Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic granulomatous enteritis of the small intestine of ruminants, taking many years for clinical symptoms to develop. Though primarily transmitted through ingestion of contaminated feces, MAP also can be transmitted through milk and colostrum. Unfortunately, many other microorganisms are present in milk along with MAP. Therefore, a decontamination protocol must be applied to the milk before culture. The objective of this research was to optimize a decontamination and culture protocol for the recovery of MAP from milk. The optimal protocol then was applied to milk and colostrum samples obtained from naturally infected dairy cows over complete 305-day lactation cycles to evaluate whether a correlation exists between stage in JD progression or point in lactation and the concentration of MAP shed into the milk. Studies found the most efficacious decontamination and culture protocol consisted of exposing milk to a solution of N–acetyl–L–cysteine–1.5% sodium hydroxide for 15 min followed by inoculation into BACTEC 12B medium. When this protocol was applied to milk and colostrum samples collected from naturally infected dairy cows in various stages of JD, it was found that cows in more advanced stages had a higher likelihood of shedding MAP into milk and colostrum. MAP was also more likely to be isolated from milk samples taken within early lactation (0–60 days in milk). This study demonstrated that milk is a viable route of MAP transmission to calves, especially in early lactation when calves are most susceptible to infection and from cows with advanced stages of JD. Dairy producers should use this valuable information to change their calf–rearing practices to decrease the chances of disseminating MAP through their herds.