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Recovery and Characterization of Mycobacterium spp. from striped bass, Morone saxatilis, from the Chesapeake
Bay Martha W.
Rhodes1, Howard Kator1,
Ilsa Kaattari1, Wolfgang Vogelbein1, Shaban Kotob1
Frederick D.Quinn2, Mitchell A. Yakrus2, Emmett B.Shotts3 1Department of
Environmental Science, Virginia Institute of Marine Science, College of William
and Mary, Gloucester Point, VA; 2Tuberculosis/Mycobacteriology
Branch, Centers for Disease Control and Prevention, Atlanta, GA; 3U.S.
Geological Survey, National Fish Health
Laboratory, 1700 Leetown Rd.,
Kearnysville, WV 25430 A method was developed to maximize
recovery of mycobacteria from striped bass, Morone
saxatilis, exhibiting external and/or internal granulomatous lesions. Tissue samples (n=68) were treated with
traditional disinfectants routinely used to decontaminate specimens submitted
for culturie of mycobacteria, i.e., 0.3% Zephiran, 2% HCl or 2% NaOH. Treated homogenates were inoculated onto
Brain Heart Infusion agar with sheep blood (5%), Lowenstein Jensen (LJ) slants,
and Middlebrook 7H10 agar (MDA) and incubated 2–3 months at 30 oC. Mycobacteria were most frequently detected
following acid (32%) or alkali (34%) treatment and plating on Lowenstein
Jensen. However, suspensions of 7 purified mycobacterial cultures isolated from
fish showed 2 to 4 log(10) reductions in culturable cell densities
when exposed to the preceding disinfectant treatments. Therefore, subsequent
analyses were performed using aseptic necropsy techniques and streaking
non-treated homogenates directly onto MDA plates. Primary tissue inoculated plates were incubated at 23 and 30 oC
for 3 months. The revised approach
facilitated detection of contamination by
non-mycobacteria, provided a semi–quantitative assessment of
mycobacterial infection, and allowed for visualization of microscopic colonies
of slow–growing mycobacteria. Moreover,
whereas initial analyses recovered a variety of mycobacterial species, 76
tissue samples were processed with the
revised technique yielded primarily two types of slow–growing
mycobacteria. One group is
scotochromogenic with a temperature optimum of 30 oC and resembles M. scrofulaceum. The other group is non-chromogenic with a
temperature optimum of 23 oC, and although unidentified at this
time, possess a mycolic acid HPLC profile similar to the M.
tuberculosis complex. All
mycobacterial isolates were characterized using traditional biochemical
techniques and growth studies.
Additional confirmatory identifications were provided for some isolates
by the CDC usingHPLC and molecular methods.
Successful recovery of mycobacterial isolates from diseased fish is the
first step to enable experimental challenging of fish and testing for the
fulfillment of Koch’s postulates.
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