TWENTY-FIFTH ANNUAL EASTERN FISH HEALTH WORKSHOP MARCH 10-13, 2000   Rapid Detection of Mycobacteriosis in Striped Bass by PCR-RFLP Analysis     Ilsa Kaattari1, M. Rhodes1, H. Kator1, S. Kotob1, R. Butler2, F. Quinn2, & W. Vogelbein1   1Department of Environmental Sciences, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia  23062, USA and 2Tuberculosis/Mycobacteriology Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia  30333, USA     The main goal of our research was to employ the rapid and highly sensitive PCR assay to diagnose and identify mycobacteriosis in striped bass (Morone saxatilis).  PCR assay was conducted on nine different mycobacterial species that have been identified using biochemical assays.  One of them has been tentatively identified, using HPLC at CDC, as a member of the M. tuberculosis complex.  PCR assay for the identification of mycobacterial  species was accomplished using primers amplifying 924 bp of the 16S rRNA gene from pure mycobacterial cultures.  The amplified PCR products were purified using a Concert Rapid Gel Extraction System kit (GibcoBRL) and then digested with two different enzymes, ApaI and BanI. Different RFLP profiles were obtained: digestion with BanI yielded two fragments (562 bp and 362 bp) for M. fortuitum and M. chelonae, while M. marinum was left intact (924 bp).  Digestion with ApaI  produced three fragments (677 bp, 132 bp, and 115  bp) for M. fortuitum and M. marinum and two fragments (812 bp and 112 bp) for M. chelonae.  Detection of mycobacterial species in tissues was also accomplished, using nested PCR to amplify a 300 bp internal fragment of the 924 bp amplicon.  PCR-RFLP analysis is a useful tool for the identification of mycobacterial infections at the species level.     Return to 25th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page