Isolation and characterization of mycobacteria from striped bass Morone saxatilis from the Chesapeake Bay

Abstract

Mycobacteriosis in striped bass Morone saxatilis of Chesapeake Bay, USA, was first diagnosed in 1997 based on the presence of granulomatous inflammation and acid-fast bacteria in skin and spleen. To confirm histopathology, bacteriological detection and identification of mycobacteria were begun using splenic tissue from fish with and without skin ulcerations. On the basis of initial studies using a variety of selective and nonselective media, decontamination, homogenization and incubation conditions, a simple and quantitative recovery method using aseptic necropsy of splenic tissue was developed. Optimal recovery was obtained by spread-plating homogenates on Middlebrook 7H10 agar with incubation for 3 mo at 23°C. Mycobacteria were recovered from 76% (n = 149/196) of fish examined. Mycobacterial densities exceeded 10 4 colony forming units · g tissue -1in 38% of samples (n = 63/168) that were examined using a quantitative approach. The most frequently recovered mycobacterium, present in 57% (n = 109/192) of characterized samples, was the recently named new species Mycobacterium shottsii. Polyinfections of M. shottsii and other mycobacteria were observed in 25% of samples (n = 47/192) with densities of M. shottsii usually 1 or more orders of magnitude higher than co-isolate(s). Other mycobacteria recovered included isolates that, based on phenotypic traits, resembled M. interjectum, M. marinum, M. scrofulaceum, M. szulgai and M. triplex. M. marinum, commonly associated with fish mycobacteriosis and human disease, was recovered infrequently (3%, n = 6/192). The presence of multiple mycobacterial types occurring at high densities suggests that a variety of mycobacteria could be causative agents of mycobacteriosis in striped bass from the Chesapeake Bay. Striped bass is the major recreational fish species in the Chesapeake Bay, and the significance of the current epizootic to human health and the potential adverse effects on fish stocks are not known. KEY WORDS: Mycobacteriosis · Fish disease · Striped bass · Morone saxatilis · Mycobacterium marinum · Mycobacterium shottsii Resale or republication not permitted without written consent of the publisherDis Aquat Org 61: [41][42][43][44][45][46][47][48][49][50][51] 2004 ded during the early to mid-1990s but by the late 1990s concern for the health of these fish returned. An epizootic in Chesapeake Bay striped bass, characterized by skin lesions and granulomatous inflammation with associated acid-fast bacteria, was first described in 1997 (Baya 1998, Vogelbein et al. 1998. Bacterial diseases in striped bass exhibiting skin lesions and scale loss have been attributed to a variety of aetiologies, including Aeromonas, Edwardsiella, Flexibacter, Pseudomonas, and Vibrio spp. (Toranzo et al. 1983, Mitchell 1984, Sindermann 1988, Plumb 1991, Baya et al. 1997. However, in the current epizootic the presence of nonbranching acid-fast bacteria within granulomas implicated mycobacteria as the causative agent and represented the first time that fish mycobacteriosis had ...

Characterization of photochromogenic Mycobacterium spp. from Chesapeake Bay striped bass Morone saxatilis

“…The 3 species of Mycobacterium traditionally associated with infections in fishes are M. marinum, M. chelonae and M. fortuitum (Gauthier & Rhodes 2009), and reports continue to implicate these species in fish disease worldwide. It was therefore surprising when bacteriological surveys of Chesapeake Bay striped bass conducted from 1997 to 2001 indicated that M. marinum comprised only about 3% of Chesapeake Bay striped bass isolates, and neither M. chelonae nor M. fortuitum was detected (Rhodes et al 2004). Since this first report, M. chelonae has been isolated from only a single striped bass in Delaware Bay (Ottinger et al 2007), and M. fortuitum has not yet been isolated from internal tissues of this fish.…”

“…Fish were placed on ice immediately and necropsy was performed within 24 h after collection. Bacterial isolation and phenotypic characterization were as described in previous work (Rhodes et al 2004, Gauthier et al 2008b. Briefly, 0.1 to 0.5 g of spleen was homogenized using a Tenbroek tissue grinder, and dilutions were made in Butterfield's buffer + 0.05% Tween-80.…”

“…Since this first report, M. chelonae has been isolated from only a single striped bass in Delaware Bay (Ottinger et al 2007), and M. fortuitum has not yet been isolated from internal tissues of this fish. Instead, a suite of biochemically diverse, primarily slow-growing mycobacteria has been isolated, including M. shottsii and M. pseudoshottsii (Rhodes et al 2001(Rhodes et al , 2003(Rhodes et al , 2004(Rhodes et al , 2005, which are related to M. marinum and M. ulcerans. M. pseudoshottsii and M. shottsii appear to be the dominant isolates, although a phenotypic grouping of non-pigmented, slow-growing mycobacteria similar to M. triplex and M. interjectum are also numerically relevant (Rhodes et al 2004).…”

“…Instead, a suite of biochemically diverse, primarily slow-growing mycobacteria has been isolated, including M. shottsii and M. pseudoshottsii (Rhodes et al 2001(Rhodes et al , 2003(Rhodes et al , 2004(Rhodes et al , 2005, which are related to M. marinum and M. ulcerans. M. pseudoshottsii and M. shottsii appear to be the dominant isolates, although a phenotypic grouping of non-pigmented, slow-growing mycobacteria similar to M. triplex and M. interjectum are also numerically relevant (Rhodes et al 2004). More recent work using fatty acid methyl ester (FAME) analysis and DNA sequence data on mycobacterial isolates recovered from Chesapeake Bay striped bass and other fishes has further demonstrated the large diversity of Mycobacterium species and/or strains capable of infecting fishes in this ecosystem (Stine et al 2010).…”

General and Specialized Media Routinely Employed for Primary Isolation of Bacterial Pathogens of Fishes

“…Primary isolation is commonly made using Lowenstein and Middlebrook media (BD Difco). Rhodes et al (2004) developed a tissue preparation and media inoculation procedure that enhances the recovery of Mycobacterium spp. from fish (i.e., striped bass M. saxatilis) using Middlebrook 7H9 medium supplemented with antimicrobials (Teska et al, 1997) to reduce overgrowth by contaminants and thereby increase recovery of the pathogen.…”