Detection of Mycobacterium bovis in bovine carcasses by multiplex-PCR

The causative agent of bovine tuberculosis (BTB) is Mycobacterium bovis, a bacterium belonging to the M. tuberculosis complex (MTC). The definitive diagnosis is achieved through isolation and identification of M. bovis from clinical samples, using a combination of traditional culture and biochemical methods, which is considered the “gold standard”. This procedure is cumbersome and time-consuming. We evaluated a multiplex-PCR (m-PCR) assay for the direct detection of M. bovis DNA from tissue with BTB-suspected lesions. A dairy herd consisting of 270 adult cattle where 34 animals were positive to the tuberculin skin test has been reported. At 30 days after the tuberculin test, all 34 reactive animals were slaughtered and subjected to a necropsy procedure. A pool of tissue samples representative of each animal (lung and mediastinal, scapular and retropharyngeal lymph nodes) were collected and subjected to bacteriological culture and m-PCR. Mycobacterium spp. was isolated in 50% (17/34) of the collected samples. When using m-PCR directly from tissue fragments, it was possible to detect M. bovis in 67.6% (23/34) of the collected samples including 15 samples isolated by bacteriological culture. High performance liquid chromatography (HPLC) was used to differentiate the 17 isolated strains of Mycobacterium spp., from the Mycobacterium tuberculosis complex (MTC) or other Mycobacterium sp. not belonging to the MTC. The use of m-PCR assays directly from tissue samples may be a valid supplementary tool for the post mortem diagnosis of BTB, since this is a a faster and more specific technique than bacterial culturing, reducing the diagnosis time for diagnosis of the disease from three months to two days. Key words: Bovine tuberculosis, high performance liquid chromatography (HPLC), Mycobacterium tuberculosis complex, multiplex polymerase chain reaction (PCR), tissues with macroscopic lesions.