Investigation of the correlation between viability and optimal oxygen demand of dormant Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (Mpt) is the causative agent of Johne’s disease, a chronic and often fatal enteritis in many ruminants. In the U.S., Mpt infections in dairy cattle are a significant cause of economic loss. It is known that clinically diseased cattle shed large numbers of Mpt; however sub-clinically infected animals excrete Mpt intermittently and may therefore pose a risk of infection to the rest of the herd. Moreover, mycobacteria can enter a dormant state in vivo. These organisms may be susceptible to oxidative damage and death due to the sudden surge in metabolic rates resulting from culture in rich, selective media. The current method for culturing Mpt from cattle feces does not take into account the reduced metabolic rate of dormant Mpt. If the optimal amount of oxygen required for Mpt to divide in the microenvironment can be determined, then reducing oxygen in the culture microenvironment may assist in culturing these organisms by allowing them to recover. The primary goal of the research is to develop a system that will provide the ability to recover viable but not culturable Mpt by determining the relationship between the percent of viable organisms and the optimal oxygen requirement of those organisms. Viability (a ratio of live to dead organisms) was measured using a flow cytometer. Broth cultures containing doubling dilutions of Oxyrase, an enzyme that depletes oxygen in the microenvironment, were used to determine the optimal oxygen demand for the resuscitation of dormant organisms