Evaluation of immunomagnetic beads and loop mediated Isothermal amplification in detecting mycobacterium tuberculosis complex in Bovine Milk

Abstract

The study evaluated the use of immunomagnetic beads (IMB) in detecting Mycobacterium tuberculosis complex (MTC) in bovine milk. A total of 161 animals from traditional small scale farmers of Mphango and Itope areas of Chisamba and Rufunsa districts of Zambia were tested for tuberculosis using the comparative intradermal tuberculin skin test. Of the tested animals, 4 (2.5%, 95% CI=0.1%-4.9%) out of the 161 animals tested positive on tuberculin skin test and 12 (7.5%) showed inconclusive results. Furthermore, milk samples from the 161 animals were tested for tuberculosis (TB) using the immunomagnetic beads technique and, DNA from the captured cells was used as samples for the loop mediated isothermal amplification (LAMP) test. A total of 24 (14.9%, 95% CI= 9.4%-20.4%) animals tested positive on IMB-LAMP. This technique was also able to detect 12 (8.3%, 95% CI=3.8%-12.8%) animals that initially tested negative with the tuberculin skin test as positives. The twenty four (24) milk samples that tested positive on IMB-LAMP were further cultured on Lowenstein-Jensen media supplemented with Tween 80. Out of the 24 samples, 4 (16.7%, 95% CI=1.8%-31.6%) showed signs of Mycobacterium growth and were considered positive. The rest 20 (83.3%) did not grow and were considered negative. The suspected culture positives were confirmed by multiplex PCR as Mycobacterium bovis. The study has demonstrated that immunomagnetic beads can be used to capture MTC from milk of naturally infected animals and that the Mycobacterium can be isolated from milk samples of animals certified negative by the comparative intradermal tuberculin skin test. The study also demonstrated that the culturing technique, though regarded as a ‘Gold standard method’ need to be conducted alongside molecular tests. The study recommends the use of IMB-LAMP as a complementary diagnostic test for detecting Mycobacterium bovis besides the intradermal tuberculin skin test and the culture method. It further suggests that milk be considered as a diagnostic specimen for tuberculosis in animals. Further studies may be carried out to establish the minimum number of mycobacterial cells that can be captured by the beads in milk samples.