Conventional and molecular Characterization of mycobacterial Strains Isolated from Cattle and Humans

Abstract

In the present study, a total of 167 caseated, enlarged and inflammed lymph nodes and tuberculous organs were collected from cattle slaughtered at Slaughterhouses in Khartoum State. Also twenty lyophilized mycobacterial strains previously isolated from both human and animal cases were examined in this study using PCR. The aim of this study was to estimate the prevalence of bovine tuberculosis and to improve identification and to compare between identification methods. The purulent materials from the suspected specimens were decontaminated with 2% NaOH and concentrated by centrifugation. The smears made from decontaminated purulent materials were stained by Ziehl-Neelsen method and culture onto Lowenstein-Jensen medium was done. Results from culture and microscopy were compared to a PCR method by amplifying an insertion element IS6110 which is specific for Mycobacterium tuberculosis complex. Out of the 167 specimens examined, 35 (20.96%) were found to have acid-fast bacteria when smears were examined microscopically. Out of the 35 acid-fast bacteria, 22 (62.86%) showed branching filaments and were identified tentatively as Mycobacterium farcinogenes. The remaining 13 (37.14%) were bacilli and were identified tentatively as Mycobacterium bovis. Of the 167 specimens, 12 (7.19%) showed visible growth on Lowenstein-Jensen medium when incubated aerobically at 37°C between 4 and 8 weeks. None of the 22 specimens with branching filaments grew using the above decontamination and growth conditions. Ten isolates were nitrate-negative and urease-positive, - 8 - and were thus identified as M. bovis, whereas the remaining tow strains were nitrate-positive and urease-negative but one was urease-positive. Polymerase chain reaction (PCR) assay for the rapid diagnosis of M. tuberculosis complex (M. tuberculosis, M. bovis, M. microti and M. africanum) was done using oligonucleotide primers TB41 and TB42 to amplify fragments of the IS6110, an insertion sequence specific for M. tuberculosis complex. Of the 12 grown culture, ten (83.3%) gave PCR product for IS6110 on agarose gel electrophoresis, which confirmed their identity as M. tuberculosis complex. Ten isolates were already found nitrate negative and thus were identified as M. bovis, none of the isolates were found to be M. tuberculosis when using this test combination. In conclusion, IS6110 amplification, urease and nitrate tests provide a species identity to M. bovis. The results of the above study is as follows: of the 167 caseated and purulent specimens examined 10 (5.99%) were M. bovis, 2 (1.2%) were mycobacterium other than tuberculosis (MOTT), 22 (13.17%) were tentatively identified as M. farcinogenes, 23 (13.77%) were acidfast bacteria (AFB) that failed to grow on L.J medium and 132 (79.04%) were caseous infections caused by non-acid-fast bacteria.