Characterization Of Mycobacterium Spp. Isolated From Striped Bass, Morone saxatilis, From The Chesapeake Bay     Martha W. Rhodes1, Howard Kator1, Ilsa Kaattari1, Shaban Kotob1, Peter van Berkum2, Wolfgang Vogelbein1 and Chris Ottinger3   1Department of Environmental Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA, 2United States Department of Agriculture, 3USGS, National Fish Health Research Laboratory, Kearneysville, WVA     Striped bass (Morone saxatilis) with and without skin ulcerations were examined for mycobacteria in splenic tissue using a method developed to maximize mycobacterial recovery.  Fish were necropsied using aseptic technique, splenic tissue excised and homogenized in phosphate buffer.  Tissue homogenates were plated onto Middlebrook 7H10 agar and incubated 3 months at 23°C.  Mycobacteria were recovered from 77% (N=37/48) of the fish examined.  Mycobacterial densities ranged between 104-105 colony-forming units per g splenic tissue.  A unique mycobacterium, hereafter referred to as M175, was isolated from 68% of samples (N=19/28) yielding only acid-fast colonies (N=28), and was thus presumed free of external contamination. Based on growth and biochemical characteristics, M175 could not be assigned to an existing species.  Sequence analysis of a PCR product (1,494 nucleotides long) of the 16S rRNA gene showed sequence similarities of 99.2% to Mycobacterium marinum and M. ulcerans and  98.7% to M. bovis and M. tuberculosis.  Despite the high similarities, the 16S gene sequence of M175 differed from M. ulcerans by 11 nucleotides and from M. marinum by 10 nucleotides. AN RFLP analysis of a large segment of the 16S rRNA gene differentiated M175 from other Mycobacterium spp. isolated from striped bass.  Co-infection of M175 with other mycobacteria was observed in 29% (N=8/28) of the samples.  In addition to the co-infected fish, 32% (N=9/28) of the positive spleen samples yielded mycobacteria other than M175.  These isolates included mycobacteria that were either (1) scotochromogenic, resembling M. scrofulaceum; (2) weakly photochromogenic resembling M. simiae, or (3) nonchromogenic.  Isolates resembling M. simiae or M. scrofulaceum belonging in separate RFLP categories.  Because each category contained multiple Mycobacterium spp., RFLP analyses are being refined to increase separation of isolates within categories.  The presence of multiple mycobacterial types occurring at high densities in samples showing no evidence of external contamination suggests that a variety of mycobacteria may be causative agents of mycobacteriosis in striped bass from the Chesapeake Bay.  Challenge studies in progress show that M175 produces granulomatous lesions in spleens of infected striped bass and only this strain can be reisolated, thus fulfilling Koch’s postulates.