Experimental mycobacteriosis in striped bass Morone saxatilis

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.     

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

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.     

Water balance in eggs of striped bass (Morone saxatilis)

Measurements of embryonic volume revealed regulation capability before hatch. Water diffusion permeability, determined as tritiated water efflux, was found to be low, but within the range for fish eggs. The perivitelline fluid is readily interchangeable with the external medium due to a high chorion permeability to both water and salt.     

Characterization of a binary tandem domain F-type lectin from striped bass (Morone saxatilis)

Among other functions, lectins play an important role in the innate immune response of vertebrates and invertebrates by recognizing exposed glycans on the surface of potential pathogens. Despite the typically weak interaction of lectin domains with their carbohydrate ligands, they usually achieve high avidity through oligomeric structures or by the presence of tandem carbohydrate-binding domains along the polypeptide. The recently described structure of the fucose-binding European eel agglutinin revealed a novel lectin fold (the "F-type" fold), which is shared with other carbohydrate-binding proteins and apparently unrelated proteins from prokaryotes to vertebrates, and a unique fucose-binding sequence motif. Here we described the biochemical and molecular characterization of a unique fucose-binding lectin (MsaFBP32) isolated from serum of the striped bass (Morone saxatilis), composed of two tandem domains that exhibit the eel carbohydrate recognition sequence motif, which we designate F-type. We also described a novel lectin family ("F-type") constituted by a large number of proteins exhibiting greater multiples of the F-type motif, either tandemly arrayed or in mosaic combinations with other domains, including a putative transmembrane receptor, that suggests an extensive functional diversification of this lectin family. Among the tandem lectins, MsaFBP32 and other tandem binary homologues appear unique in that although their N-terminal domain shows close similarity to the fucose recognition domain of the eel agglutinin, their C-terminal domain exhibits changes that potentially could confer a distinct specificity for fucosylated ligands. In contrast with the amniotes, in which the F-type lectins appear conspicuously absent, the widespread gene duplication in the teleost fish suggests these F-type lectins acquired increasing evolutionary value within this taxon.     

Thermal tolerance of a northern population of striped bass Morone saxatilis

Thermal tolerance of age 0+ year Shubenacadie River (Nova Scotia, Canada) striped bass Morone saxatilis juveniles (mean ± s . e . fork length, L _F, 19·2 ± 0·2 cm) acclimated in fresh water to six temperatures from 5 to 30° C was measured by both the incipient lethal technique (72 h assay), and the critical thermal method ( C _m). The lower incipient lethal temperature ranged from 2·4 to 11·3° C, and the upper incipient lethal temperature ( I _U) from 24·4 to 33·9° C. The area of thermal tolerance was 618° C². In a separate experiment, the I _U of large age 2+ year fish (34·4 ± 0·5 cm L _F) was 1·2 and 0·6° C lower ( P < 0·01) than smaller age 1+ year fish (21·8 ± 0·5 cm L _F) at acclimation temperatures of 16 and 23° C. Using the C _m, loss of equilibrium occurred at 27·4–37·7° C, loss of righting response at 28·1–38·4° C and onset of spasms at 28·5–38·8° C, depending on acclimation temperature. The linear regression slopes for these three responses were statistically similar (0·41; P > 0·05), but the intercepts differed (25·3, 26·0 and 26·5° C; P < 0·01). The thermal tolerance of this northern population appears to be broader than southern populations.     

Ultrastructure of Mycobacterium marinum granuloma in striped bass Morone saxatilis

An emerging epizootic of mycobacteriosis currently threatens striped bass Morone saxatilis populations in Chesapeake Bay, USA. Several species of mycobacteria, including Mycobacterium marinum, species resembling M. avium, M. gordonae, M. peregrinum, M. scrofulaceum and M. terrae, and the new species M. shottsii have been isolated from diseased and healthy bass. In this study, we describe the ultrastructure of developing M. marinum granulomas in experimentally infected bass over a period of 45 wk. The primary host response to injected mycobacteria was formation of large macrophage aggregations containing phagocytosed bacilli. M. marinum were always contained within phagosomes. Close association of lysosomes with mycobacterial phagosomes, as well as the presence of electron-opaque material within phagosomes, suggested phagolysosomal fusion. Development of granulomas involved epithelioid transformation of macrophages, followed by appearance of central necrosis. Desmosomes were present between mature epithelioid cells. The necrotic core region of M. marinum granulomas was separated from overlying epithelioid cells by several layers of flattened, electron-opaque spindle-shaped cells. These cells appeared to be formed by compression of epithelioid cells and, aside from a flattened nucleus, did not possess recognizable organelles. Following the development of well-defined, paucibacillary granulomas, secondary disease was observed. Recrudescence was marked by bacterial replication followed by disruption of granuloma architecture, including loss of epithelioid and spindle cell layers. In advanced recrudescent lesions, normal tissue was replaced by macrophages, fibroblasts, and other inflammatory leukocytes. Large numbers of mycobacteria were observed, both intracellular and suspended in cellular debris.     

Apolipoprotein A-I from striped bass (Morone saxatilis) demonstrates antibacterial activity in vitro

HDL and apolipoprotein A-I from teleostean fishes demonstrate in vitro activity against gram-positive and gram-negative bacteria. In this study, we purified ApoA-1 from striped bass (Morone saxatilis) plasma and examined its in vitro antibacterial activity against Streptococcus sp., Escherichia coli, and Mycobacterium marinum. In addition, we obtained sequence for a putative striped bass ApoA-1 gene, which when translated contained the identical sequence generated from N-terminal sequencing of the purified ApoA-1. The predicted secondary and tertiary structures contained the characteristic proline residues and high alpha-helical content conserved between mammals and fishes. Purified ApoA-1 exhibited antibacterial activity against the bacteria assayed. Concentrations of 125 microg/mL for E. coli, 250 microg/mL for Streptococcus sp., and 250 microg/mL for M. marinum, inhibited bacterial growth by 50% compared to control. ApoA-1 plasma concentrations in experimental and wild fish ranged from undetectable levels to greater than 5 mg/mL, indicating that striped bass ApoA-1 is an effective antibacterial agent at concentrations below the range of physiological concentrations in striped bass plasma. We therefore conclude that ApoA-1 could play a role in innate defense against bacterial pathogens in striped bass.     

Short-term infection of striped bass Morone saxatilis with Mycobacterium marinum

Striped bass Morone saxatilis were studied in order to characterize their immune responses over the short term following challenge with Mycobacterium marinum. The expression of immunity-related genes (IL-1beta, TNF-alpha, Nramp and TGF-beta) quickly increased following infection with M. marinum, but these genes were subsequently down-regulated despite the fact that bacterial counts remained high. The number of monocytes and neutrophils also initially increased at 1 d postinfection. This confirms the importance of these types of cells in initial inflammation and mycobacterial infection in striped bass. The phagocytic index of splenic leukocytes over these same time frames did not change significantly following infection. The discrete window in which inflammatory mechanisms were stimulated in striped bass may be related to the intracellular nature of this pathogen.     

Epitheliocystis disease in the striped bass Morone saxatilis from the Chesapeake Bay.

Epitheliocystis disease in the gills of the striped bass Morone saxatilis from Chesapeake Bay was studied using light and electron microscopy. The epitheliocystis infection appeared synchronous in that all capsules on a single host were at the same stage of development. The disease appeared to begin in a single cell on the gill lamella, which gradually enlarged to form a large cyst, encapsulated by a thick cellular capsule of epithelial origin. The epitheliocystis inclusion was filled with cells of the general morphology and size of rickettsiae; however, the infection was atypical for rickettsiae in that the cells had a dense central nucleoid region and they developed within an inclusion separated from the host cytoplasm.     

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

A large diversity of Mycobacterium spp. has been isolated from striped bass Morone saxatilis in Chesapeake Bay, USA. The new species M. shottsii and M. pseudoshottsii are the dominant isolates, while the classical fish pathogen M. marinum is found much less frequently. M. fortuitum and M. chelonae, other Mycobacterium spp. known to commonly infect fishes, have not yet been aseptically isolated from striped bass within Chesapeake Bay. While M. pseudoshottsii and M. shottsii have been phenotypically and genotypically characterized, other less common mycobacterial isolates have not. In the present study, we describe 17 photochromogenic isolates from Chesapeake Bay striped bass using phenotypic characterization and multilocus sequencing of 16S rRNA, hsp65 and rpoB genes. Genetic characterization reveals that these isolates are related to widely divergent portions of the mycobacterial phylogeny; however, some interesting trends are observed, such as a majority of isolates (10/17) belonging to the M. simiae-related grouping. Five additional isolates were assigned to the slow-growing mycobacteria (including 2 identified as M. marinum), while 2 are clearly shown to belong genetically to the fast-growing mycobacteria.     

Hypoxia tolerance variance between swimming and resting striped bass Morone saxatilis

Individual striped bass Morone saxatilis were each exposed in random order to aquatic hypoxia (10% air saturation) either while swimming at 50% of their estimated critical swimming speed (U_crit) or while at rest until they lost equilibrium. Individuals were always less tolerant of hypoxia when swimming (P < 0·01); the average fish was over five times more tolerant to the same hypoxia exposure when not swimming. There was no relationship between an individual's rank order of hypoxia tolerance (HT) under the two flow regimes, suggesting that different factors determine an individual's HT when at rest than when swimming.     

Comparative analysis of mycobacterial infections in wild striped bass Morone saxatilis from Chesapeake Bay

During an ongoing epizootic of mycobacteriosis, wild striped bass Morone saxatilis from Chesapeake Bay were analyzed using 3 methods for detection of either mycobacterial infection or associated granulomatous pathology. The specific detection techniques, which utilized aseptically collected splenic tissue, were histology, quantitative culture and nested PCR. Based on analysis of 118 samples, detection of infection differed significantly between the 3 methods (chi-square, p = 0.0007). Quantitative culture and nested PCR detected similar, higher rates of infection (69 and 75%, respectively) than the histological method (52%). Although primary PCR assays for a 924 to 940 bp segment of the mycobacterial 16S rRNA gene were positive for genomic DNA from mycobacterial cultures, a secondary, nested PCR reaction for an internal 300 bp gene segment was required in order to detect mycobacteria within splenic tissue. A similar rate of mycobacterial infection was present in fish collected from all sites tested. Although all detection methods found that striped bass age 4.0 to 4.9 yr had the highest positive incidence, nested PCR detected a higher frequency of mycobacterial infection in fish > or = 6.0 yr of age than the other 2 methods. Quantitative bacteriology was a more sensitive detection technique when the fish tissue contained < or = 10(3) mycobacteria g(-1).     

Parasites and diseases of striped bass, Morone saxatilis (Walbaum), from the lower Chesapeake Bay

A survey to determine presence of parasitic and disease organisms and their effect on estuarine populations of striped bass, Morone saxatilis (Walbaum), was conducted monthly from May 1972 to May 1973. A total of 514 fish over 1 year old and 140 young-of-the-year were examined using standard necropsy and histological procedures. Other species of fishes were studied to determine the specificity of striped bass parasites and to determine if other fishes were reservoirs for striped bass pathogens. Forty-five species of parasitic organisms from viruses to Metazoa were recognized from striped bass. Heavy infections by some were associated with definite pathological conditions.     

An in vitro assay to measure phagocytosis in striped bass hybrids (Morone saxatilis x Morone chrysops)

An in vitro phagocytosis assay was developed for hybrid striped bass (Morone saxatilis x Morone chrysops), using cells collected from the peritoneal cavity of this fish. The findings indicated that: (1) 10 days following a single intraperitoneal injection (1 ml) of Freund's incomplete adjuvant (FIA) was an appropriate time for collecting suitable working concentrations (5.3+/-4.0 x 10(7) cells ml(-1)) of peritoneal phagocytes (83.7+/-1.5% macrophages) from these hybrids held at 23 degrees C; (2) these cells phagocytosed latex beads (polystyrene microspheres 3.12 microm in diameter) after 30 min of in vitro incubation at room temperature (25+/-1 degrees C). The phagocytic ability and phagocytic capacity in a washed adherent layer exposure system were 67.2+/-2.76% and 4.14+/-0.35 beads phagocyte(-1), respectively. These results strongly suggest that a simple methodology, including baseline data serving as guidelines, is now available for conducting in vitro phagocytosis assays in this hybrid.     

Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis)

The time course of osmoregulatory adjustments and expressional changes of three key ion transporters in the gill were investigated in the striped bass during salinity acclimations. In three experiments, fish were transferred from fresh water (FW) to seawater (SW), from SW to FW, and from 15-ppt brackish water (BW) to either FW or SW, respectively. Each transfer induced minor deflections in serum [Na+] and muscle water content, both being corrected rapidly (24 hr). Transfer from FW to SW increased gill Na+,K+-ATPase activity and Na+,K+,2Cl- co-transporter expression after 3 days. Abundance of Na+,K+-ATPase alpha-subunit mRNA and protein was unchanged. Changes in Na+,K+,2Cl- co-transporter protein were preceded by increased mRNA expression after 24 hr. Expression of V-type H+-ATPase mRNA decreased after 3 days. Transfer from SW to FW induced no change in expression of gill Na+,K+-ATPase. However, Na+,K+,2Cl- co-transporter mRNA and protein levels decreased after 24 hr and 7 days, respectively. Expression of H+-ATPase mRNA increased in response to FW after 7 days. In BW fish transferred to FW and SW, gill Na+,K+-ATPase activity was stimulated by both challenges, suggesting both a hyper- and a hypo-osmoregulatory response of the enzyme. Acclimation of striped bass to SW occurs on a rapid time scale. This seems partly to rely on the relative high abundance of gill Na+,K+-ATPase and Na+,K+,2Cl- co-transporter in FW fish. In a separate study, we found a smaller response to SW in expression of these ion transport proteins in striped bass when compared with the less euryhaline brown trout. In both FW and SW, NEM-sensitive gill H+-ATPase activity was negligible in striped bass and approximately 10-fold higher in brown trout. This suggests that in striped bass Na+-uptake in FW may rely more on a relatively high abundance/activity of Na+,K+-ATPase compared to trout, where H+-ATPase is critical for establishing a thermodynamically favorable gradient for Na+-uptake.     

Blood oxygen transport in stressed striped bass (Morone saxatilis): role of beta-adrenergic responses

Summary Strenuous 5-min exercise resulted in a 0.3 unit drop in the dorsal aortic pH of striped bass. The acidosis was metabolic: the blood lactate concentration increased during the exercise, whereas blood CO₂ tension decreased. Dorsal aortic oxygen content was maintained despite the acidosis. This was a result of increased blood O₂ tension, haemoglobin concentration and red cell volume, decreased cellular nucleoside triphosphate (NTP) concentration, and decreased proton gradient across the red cell membrane. When the fish were treated with the beta-antagonist, propranolol, before the exercise, the arterial oxygen content decreased significantly in the stress. The mean cellular haemoglobin concentration and cellular NTP concentration increased slightly, and the proton gradient across the red cell membrane decreased less than in control exercise. These results show that the beta-adrenergic responses of striped bass red cells play an important role in maintaining the arterial O₂ content in stress.Abbreviation NTP nucleoside triphosphates     

Biochemical responses to temperature in the contractile protein complex of striped bass Morone saxatilis

The effects of acute and long-term changes in temperature upon catalytic and calcium regulatory function of red (slow oxidative) and white (fast glycolytic) muscle from striped bass (Morone saxatilis) were determined. Acclimation to 5 degrees C or 25 degrees C had no significant effect on catalytic function (ATPase activity) or regulatory sensitivity (Ca++-activation) of myofibrils from either muscle type. Substantial differences between red and white muscle were found in the intrinsic thermal sensitivity of maximally-activated Mg++-Ca++ myofibrillar ATPase. Arrhenius plots of myofibrillar ATPase from white muscle show one significant breakpoint at 29 degrees C, with activation energies (Ea) of 2.3 and 23.4 kcal mole-1 at temperatures above and below this transition, respectively. Arrhenius plots of myofibrillar ATPase from red muscle show two transitions occurring at 22 and 9 degrees C, with Ea of 7.6 kcal mole-1 above 22 degrees C and 18.3 kcal mole-1 between 9 and 22 degrees C. Activation energies for myofibrils from red muscle increase substantially to approximately 107.3 kcal mole-1 below the 9 degrees C breakpoint. Differences in the intrinsic thermal sensitivity of red and white muscle catalytic function are apparently due to interaction of actomyosins and calcium regulatory proteins which are specific to each muscle type. The results suggest that capacity for sustained swimming in striped bass, which is powered exclusively by red muscle, will be severely impaired at cold temperature unless compensations occur above the level of contractile proteins.