A combination of immunomagnetic separation separation and PCR have been used to improve the specificity and early detection of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in raw cow's milk. An assay for sensitive and early detection of MAP is critical to improving the health of the cows and the dairy industry. A new assay has been developed called IMS-IS1PCR to reflect its two components: immunomagnetic separation (IMS) and IS900 PCR (IS1 PCR). Immunomagnetic separation selectively enriches the population of MAP in milk samples prior to DNA amplification and detection by PCR. The use of magnetic separation is the key component to the success of this new assay. For more information about the magnetic separation process read this article.
Immunomagnetic separation is necessary for early diagnosis of paratuberculosis
The previous assays for MAP were fecal culture and serum ELISA. These were time-consuming and/or not sensitive enough for preclinical MAP detection. The MAP bacteria causes a chronic inflammatory wasting disease called paratuberculosis or Johne's disease, which is responsible for the premature death of milk cows. It is a major economic loss for the dairy industry, and can devastate a herd if not detected early because MAP is readily transmitted from mother to calf and between adult cows by environmental contamination. Early diagnosis is critical for controlling the spread of the bacteria between cows, but early detection must be accomplished before clinical disease symptoms are apparent and before major shedding of bacteria into the environment. Detection by fecal culture is time-consuming and has a low senstitivity of 23-29%. The serum ELISA method is faster but has a lower sensitivity of 15% in the preclinical stages of infection. PCR has been used previously to detect MAP DNA in raw milk samples, but the sensitivity of this is also low at 23%. The problem with directly using milk samples is that there are a lot of PCR inhibitory substances present. The assay sensitivity can be greatly improved by selectively enriching the population of MAP in milk samples prior to PCR.
How it works: IMS-IS1PCR
By this method, antibody-coated magnetic beads bind to the MAP bacteria and are collected by magnetic separation while the PCR inhibitory substances are washed out of the sample. This added step leads to greater amplification of MAP DNA by PCR and improved detection of the bacteria at preclinical stages. The magnetic beads were coated with anti-Map antibodies and incubated with the milk samples for one hour. Then the bead conjugates were separated from solution in 10 minutes by using a magnetic rack before further characterization by IS900 PCR.
Immunomagnetic Separation improves pathogen detection
The new IMS-IS1PCR technique correctly diagnosed 80 out of 147 cows that were MAP positive (55%), which is significantly better than previous methods. Only 2 of those 147 cows were diagnosed by milk culture (1.4%), 15 by fecal culture (10%), and 20 by serum ELISA (14%). In the 118 uninfected cows there were no false positives by IMS-IS1PCR as they all tested negative for MAP. The use of immunomagnetic separation greatly improved detection of MAP in raw cow milk samples, and this work suggests that IMS could be useful in the detection of other pathogens as well.