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In mammals, the natural resistance-associated macrophage protein 1 gene,Nramp1, plays a major role in resistance to mycobacterial infections. Chesapeake Bay striped bass (Morone saxatilis) is currently experiencing an epizootic of mycobacteriosis that threatens the health of this ecologically and economically important species. In the present study, we characterized anNrampgene in this species and obtained evidence that there is induction followingMycobacteriumexposure. The striped bassNrampgene (MsNramp) and a 554-amino-acid sequence contain all the signal features of theNrampfamily, including a topology of 12 transmembrane domains (TM), the transport protein-specific binding-protein-dependent transport system inner membrane component signature, three N-linked glycosylation sites between TM 7 and TM 8, sites of casein kinase and protein kinase C phosphorylation in the amino and carboxy termini, and a tyrosine kinase phosphorylation site between TM 6 and TM 7. Phylogenetic analysis most closely groupedMsNrampwith other teleostNrampgenes and revealed high sequence similarity with mammalianNramp2. MsNrampexpression was present in all tissues assayed by reverse transcription-PCR. Within 1 day of injection ofMycobacterium marinum,MsNrampexpression was highly induced (17-fold higher) in peritoneal exudate (PE) cells compared to the expression in controls. The levels ofMsNrampwere three- and sixfold higher on days 3 and 15, respectively. Injection ofMycobacterium shottsiiresulted in two-, five-, and threefold increases in gene expression in PE cells over the time course. This report is the first report of induction of anNrampgene by mycobacteria in a poikilothermic vertebrate.

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.

A group of slowly growing photochromogenic mycobacteria was isolated from Chesapeake Bay striped bass (Morone saxatilis) during an epizootic of mycobacteriosis. Growth characteristics, acid-fastness and 16S rRNA gene sequencing results were consistent with those of the genus Mycobacterium. Biochemical reactions, growth characteristics and mycolic acid profiles (HPLC) resembled those of Mycobacterium shottsii, a non-pigmented mycobacterium also isolated during the same epizootic. Sequencing of the 16S rRNA genes, the gene encoding the exported repeated protein (erp) and the gene encoding the 65 kDa heat-shock protein (hsp65) and restriction enzyme analysis of the hsp65 gene demonstrated that this group of isolates is unique. Insertion sequences associated with Mycobacterium ulcerans, IS2404 and IS2606, were detected by PCR. These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 °C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 μg ml−1), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide. On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, Mycobacterium pseudoshottsii sp. nov. The type strain, L15T, has been deposited in the American Type Culture Collection as ATCC BAA-883T and the National Collection of Type Cultures (UK) as NCTC 13318T.

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.

Striped bass Morone saxatilis were infected intraperitoneally with approximately 10(5) Mycobacterium marinum, M. shottsii sp. nov., or M. gordonae. Infected fish were maintained in a flow-through freshwater system at 18 to 21 degrees C, and were examined histologically and bacteriologically at 2, 4, 6, 8, 17, 26, 36 and 45 wk post-infection (p.i.). M. marinum caused acute peritonitis, followed by extensive granuloma development in the mesenteries, spleen and anterior kidney. Granulomas in these tissues underwent a temporal progression of distinct morphological stages, culminating in well-circumscribed lesions surrounded by normal or healing tissue. Mycobacteria were cultured in high numbers from splenic tissue at all times p.i. Standard Ziehl-Neelsen staining, however, did not demonstrate acid-fast rods in most early inflammatory foci and granulomas. Large numbers of acid-fast rods were present in granulomas beginning at 8 wk p.i. Between 26 and 45 wk p.i., reactivation of disease was observed in some fish, with disintegration of granulomas, renewed inflammation, and elevated splenic bacterial densities approaching 10(9) colony-forming units g(-1). Infection with M. shottsii or M. gordonae did not produce severe pathology. Mild peritonitis was followed by granuloma formation in the mesenteries, but, with 1 exception, granulomas were not observed in the spleen or anterior kidney. M. shottsii and M. gordonae both established persistent infections in the spleen, but were present at densities at least 2 orders of magnitude less than M. marinum at all time points observed. Granulomas in the mesenteries of M. shottsii- and M. gordonae-infected fish resolved over time, and no reactivation of disease was observed.