Comparison of antimicrobial activity in the epidermal mucus extracts of fish

The mucus layer on the surface of fish consists of several antimicrobial agents that provide a first line of defense against invading pathogens. To date, little is known about the antimicrobial properties of the mucus of Arctic char (Salvelinus alpinus), brook trout (S. fontinalis), koi carp (Cyprinus carpio sub sp. koi), striped bass (Morone saxatilis), haddock (Melanogrammus aeglefinus) and hagfish (Myxine glutinosa). The epidermal mucus samples from these fish were extracted with acidic, organic and aqueous solvents to identify potential antimicrobial agents including basic peptides, secondary metabolites, aqueous and acid soluble compounds. Initial screening of the mucus extracts against a susceptible strain of Salmonella enterica C610, showed a significant variation in antimicrobial activity among the fish species examined. The acidic mucus extracts of brook trout, haddock and hagfish exhibited bactericidal activity. The organic mucus extracts of brook trout, striped bass and koi carp showed bacteriostatic activity. There was no detectable activity in the aqueous mucus extracts. Further investigations of the activity of the acidic mucus extracts of brook trout, haddock and hagfish showed that these fish species had specific activity for fish and human pathogens, demonstrating the role of fish mucus in antimicrobial protection. In comparison to brook trout and haddock, the minimum bactericidal concentrations of hagfish acidic mucus extracts were found to be approximately 1.5 to 3.0 times lower against fish pathogens and approximately 1.6 to 6.6 folds lower for human pathogens. This preliminary information suggests that the mucus from these fish species may be a source of novel antimicrobial agents for fish and human health related applications.     

Evidence for involvement of penicillin-binding protein 3 in murein synthesis during septation but not during cell elongation

The effect of growth temperature on the loss of virulence of the fish pathogen Aeromonas salmonicida was investigated. Three virulent strains were grown in Trypticase soy broth at temperatures ranging from 22 to 30 degrees C. Growth at a higher-than-optimal temperature (26 to 27 degrees C for the three strains studied) resulted in the selection of spontaneous attenuated derivatives in the initial bacterial population. For example, virulent bacteria represented less than 10% of the population of a culture grown at 30 degrees C, and attenuated derivatives were easily isolated by streaking the culture on solid medium and picking single colonies. Virulent strains autoaggregated during growth and possessed a cell wall layer (A-layer) external to the outer membrane, as previously described. Attenuated strains did not autoaggregate and did not possess the A-layer. The A-layer apparently shielded bacteriophage receptors and a mannose-specific yeast agglutinin located in the outer membrane. Thus, virulent strains exhibited impaired adsorption of phages, whereas attenuated strains were phage sensitive. Furthermore, attenuated strains agglutinated yeast cells but virulent strains did not. The attenuated strains had higher maximum growth temperatures than their virulent parent strains, and this accounts for their selection at high temperatures. It is proposed that the A-layer contributes significantly to the physical properties of the A. salmonicida cell envelope and that these physical properties of the A. salmonicida cell envelope and that these physical change upon loss of the A-layer to permit growth at a higher-than-usual temperature.     

Human serum bactericidal activity against Haemophilus influenzae type b

We examined bactericidal and opsonizing activity of pooled adult 'immune' serum against Haemophilus influenzae type b with and without the addition of phagocytes. Four type b strains from cerebrospinal fluid (CSF) and three such strains from the nasopharynx (NP) of healthy children were examined. Duplicate reaction mixtures contained organisms in exponential (E) or stationary phase (S) of growth, serum, a complement source (human agammaglobulinaemic serum), and culture medium (bactericidal assay); separate assays contained the above components and polymorphonuclear leucocytes (opsonization system). A decrease in bacterial density of greater than or equal to 1 log10 unit was considered significant. All four S-CSF strains, three of four E-CSF strains and one of three S-NP strains were sensitive to the bactericidal activity of pooled serum. The other E-CSF strain, two S-NP strains and all three E-NP strains were resistant to the bactericidal activity of pooled serum. Two of three E-NP strains were opsonized by pooled serum; the other strains resistant to the bactericidal activity of pooled serum were also resistant to opsonization. Bactericidal and opsonizing activity of serum from an immunized adult was greater than or equal to that of pooled serum against each strain. Assuming normal adults are immune to invasive H. influenzae type b infection, an experimental test reflecting this immunity is the bactericidal activity against CSF isolates tested in stationary phase. We conclude that protection against invasive disease due to H. influenzae type b appears more complex than the presence of bactericidal and opsonizing activity in serum.     

Cell surface hydrophobicity and macrophage association ofAeromonas salmonicida

Hydrophobic interaction chromatography and salt aggregation were used to compare the call surface hydrophobicity of strains of the fish pathogenAeromonas salmonicida which differed in their ability to produce the surface protein array known as A-layer. Presence of this superficial protein layer is crucial to the virulence of this organism and was found to coincide with a dramatic increase in cell surface hydrophobicity. Assays with in vitro cultured macrophages from either rainbow trout or mice revealed that this hydrophobic A-layer providedA. salmonicida cells with an enhanced ability to associate with phagocytic monocytes. This enhanced association was demonstrated in the absence of opsonizing antibody and may have important implications in the virulence ofA. salmonicida for fish.     

Serine proteinase inhibitors of seminal plasma of teleost fish: distribution of activity, electrophoretic profiles and relation to proteinase inhibitors of blood

Anti-proteinase activity has been found in seminal plasma of eight teleost fish species: brown trout, rainbow trout, brook trout, lake whitefish, bream, northern pike, Danube salmon and burbot. This activity correlated with seminal plasma protein and sperm concentrations. Using a mammalian (bovine) trypsin for detecting proteinase inhibitors it was found for the first time that there are species-specific electrophoretic profiles of anti-proteinase activity. One to three bands could be identified by this method. However, additional proteinase inhibitors could be identified by using fish (cod) trypsin. These inhibitors were detected in seminal plasma of salmonids and coregonids and have a slow migration rate. Fast-migrating proteinase inhibitors were present in rainbow, brown and brook trout, northern pike, whitefish and burbot. These inhibitors could be detected in brook and brown trout by using either trypsins. However, they were detected only with bovine trypsin in rainbow trout, northern pike, whitefish and burbot. These results suggest that multiple forms of serine proteinase inhibitors exist in seminal plasma of teleost fish and they differ in their affinity toward serine proteinases. Seminal plasma serine proteinase inhibitors of rainbow trout migrated during electrophoresis similarly to blood plasma proteinase inhibitors, and suggests that the two inhibitors may be similar or the same. Anti-proteinase specific activity was similar in blood and seminal plasma. Proteinase inhibitors of fish seminal plasma seem to be an important part of sperm physiology, possibly related to protection of spermatozoa. Staining for detection of serine proteinase inhibitors also allowed detection of presence of nonspecific esterase in seminal plasma of most species.     

A temperate bacteriophage specific for strains ofAeromonas salmonicida possessing A-layer,a cell surface virulence factor

A temperate bacteriophage designated TP446 was isolated from culture supernatants ofAeromonas salmonicida strain A446. Phage TP446 adsorbed to all of the typical and atypical strains ofA. salmonicida tested that possessed A-layer, the surface protein array that represents the primary virulence factor of this fish pathogen. In contrast, TP446 failed to adsorb to mutants lacking A-layer. These results indicate that the A-layer is a component of the receptor for phage TP446.     

Effect of gonadotropin-releasing hormone on phagocytic leucocytes of rainbow trout

To clarify the role of gonadotropin-releasing hormone (GnRH) in the fish immune system, in vitro effect of GnRH was examined in phagocytic leucocytes of rainbow trout (Oncorhynchus mykiss). Gene expression of GnRH-receptor was detected by RT-PCR in leucocytes from head kidney. Administration of sGnRH increased proliferation and mRNA levels of a proinflammatory cytokine, tumor necrosis factor (TNF)-α, in trout leucocytes. Superoxide production in zymosan-stimulated phagocytic leucocytes was also increased by sGnRH in a dose-related manner from 0.01 to 100 nM. There was no significant effect of sGnRH on mRNA levels of growth hormone (GH) expressed in trout phagocytic leucocytes. Immunoneutralization of GH by addition of anti-salmon GH serum into the medium could not block the stimulatory effect of sGnRH on superoxide production. These results indicate that GnRH stimulates phagocytosis in fish leucocytes through a GnRH-receptor-dependent pathway, and that the effect of GnRH is not mediated through paracrine GH in leucocytes.     

Cytogenetic identification of genomic elements of brook trout (Salvelinus fontinalis) in the karyotype of Arctic char (Salvelinus alpinus) from the aquaculture broodstock

Growing interest of Arctic char (Salvelinus alpinus) aquaculture in Europe, and the fact that it can easily hybridize with brook trout (Salvelinus fontinalis) resulting in fertile progeny, led us to investigate fish from the farmed stocks. Chromosomes of sampled Arctic char were examined using conventional and molecular cytogenetic (FISH) techniques in order to determine possible contamination of genomic elements of brook trout. Investigated fish possessed karyotypes composed of 80–82 chromosomes and up to three chromosome fragments. Using staining methods and FISH approach enabled identification of the brook trout chromosomes in the eight out of twenty‐two examined Arctic char. Specific location of AT‐, GC‐ positive and NOR sites observed on chromosomes as well as chromosome fragments in the karyotypes of several individuals points on past chromosomal rearrangements in fish from examined broodstock. Based on our results, it may be assumed that individuals with the brook trout genomic elements, although phenotypically identified as Arctic chars, were hybrids. Our results highlights that special care should be taken to protect gene pools of brook trout and Arctic char in farms where both species are cultured.     

Effect of nutritional factors and cortisol on tyrosine aminotransferase activity in liver of brook trout, Salvelinus fontinalis Mitchill.

1. Starvation for over 50 days increased the activity of Tyrosine aminotransferase (TAT) from brook trout liver and decreased liver glycogen. 2. Cortisol (6-40 mg/100 g body wt) increased TAT activity, optimally 72 hr post-injection; liver glycogen response to cortisol was variable while tissue water either increased or remained constant. 3. Fish fed high-protein/low-carbohydrate had greater TAT activity than fish fed low-protein/high carbohydrate. There was food-induced peak of activity indicating a "diurnal rhythm" analogous to that of rat TAT. 4. TAT activity in brook trout appears to be correlated with protein catabolism.     

Fall synchrony between leaf color change and brook trout spawning in the Laurentides Wildlife Reserve (Québec,Canada) as potential environmental integrators

Fall biological processes are driven by a combination of environmental factors, with cumulative effects over the length of the growing season, which are currently difficult to model. This study evaluated if leaf color change in fall (i.e., leaf yellowing) and brook trout spawning could be two biological processes that are synchronized at a regional scale and if leaf yellowing could be used to determine the peak of spawning activity of this species. To this end, we surveyed 551 brook trout redds and examined 193 digital images of forest trees in the Laurentides Wildlife Reserve (Québec, Canada) over the fall season. Results showed that leaf yellowing and brook trout spawning were synchronized, providing one of the first examples of temporal matching between freshwater and terrestrial biological processes at the regional scale. Considering the difficulty of monitoring the phenology of freshwater fish spawning at high spatial and temporal resolution, terrestrial integrators of environmental conditions, such as leaf color change, may prove to be promising predictors of spawning activity in the management of fish populations.     

Non-indigenous brook trout and the demise of Pacific salmon: a forgotten threat?

Non-indigenous species may be the most severe environmental threat the world now faces. Fishes, in particular, have been intentionally introduced worldwide and have commonly caused the local extinction of native fish. Despite their importance, the impact of introduced fishes on threatened populations of Pacific salmon has never been systemically examined. Here, we take advantage of several unique datasets from the Columbia River Basin to address the impact of non-indigenous brook trout, Salvelinus fontinalis, on threatened spring/summer-run chinook salmon, Oncorhynchus tshawytscha. More than 41 000 juvenile chinook were individually marked, and their survival in streams without brook trout was nearly double the survival in streams with brook trout. Furthermore, when brook trout were absent, habitat quality was positively associated with chinook survival, but when brook trout were present no relationship between chinook survival and habitat quality was evident. The difference in juvenile chinook survival between sites with, and without, brook trout would increase population growth rate (lambda) by ca. 2.5%. This increase in lambda would be sufficient to reverse the negative population growth observed in many chinook populations. Because many of the populations we investigated occur in wilderness areas, their habitat has been considered pristine; however, our results emphasize that non-indigenous species are present and may have a dramatic impact, even in remote regions that otherwise appear pristine.     

Effects of immunopotentiating agents on the immune response of rainbow trout, Salmo gair dneri Richardson, to ERM vaccine

The use of Bacille Calmette Guerin (BCG) and the saponin Quil-A as a means of potentiating the immune response elicited by immersion vaccination of Salmo gairdneri with a commercial enteric redmouth (ERM) vaccine was investigated. The bactericidal activity of trout serum to Yersinia ruckeri , the causative agent of ERM, was enhanced by vaccination, although non-specific factors appear to serve an important defensive function. Whilst the use of BCG and Quil-A produced no conclusive improvement of the immune response, as determined by bacterial agglutination and serum bactericidal activity, some enhancement of in vivo bacterial clearance was apparent. Differences were observed in the susceptibility to trout serum of the three strains of Y. ruckeri tested.     

Sublethal effects of three ectoparasites on fish

Vulnerability to predation, resistance to high temperature, weight-length relationships, and fecundity were compared for fish with and without or with light and heavy infestations of three ectoparasites. The fish and parasites were fathead minnow ( Pimephales promelas ) with anchor worm ( Lernaea cyprinaceae); brook trout ( Salvelinus fontinalis ) with gill lice ( Salmincola edwardsii); and yellow perch ( Perca flavescens ) with black-spot ( Neascus of Crassiphiala bulboglossa). The only sublethal effect found was that of gill lice, which reduced resistance of brook trout to high temperature. None of the three ectoparasites increased vulnerability offish to predation by piscivorous fish.     

Sympatric relationship between redband trout and non-native brook trout in the Southeastern Oregon Great Basin

Brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss) have been widely introduced outside their respective ranges within North America causing declines and displacement of native trout. Yet, successful coexistence of native and non-native trout has received little attention. Here we evaluated the effect of introduced brook trout on the size and density of native redband trout in two invaded sub-basins in southeastern Oregon. In a multi-year study, we investigated whether habitat and fish communities differed between streams and stream reaches where redband trout were allopatric versus where redband trout were sympatric with brook trout. We hypothesized that redband trout would be less dense and have smaller total length in sympatry with brook trout than in allopatry, but that total trout density would not differ. We investigated whether differences in habitat existed between sympatric and allopatric locations that would indicate differentiation in site level habitat preferences for each trout species. We found that sympatric locations had more wood but similar fish community structure. Mean length and densities of redband trout were higher at allopatric locations. However, in most years at sympatric locations total trout density was twice that of allopatric redband trout sites. Using comparable data from an eastern United States system where brook trout are native, sympatric sites had lower densities of brook trout; however, total trout density did not differ. We conclude that invading trout negatively impact native trout densities; but in southeastern Oregon system the negative impact is minimized.     

Species-specific probe,based on 18S rDNA sequence,could be used for identification of the mucilage producer microalga <Emphasis Type="Italic">Gonyaulax fragilis</Emphasis> (Dinophyta)

Brook trout (Salvelinus fontinalis) in Appalachia experience prolonged periods of poor feeding conditions, particularly during summer and fall. To determine which prey organisms are important in sustaining brook trout populations, we monitored the feeding patterns of a population of brook trout over the course of 2 years with an emphasis on seasonal change. We employed a bioenergetics model to estimate whether or not each fish had obtained enough energy to meet daily metabolic demand. As a result, qualitative comparisons between fish feeding above maintenance ration (successfully feeding fish) and fish feeding below maintenance ration (unsuccessfully feeding fish) were possible. With the exception of winter, brook trout derived significantly more energy from terrestrial organisms than aquatic organisms. During each season, successfully feeding brook trout fed on greater proportions of specific prey types. Terrestrial Coleoptera and Lepidoptera consistently proved to be important prey during warmer seasons, while large organisms such as vertebrates and crayfish appeared to be important during winter. Our findings suggest that terrestrial organisms are more important than aquatic organisms in sustaining brook trout populations. Further, certain large and abundant terrestrial taxa are critical in providing energy to brook trout.     

Study of Vibrio anguillarum strains from different sources with emphasis on ecological and pathobiological properties.

A total of 317 Vibrio anguillarum strains were isolated from water, sediment, and diseased as well as healthy rainbow trout at a Danish mariculture farm and from feral fish caught close to the farm. All strains were examined serologically. Ten sera permitted determination of the O group in 66.7% of the strains from diseased rainbow trout. Furthermore, the O group could be determined in 45.1 to 65.4% of the strains from mucus, gills, and intestinal contents of healthy rainbow trout, while only 22.2 to 28.8% of the isolates from water, sediment, and gills or mucus of feral fish were groupable. Serogroup O1 and to some extent O2 appeared to be associated with trout. Strains from these serogroups were selected for analyses of hemagglutinating activity and surface hydrophobicity. Serogroup O1 comprised hemagglutinating as well as nonhemagglutinating strains; from cases of vibriosis, all O1 strains were nonhemagglutinating. The strains belonging to serogroup O2 were generally hemagglutinating. Examinations of surface hydrophobicity by salt aggregation and hydrophobic interaction chromatography suggested that the O1 strains were more hydrophobic than the O2 strains. In pathogenicity tests, O1 strains isolated from gills and mucus of healthy rainbow trout killed all trout in the test groups. A strain from the intestinal contents of healthy rainbow trout did not produce significant mortality. This strain could, however, be frequently reisolated from the pronephros of fish in the test group concerned. After challenge with strains from eel mucus and seawater, mortality was not produced, and furthermore, these strains could not be reisolated from the pronephros.     

Study of Vibrio anguillarum strains from different sources with emphasis on ecological and pathobiological properties

A total of 317 Vibrio anguillarum strains were isolated from water, sediment, and diseased as well as healthy rainbow trout at a Danish mariculture farm and from feral fish caught close to the farm. All strains were examined serologically. Ten sera permitted determination of the O group in 66.7% of the strains from diseased rainbow trout. Furthermore, the O group could be determined in 45.1 to 65.4% of the strains from mucus, gills, and intestinal contents of healthy rainbow trout, while only 22.2 to 28.8% of the isolates from water, sediment, and gills or mucus of feral fish were groupable. Serogroup O1 and to some extent O2 appeared to be associated with trout. Strains from these serogroups were selected for analyses of hemagglutinating activity and surface hydrophobicity. Serogroup O1 comprised hemagglutinating as well as nonhemagglutinating strains; from cases of vibriosis, all O1 strains were nonhemagglutinating. The strains belonging to serogroup O2 were generally hemagglutinating. Examinations of surface hydrophobicity by salt aggregation and hydrophobic interaction chromatography suggested that the O1 strains were more hydrophobic than the O2 strains. In pathogenicity tests, O1 strains isolated from gills and mucus of healthy rainbow trout killed all trout in the test groups. A strain from the intestinal contents of healthy rainbow trout did not produce significant mortality. This strain could, however, be frequently reisolated from the pronephros of fish in the test group concerned. After challenge with strains from eel mucus and seawater, mortality was not produced, and furthermore, these strains could not be reisolated from the pronephros.     

Dietary vitamin E and rainbow trout (Oncorhynchus mykiss) phagocyte functions: effect on gut and on head kidney leucocytes

The effects of vitamin E (deficiency or supplementation) on the non-specific immune system in rainbow trout, Oncorhynchus mykiss, were evaluated. Rainbow trout were fed daily a semi-purified diet supplemented with vitamin E at 0, 28 and 295 mg x kg(-1) of diet. After 80 days of experimental feeding, the phagocytic function (respiratory burst evaluated by the CL response, phagocytosis) from gut leucocytes and head kidney enriched macrophages was measured; head kidney cell pinocytosis and serum lysozyme activity were also analysed. The results showed that some phagocyte functions were influenced by dietary vitamin E. When fish were fed the high dietary dose of vitamin E an enhancement of phagocytosis was found, but only significantly for the leucocytes isolated from the gut of rainbow trout; moreover, an impaired response was also observed in the fish fed no vitamin E for 80 days. However, no significant differences were noticed on the oxidative burst (CL) response of both gut and head kidney cells according to the dietary dose of vitamin E. Pinocytosis evaluated on head kidney cells was not influenced by dietary vitamin E. Fish fed vitamin E at 295 mg x kg(-1) had a lower serum lysozyme activity than those fed with vitamin E at 28 mg x kg(-1) and the fish fed no vitamin E for 80 days had an impaired activity. Thus, the present results demonstrate that altered dietary levels of vitamin E modulates the phagocytic functions of gut leucocytes in rainbow trout; moreover, the vitamin E diet effect seems to be greater on the local intestinal response as compared to systemic (head kidney). Taken together, this study confirms the crucial role of gut phagocytes in mucosal non-lymphoid defences in fish.     

Do fish lymphocytes secrete interferon-γ?

Macrophage activating factor (MAF)-containing supernatants, generated by mitogen (Con A/PMA) stimulation of rainbow trout leucocytes, were found to confer viral resistance (interferon, IFN, activity) on a rainbow trout epithelial cell line challenged with infectious pancreatic necrosis virus. Both the MAF and IFN activities co-eluted by HPLC size exclusion chromatography and showed similar sensitivities to acid (pH 2), temperature (60° C) and trypsin. The mode of induction of this IFN, its acid and temperature sensitivity and its possible MAF activity suggest that fish leucocytes can secrete an IFN-γ like molecule.     

Cells, proteins, and certain physical-chemical properties of brook trout (Salvelinus fontinalis) blood

Laboratory brook trout were used to evaluate, refine, or develop biochemical procedures for the analysis of fish blood. Analytical values were obtained for the following blood properties: total and differential leucocytes and erythrocytes; erythrocyte and plasma proteins (by electrophoresis); plasma refractive index; erythrocyte sedimentation rate; erythrocyte osmotic fragility; blood surface tension and density; haemoglobin; and packed cell volume. These blood factors are discussed with reference to fish health and disease and to changes caused by deleterious quantities of water pollutants.