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.     

Biochemical, molecular, and virulence characteristics of select Mycobacterium marinum isolates in hybrid striped bass Morone chrysops x M. saxatilis and zebrafish Danio rerio

A panel of 15 Mycobacterium marinum isolates was characterized by biochemical tests, sequencing the ribosomal DNA intergenic spacer (ITS) region and the heat shock protein 65 gene (hsp65) and pulsed-field gel electrophoresis (PFGE). The biochemical characteristics of all isolates were similar, except for Tween 80 hydrolysis. DNA sequence of hsp65 for a subset of isolates were identical; however, at position 5 of the ITS rDNA, a single nucleotide polymorphism was identified. Isolates possessing a guanine residue at this position (G strains) were unable to hydrolyze Tween 80, while isolates that contained an adenine residue at this position (A strains) were positive for Tween 80 hydrolysis. PFGE successfully discriminated between the G and A strains; all G strains had identical AseI restriction enzyme-cutting patterns while the A strains exhibited a variety of cutting patterns. Eight isolates (4 G and 4 A strains) were further characterized for virulence by experimental infection of hybrid striped bass (HSB) Morone chrysops x M. saxatilis and zebrafish Danio rerio. Seven of the 8 strains produced cumulative mortality ranging from 13.3 to 83.3% in the HSB virulence trial. The M. marinum reference strain ATCC 927T did not produce mortality in HSB. HSB exposed to the G strains had significantly higher cumulative mortality than those exposed to the A strains. When these same isolates were tested in zebrafish, 6 of the 8 strains caused 100% cumulative mortality, with 2 of the A strains being the most pathogenic. In zebrafish, however, ATCC 927T was virulent and produced 28.5% mortality. Collectively, we conclude that the M. marinum G strains are unique and may represent a distinct virulence phenotype in HSB, but this trend was not consistent in zebrafish.     

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.     

Aquatic Francisella-like bacterium associated with mortality of intensively cultured hybrid striped bass Morone chrysops x M. saxatilis

The present study identifies an emerging disease associated with an aquatic Francisella-like bacterium that can cause mortality in hybrid striped bass Morone chrysops x M. saxatilis reared intensively in freshwater. Clinically affected fish were lethargic, had scattered haemorrhagic cutaneous lesions and diffuse gill pallor. The head kidney and spleen were markedly swollen and contained numerous interstitial granulomas; histological examination revealed small, pleomorphic Gram-negative coccobacilli within vacuolated cells. The bacterium could not be cultured from head kidney homogenates either with standard or enriched microbiological media or following inoculation of a Chinook salmon embryo (CHSE)-214 cell line. No amplification product was obtained from head kidney DNA by polymerase chain reaction (PCR) assay using Piscirickettsia salmonis-specific primers. PCR analysis of infected head kidney homogenate with primers designed for the eubacterial 16S rRNA produced a single amplicon. Phylogenetic analysis of this DNA sequence demonstrated that the sequence aligned most closely with members of the genus Francisella, identified from tilapia Oreochromis spp. in Taiwan and an aquatic Francisella species that was recently isolated from the three-line grunt Parapristipoma trilineatum in Japan. This Francisella-like disease was transmitted to naive hybrid striped bass fingerlings by intraperitoneal injection of tissue homogenates prepared from a natural outbreak. All fish developed gross and histological lesions identical to those from natural outbreaks. Intracellular Gram-negative bacteria were observed within the cytoplasm of cells (presumably macrophages) within the granulomas, but bacteria were not recovered. The 16S DNA sequence of the bacterium obtained from tissues of experimentally infected fish was identical to that obtained from the fish used as infected donor tissue.     

Dietary oligonucleotides from yeast RNA influence immune responses and resistance of hybrid striped bass (Morone chrysops x Morone saxatilis) to Streptococcus iniae infection

Three feeding trials were conducted to evaluate potential immunomodulatory effects of nucleotides in the diet of hybrid striped bass. A basal diet was formulated from menhaden fish meal to contain 40% crude protein and 10% lipid. An oligonucleotide product (Ascogen P) from brewer's yeast was added to the basal formulation at the manufacturer's recommended rate of 0.5% to produce the experimental diet. Each diet was fed to four replicate groups of juvenile hybrid striped bass for seven or eight weeks in two separate trials. After Trials 1 and 2, a Streptococcus iniae bath challenge was executed to test the effects of diet on disease resistance. No significant difference in growth performance was observed between fish fed the basal and experimental diets. Body composition of whole fish, hematocrit and serum lysozyme levels were observed to be within normal ranges and not influenced by dietary nucleotides. Neutrophil oxidative radical production of fish fed the nucleotide-supplemented diet was significantly (P=0.011) higher than in fish fed the basal diet. Significantly (P<0.05) enhanced survival after exposure to S. iniae also was generally observed in fish fed the nucleotide-supplemented diet. In addition, fish fed the nucleotide-supplemented diet tended to have a higher antibody response based on microtitration agglutination; however, the difference was not statistically significant because of high variation between individual fish. Long-term (16 weeks) administration of oligonucleotides in Trial 3 failed to show enhancement of immune responses between treatments. It is concluded that dietary oligonucleotides positively influenced immune responses and resistance of juvenile hybrid striped bass to S. iniae infection.     

Temperature affects physiological stress responses to acute confinement in sunshine bass (Morone chrysops x Morone saxatilis)

Sunshine bass (Morone chrysopsxMorone saxatilis) were subjected to a 15-min low-water confinement stressor at temperatures ranging from 5 to 30 degrees C. Physiological responses were evaluated by measuring hematocrit, and plasma chloride, glucose and cortisol. Fish acclimated to 30 degrees C had initial glucose concentrations of 3.13 mM (564 mg/L) which were significantly lower than in fish acclimated to 5 and 10 degrees C (4.32 and 4.82 mM or 779 and 868 mg/l, respectively). Fish survived the conditions imposed at every temperature except 30 degrees C, where 15 out of 42 fish died during the stress and recovery protocol. The general pattern was an initial increase in hematocrit, followed by a delayed decrease in hematocrit and chloride, and an increase in plasma glucose and cortisol. In general, fish stressed at temperatures below 20 degrees C had lower and more delayed changes in plasma glucose and cortisol than fish tested at 20, 25 and 30 degrees C. Initial cortisol concentrations were 65 ng/ml and increased to above 200 ng/ml in fish held at 20 degrees C and above. At the higher temperatures, glucose concentrations were twice the initial concentration after stress and cortisol changes were four to five times the initial concentration after the stress. Quantitative responses for glucose and cortisol were moderate and recovery rapid in fish stressed at 10 and 15 degrees C; therefore, this range of water temperature is recommended when handling sunshine bass.     

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.     

Tissue essential fatty acid composition and competitive response to dietary manipulations in white bass (Morone chrysops), striped bass (M. saxatilis) and hybrid striped bass (M. chrysopsxM. saxatilis)

The effects of wide changes in dietary levels of docosahexaenoic (DHA) or arachidonic (ArA) acids on growth, survival and fatty acid composition in body tissues of Morone larvae were examined. White bass (WB, Morone chrysops), striped bass (SB, Morone saxatilis) and sunshine hybrid bass (HSB, M. chrysopsxM. saxatilis) larvae (day 24-46) were fed Artemia nauplii enriched with algal sources of varying proportions of DHA and ArA (from 0 to over 20% of total fatty acids). WB larvae fed DHA-deficient Artemia diet retarded over 50% of their potential growth, however, increasing dietary DHA/ArA ratios were associated with a significant growth improvement. The highest proportion of polyunsaturated fatty acids was found in WB neural tissue (approx. 50% of total fatty acids), while HSB neural tissue contained the highest proportion of saturated fatty acids (approx. 35% of total fatty acids). Within the neural tissues of all Morone larvae, both DHA and ArA were generally the most dominant as well as the most responding fatty acids to dietary manipulations (except in WB fed DHA or ArA deficient diets). HSB neural tissue was particularly efficient in retaining a significant amount of DHA in the face of dietary deficiency. However, WB neural tissue was the most responsive to dietary increase in DHA, accumulating a significantly higher amount of DHA (P<0.05) than SB or HSB. Results demonstrate significant differences in fatty acid composition and growth responsiveness to dietary manipulations between Morone larvae species and within specific tissues. WB weight gain and neural tissue composition was affected most by dietary changes in both DHA and ArA whereas SB and HSB tissue compositions were generally less affected by dietary manipulations.     

The effect of storing hybrid bass (Morone saxatilis x M. chrysops) sera at freezing temperatures on antibody titers

The effect of storing hybrid bass sera at freezing temperatures on their enzyme-linked immunosorbent assay (ELISA) titers was determined, and the precipitate formed during storage was characterized. Frozen bass sera from fish vaccinated with bacterial sonicate showed no titer in either of two groups-one immunized with bacterial sonicate and the other with sonicate emulsified in adjuvant. Fresh sera exhibited average titers of 1:32 in both groups and 1:20 in convalescent serum.Hybrid bass antisera to bovine serum albumin showed maximum ELISA OD when sera were used after a short storage at 4 degrees C. Storage at freezing temperatures (-20 and -73 degrees C) reduced the OD value substantially. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of the cryoprecipitate formed after freezing hybrid bass sera showed protein bands at 85 and 38 kDa, most probably the heavy and light chains, respectively, of bass IgM. Bands at 55, 52, 28, and 26 kDa may represent fibrinogen subunits. It is possible that the cryoprecipitate is composed of a fibrinogen net, which traps the tetrameric IgM molecules and precipitates them during frozen storage.     

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.     

In vitro manipulations of vitamin C and vitamin E concentrations alter intracellular O2- production of hybrid striped bass (Morone chrysops x Morone saxatilis) head-kidney cells

To examine the mechanism by which vitamins C and E alter phagocyte function, a series of in vitro manipulations were conducted with cells isolated from the head-kidney of hybrid striped bass (average weight 680 g) fed a diet supplemented with minimum requirement levels of vitamins C and E for 2 weeks. Head-kidney phagocytes were cultured in media containing physiologically deficient (23 microM, adequate (45 microM) or excessive (182 microM) concentrations of vitamin C, and physiologically deficient (5 microM), adequate (9 microM) or excessive (32 microM) concentrations of vitamin E for 18 h. Following culture and stimulation, levels of reactive oxygen intermediates and hydrogen peroxide were determined. There were no effects of vitamin C or vitamin E concentrations on hydrogen peroxide or extracellular O2- generation. Intracellular O2- production, however, was significantly (P < or = 0.05) affected. When vitamin C was supplied at deficient levels to the medium, vitamin E elevated O2- production to levels not different from those of cells incubated with requirement levels of both vitamins. Similarly, when vitamin E was deficient in the media, vitamin C supplementation at requirement levels normalised intracellular O2- production. This data provides support for the presence of a vitamin C and vitamin E sparing mechanism in phagocytic head-kidney cells of hybrid striped bass and yield some insight into the mechanisms by which vitamin C and vitamin E function in immunomodulation.     

The effect of growth medium salinity of Photobacterium damselae subsp. piscicida on the immune response of hybrid bass (Morone saxatilis x M. chrysops)

Photobacterium damselae subsp. piscicida (P. damselae) was grown on various media and the effect of media salinity on certain immune responses of hybrid bass was studied. In Israel, pasteurellosis outbreaks have not been reported at water salinities below 1.38 per thousand. During vaccination experiments the salinity of the medium on which P. damselae is grown, was shown to affect stimulation of the immune system. No correlation was found between antibody response and protection. Bacterial envelopes separated by electrophoresis and subjected to western blot analysis revealed an antibody response against some protein bands. Band sequencing was performed to identify the protein stimulating the immune response. Sequence identity of 80% was seen in 10-amino-acid overlap of the 36-kDa band with a specific gene of alkalophilic Bacillus firmus. A preparation of P. damselae grown in a 2.5% NaCl medium at 25 degrees C is the most effective vaccine against pasteurellosis, providing hybrid bass with quite good protection.     

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.     

Isolation and characterization of 149 novel microsatellite DNA markers for striped bass,Morone saxatilis,and cross‐species amplification in white bass,Morone chrysops,and their hybrid

To support detailed genetic analysis of striped bass (Morone saxatilis) and white bass (Morone chrysops), we isolated 153 microsatellite loci from repeat‐enriched striped bass DNA libraries. Of these, 147 markers amplified in striped bass (average 4.7 alleles per locus) and 133 in white bass (average 2.2 alleles per locus). One hundred twenty‐two markers amplified in their hybrid. Development of new microsatellite markers will facilitate evaluations of genetic structure in wild populations and will support pedigree analysis and linkage mapping for selective breeding.     

Comparative severity of experimentally induced mycobacteriosis in striped bass Morone saxatilis and hybrid tilapia Oreochromis spp

Twenty striped bass Morone saxatilis and 20 hybrid tilapia Oreochromis niloticus x O. mossambicus x O. aureus each received a single intramuscular injection of 1.6 x 10(6) colony forming units per gram body weight of Mycobacterium marinum. Striped bass manifested significantly greater clinical and microscopic disease compared to tilapia. Whereas all the striped bass had died or were clinically ill by Day 8 post-infection, there was no apparent disruption of normal behaviour, physical appearance, or growth in any of the sacrificed or surviving tilapia. Histologically, granulomas in striped bass were generally larger and less discrete, with a higher proportion of heavily vacuolated macrophages, and large cores of necrotic cells. Visceral granulomas in tilapia were smaller, with a higher proportion of epithelioid macrophages, more pigment-containing cells, more peripheral lymphocytes, and virtually no central necrosis. Visceral granulomas were 18-fold more numerous in striped bass than in tilapia. Based upon histomorphometric data, mean proportions of acid-fast bacteria within pronephros granulomas were 4-fold greater in striped bass than tilapia, and striped bass granulomas averaged more than twice as large as tilapia granulomas. In the anterior kidney of striped bass, a positive correlation existed between mean mycobacterial proportions and mean necrosis scores. In tilapia, mean mycobacterial proportions correlated negatively with mean granuloma numbers, whereas there was no correlation between these parameters in striped bass. Results suggest that intrinsic functional differences in the immunologic systems of striped bass and hybrid tilapia may contribute to inter-species variation in mycobacteriosis susceptibility.     

Piscidin-1, an antimicrobial peptide from fish (hybrid striped bass morone saxatilis x M. chrysops), induces apoptotic and necrotic activity in HT1080 cells

Piscidin-1, a 22-residue cationic peptide isolated from mast cells of a hybrid striped bass, has potent antimicrobial activities against both gram-positive and -negative bacteria. To date, there is no report of its antitumor activity on any tumor cell lines. In this study, we examined the antitumor activity of a synthetic piscidin-1 peptide against several human cancer cell lines using an MTS assay and soft-agar colony-formation assay. We found that a low dose of piscidin induces both apoptosis and necrosis in HT1080 cells, as shown by annexin-V/propidium iodide and acridine orange/ethidium bromide staining, and triggers a necrotic cell death pathway in a short period with high-dose treatment. The destruction of cell membranes by piscidin-1 was demonstrated by transmission electron microscopy. Furthermore, piscidin-1 also inhibits the migration of HT1080 cells in a dose-dependent manner. This study provides the first evidence of the anticancer activity of the antimicrobial peptide, piscidin-1, with potential implications for the treatment of cancer.     

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     

Microsatellite DNA markers for parental assignment in hybrid striped bass (Morone saxatilis × Morone chrysops)

Development of nine polymorphic microsatellites from a genomic library of hybrid striped bass (female Morone chrysops × male Morone saxatilus) DNA is described. Breeding of hybrid striped bass for aquaculture is based largely on breeding wild fish. Molecular markers such as microsatellites will be useful tools for developing broodstock, estimating heritability for production traits, and selective breeding via marker‐assisted selection. The nine polymorphic microsatellites include six dinucleotide and three complex repeat motifs. The number of alleles detected among a sample of 10 individuals of each species was relatively low. All polymerase chain reaction primer pairs also amplified products in the sea bass Dicentrarchus labrax.