct phenotypes based on acid-fast staining properties, and fractions of SDCM were obtained from cultures of similar phenotype. Only higher dilutions of fractions pooled from cultures with preserved acid-fastness had a resuscitative effect, which disappeared with higher concentration of the fraction. Fractions derived from cultures with complete or partial loss of acid-fastness either had no effect or inhibitory effect on MAP recovery. The concentrates of resuscitative fractions were comparatively analyzed with non-resuscitative fractions of SDCM, fractions of ADCM and growth media for protein/peptide concentration and lipid profile using BCA assay, SDS-PAGE gel electrophoresis, and thin layer chromatography. No proteins of 5kDa or smaller were detected in any of the samples. Resuscitative fractions of SDCM had a different mycolic acid profile compared to non-resuscitative, ADCM fractions, and cell-wall bound mycolic acids from exponentially growing MAP. Further analysis of lipid composition and lipid purification are needed in order to test whether lipids are involved in resuscitation of the MAP cells from the dormancy