Detection of Mycobacterium avium subspecies paratuberculosis excretion in bovine feces, using quantitative real time polymerase chain reaction (Q-PCR)

Mycobacterium avium subspecies paratuberculosis (Map) causes paratuberculosis, an infectious enteritis that affects domestic ruminants. The main source of infection for herds comes from the elimination of the bacilli through feces. The objective of this study was to determine Map excretion in bovine feces. The study included forty, one-year-old bovine, Holstein breed females, from a herd that had >25% paratuberculosis prevalence. Samples of blood and feces were obtained four times with three-month intervals. Feces samples were subjected to bacteriological culture and DNA extraction for IS900 quantitative real time polymerase chain reaction (Q-PCR). Enzyme-linked immunosorbent assay (ELISA) was done from serum samples to detect the presence of anti-Map antibodies. Correlation between IS900 Q-PCR, culture and ELISA was established by the Kappa (K) test; statistical analysis was carried out by the Pearson Chi2 test. On the first sampling, five animals were detected by IS900 Q-PCR, shed 1 × 102 to 2.6 × 103 Map copies per gram of feces; on the second sampling, 6 animals shed 3.25 × 104 to 8.5 × 108; on the third sampling two animals shed 2.03 × 105 to 1.10 × 106 and on the fourth, three animals shed 7.92 × 104 to 1.4 × 107 Map copies. Correlation between tests in samplings first, second and fourth, was 0.53 to 0.73, and for third was 0.22. With the use of IS900 Q-PCR, it was possible to detect animals that were eliminating Map in feces from 12 months of age without clinical manifestations. The IS900 Q-PCR is an alternative method to carry out programs of control of paratuberculosis that allow the detection of animals that shed Map in the early stages of the infection.  Key words: Paratuberculosis, Mycobacterium avium subspecies paratuberculosis, excretion, quantitative polymerase chain reaction (Q-PCR), bacteriological culture, enzyme-linked immunosorbent assay (ELISA).