Detection of Mycobacterium bovis in whey, by multiplex polymerase chain reaction (PCR) and bacteriological culture

Abstract

The objective of this study was to detect the presence of Mycobacterium bovis (M. bovis) in whey. A total of 233 cow milk samples were analyzed together with 26 tank milk samples that came from dairy herds of several states of the Mexican Republic (Queretaro, San Luis Potosi, Guanajuato, Hidalgo, Coahuila). DNA was obtained from whey and used for polymerase chain reaction-multiplex (PCR-M). Tuberculosis complex was first identified through the detection of gene RD1. Positive samples were subjected to a second PCR-M with the primers for gene RD9 to identify M. bovis. Samples were bacteriologically cultured using conventional techniques for the isolation of mycobacteria. Cohen’s Kappa test (k) and Pearson’s Chi2 were carried out for statistical analysis. A 150 bp amplification product of the RD1 region was obtained, which corresponds to the tuberculosis complex, in 34/233 (14.59%) of the individual milk samples and in 4/26 (15.38%) of the tank milk samples. PCR-M with primer RD9, of the 34 individual samples and the 4 tank milk samples, gave an amplification product of 200 pb, which is the expected product for M. bovis. By bacteriological culture, six isolates were obtained; four in individual whey samples and two from tank milk samples, which were then classified by biochemical tests as M. bovis. The concordance between RD1, RD9 PCR-M and bacteriological culture was low, but there was a significant difference between diagnostic techniques with a P = 0.000. The results showed the potential of the PCR-M as a confirmatory test for the diagnosis of tuberculosis in cattle, as well as the advantage of using whey samples, that may be a possible source of infection for the herd and/or humans. Key words: Bovine tuberculosis, whey, polymerase chain reaction-multiplex (PCR-M), RD1, RD9, milk, isolation.