Detection of Mycobacterium avium subsp. paratuberculosis in bulk tank milk by combined phage-PCR assay: Evidence that plaque number is a good predictor of MAP

• We evaluate a rapid, phage-PCR detection of Mycobacterium avium subsp. paratuberculosis in milk.
• The assay is more sensitive than culture and BTM results were gained within 48 h.
• A correlation was identified between plaque number and the presence of MAP.
• A cut off value was determined to simplify the test (Sn = 90%; Sp = 99%).
• This test could be used to screen BTM and reduce human exposure to MAP.

Conventional culture and a rapid phage-PCR method were used to detect Mycobacterium avium subsp. paratuberculosis (MAP) in bulk tank milk (BTM) samples. Only two of 225 samples (0.9%) were found to contain MAP by culture whereas 50 (22%) MAP-positive samples were identified using the phage-PCR assay, including both samples that were MAP-culture positive. Results using the phage-based method for independently tested duplicate samples indicated that the assay is very reproducible (r2 = 0.897), especially when low levels of mycobacteria are present. A relationship was established between plaque number and the presence of MAP in a sample. A cut-off value was determined allowing identification of MAP-positive samples based on plaque number alone (90% sensitivity, 99% specificity; area under the curve = 0.976). These results indicate that the assay is a robust method for screening BTM, providing results within 24 h.