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Amoebae serve as environmental hosts to a variety of mycobacteria, including Mycobacterium avium and Mycobacterium marinum. Mycobacterium shottsii and Mycobacterium pseudoshottsii are waterborne species isolated from the spleens and dermal lesions of striped bass (Morone saxatilis) from the Chesapeake Bay. The optimal growth temperature for these fish isolates is 25 °C. In the present study, amoebae were examined as a potential environmental reservoir for these fish pathogens. Several studies demonstrated that M. avium bacilli replicate within the trophozoite stage and reside in large numbers within the cytosol of the cyst of the free-living amoeba Acanthamoeba polyphaga. Results from the present study showed that M. shottsii, M. pseudoshottsii, and M. marinum bacilli were internalized by A. polyphaga trophozoites within 6 h but that intracellular viability decreased by 2 to 3 logs over 10 days. While an average of 25 M. marinum bacilli were identified by electron microscopy in the cytosol of the cyst

Wild striped bass Morone saxatilis in Chesapeake Bay are experiencing a high prevalence of mycobacteriosis, which produces granulomatous lesions of the skin and visceral organs. Culture-based studies have indicated that the newly described species Mycobacterium shottsii and M. pseudoshottsii are the dominant isolates from diseased fish. The classical fish pathogen M. marinum is also found, albeit at much lower frequencies. Both M. shottsii and M. pseudoshottsii are extremely slow-growing on standard selective media, and up to 12 months may be required for isolation and characterization. Epidemiological studies of mycobacteriosis in Chesapeake Bay would therefore benefit from rapid molecular assays with which to detect these species in fish. In this paper, we describe the development of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays capable of detecting M. shottsii, M. pseudoshottsii, and, in most instances, coinfections thereof in striped bass tissues. In addition, PCR-RFLP assays were designed to detect M. marinum and other as-yet-undescribed Mycobacterium spp. present in Chesapeake Bay striped bass. Comparison of these molecular assays with culture-based techniques using splenic tissue from wild striped bass yielded generally concordant results and demonstrated the applicability of these techniques to the study of wild fish.

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.

Mycobacterium shottsii and M. pseudoshottsii are recently described mycobacteria commonly isolated from Chesapeake Bay striped bass Morone saxatilis. However, their distribution in striped bass outside of the Chesapeake region and their ability to infect alternative hosts have not been described. Mycobacteria identified as M. shottsii (based on fatty acid methyl ester analysis and multigene sequencing) were isolated from striped bass collected in Albemarle Sound, North Carolina, and white perch Morone americana in the Rhode River, Maryland, and detected in striped bass from the New York Bight off Long Island, New York. Mycobacterium pseudoshottsii were isolated from white perch in the Rhode and Corsica rivers, Maryland, and detected in striped bass in the New York Bight. This work demonstrates that these mycobacteria can be found outside of the Chesapeake Bay as well as in hosts other than striped bass.

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 degrees C. Mycobacteria were recovered from 76% (n = 149/196) of fish examined. Mycobacterial densities exceeded 10(4) colony forming units x g tissue(-1) in 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.