Quantitative PCR Assay for Mycobacterium pseudoshottsii and Mycobacterium shottsii and Application to Environmental Samples and Fishes from the Chesapeake Bay

Striped bass (Morone saxatilis) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden (Brevoortia tyrannus) and bay anchovy (Anchoa mitchilli). Mycobacterium pseudoshottsii was found to be ubiquitous in water samples from the main stem of the Chesapeake Bay and was also present in water and sediments from the Rappahannock River, Virginia. M. pseudoshottsii was also detected in menhaden and anchovy tissues. In contrast, M. shottsii was not detected in water, sediment, or prey fish tissues. In conjunction with its nonpigmented phenotype, which is frequently found in obligately pathogenic mycobacteria of humans, this pattern of occurrence suggests that M. shottsii may be an obligate pathogen of striped bass.Mycobacteriosis is a common disease affecting a large variety of wild and aquacultured fishes worldwide (9). Chronic disease is most commonly observed and is characterized by granulomatous inflammation that may affect all host tissues. External clinical signs include scale loss, dermal ulceration, spinal defects, emaciation, and ascites (5, 6, 16, 25, 31).Mycobacteriosis in Chesapeake Bay striped bass (Morone saxatilis) was first observed in 1997 from histologic findings of acid-fast bacilli in granulomatous lesions (W. Vogelbein, unpublished data). Since the initial finding, surveys have demonstrated a very high prevalence of this disease in Chesapeake Bay striped bass, exceeding 50% in many samples (8, 17). Concomitantly with detection of high prevalence, tag recapture analysis has indicated that natural, nonfishing mortality of Chesapeake Bay striped bass has increased since 1999 (13), and modeling of apparent prevalence data has indicated that some mortality is associated with disease (8). Because the striped bass is an ecologically and economically important finfish along the U.S. Atlantic coast, the high prevalence of this disease creates considerable concern about the continuing health of the resource.Mycobacteriosis of fishes has traditionally been considered to be caused by Mycobacterium marinum, M. fortuitum, or M. chelonae; however, the recognized diversity of Mycobacterium spp. infecting fishes has increased markedly in recent years (9). To date, neither M. fortuitum nor M. chelonae have been isolated from internal tissues of striped bass in the Chesapeake Bay, and M. marinum has been cultured from only a small fraction (3%) of fish (20). Instead, a variety of slow-growing mycobacteria have been isolated, dominated by the recently described species M. pseudoshottsii and M. shottsii (9, 20-22). The 16S rRNA gene sequences of M. pseudoshottsii, M. shottsii, M. marinum, and M. ulcerans are highly similar (≥99.4%), and like M. ulcerans, M. pseudoshottsii possesses the insertion sequences IS2404 and IS2606 and produces mycolactone toxin (19). M. shottsii has been reported to be positive for IS2404 under specific PCR conditions by some authors (22), but not by others (10), and this species is not known to produce mycolactone. IS2606 has been reported to amplify weakly or not at all in M. shottsii (22). M. pseudoshottsii and M. shottsii differ in pigment production, with the former being a photochromogen and the latter being nonpigmented (22).In this study, we performed a quantitative real-time PCR-based survey of the presence and density of M. pseudoshottsii and M. shottsii in water and sediments of Chesapeake Bay, as well as in two dominant prey of striped bass, the Atlantic menhaden (Brevoortia tyrannus) and the bay anchovy (Anchoa mitchilli) (12, 30). Mycobacterium pseudoshottsii was detected by amplification of IS2404 in a manner similar to that used in previous studies (7, 24). We also amplified and sequenced mycobacterial interspersed repetitive unit (MIRU) loci from menhaden, water, and sediment samples in order to confirm that IS2404 amplification in these samples was likely to represent the presence of M. pseudoshottsii and not another IS2404-positive bacterium.No unique insertion sequences have yet been described for M. shottsii, and the high degree of similarity between M. pseudoshottsii and M. shottsii in genes for which sequences are available (e.g., hsp60, erp, 16S rRNA, 23S rRNA, internal transcribed spacer ITS]) makes development of M. shottsii-specific assays problematic. We therefore performed genomic subtractive hybridization in a manner similar to that originally described by Akopyants et al. (3) to characterize sequences specific to M. shottsii relative to M. pseudoshottsii. An M. shottsii-specific quantitative PCR (qPCR) assay was developed to target sequences identified in this manner.