Purification and characteristics of trypsin from masu salmon (<Emphasis Type="Italic">Oncorhynchus masou</Emphasis>) cultured in fresh-water

Trypsin from the pyloric ceca of masu salmon (Oncorhynchus masou) cultured in fresh water was purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl cellulose to obtain a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and native PAGE. The molecular mass of the purified trypsin was estimated to be approximately 24,000 Da by SDS–PAGE. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and N ^α -p-tosyl-l-lysine chloromethyl ketone. Masu salmon trypsin was stabilized by calcium ion. The optimum pH of the masu salmon trypsin was around pH 8.5, and the trypsin was unstable below pH 5.0. The optimum temperature of the masu salmon trypsin was around 60°C, and the trypsin was stable below 50°C, like temperate-zone and tropical-zone fish trypsins. The N-terminal 20 amino acid sequence of the masu salmon trypsin was IVGGYECKAYSQPHQVSLNS, and its charged amino acid content was lower than those of trypsins from frigid-zone fish and similar to those of trypsins from temperate-zone and tropical-zone fish. In the phylogenetic tree, the masu salmon trypsin was classified into the group of the temperate-zone fish trypsin.     

Glycerophospholipid:cholesterol acyltransferase complexed with lipopolysaccharide (GCAT-LPS) of Aeromonas salmonicida produces lysophospholipids in salmonid red cell membranes: a probable haemolytic mechanism

Abstract. The enzymic activity of GCAT-LPS was studied using whole blood ( in vivo ), citrated blood or washed red blood cells as substrate. Results demonstrated in vitro and in vivo haemolysis of Atlantic salmon, Salmo salar L., red blood cells, and in vitro haemolysis of rainbow trout, Oncorhynchus mykiss (Walbaum), red blood cells, by purified GCAT-LPS. Haemolysis coincided with 10% or more lysophospholipids, as a per cent of total phospholipids, in the red cell membranes. Addition of soybean lysophosphatidylcholine to citrated whole blood from salmon caused haemolysis. This suggests that it is the observed accumulation of lysophospholipids, caused by the enzymic activity of GCAT-LPS, which leads to lysis of salmonid red blood cells. When membranes were used as substrate, GCAT-LPS seemed to express acyltransferase and possibly some phospholipase activity, while accumulation of lysophospholipids suggests that the lysophospholipase activity is inhibited or severely suppressed. Deacylation of both phosphatidylcholine and phosphatidylethanolamine was observed, indicating that both phospholipids are substrates for the enzyme.     

Maintenance ration, protein synthesis capacity, plasma insulin and growth of Atlantic salmon (Salmo salar L.) with genetically different trypsin isozymes

Growth was found to be associated with the changes of trypsin activity in the pyloric caecal tissues and the level of plasma insulin in Atlantic salmon (Salmo salar L.). A decrease in trypsin activity accompanied by an increase in plasma insulin was detected one month before an enhanced growth was observed. There were significant relationships between weight specific consumption rate, plasma insulin levels and fish growth. The correlation of weight specific consumption rate was higher with growth rate (R²=0.7, p<0.0001) than with plasma insulin concentration (R²=0.4, p<0.0001).When the comparison was made between Atlantic salmon carrying and lacking the trypsin variant TRP-2*92, the fish with the variant had lower maintenance ration (p<0.05), higher capacity for protein synthesis in the white muscle (p<0.02), and a greater ability to utilize the feed at a restricted ration than the fish without the variant. In Atlantic salmon lacking the variant, both plasma insulin concentrations and growth rates were significantly lower (p<0.05) in the fish fed 0.5% bw day^–1 than those fed 1% bw day^–1. Whilst the growth rates of TRP-2*92 salmon fed the different rations became similar one month after similar levels of plasma insulin were observed between them. The TRP-2*92 salmon may be defined as a high protein growth efficiency fish with low protein turnover rate.Genetic variation in trypsin isozyme pattern affects feed utilization, plasma insulin levels and growth in Atlantic salmon.     

Effects of varying rearing temperatures on expression of different trypsin isozymes, feed conversion efficiency and growth in Atlantic salmon (shape Salmo salar L.)

Hatching and start-feeding temperatures affected the expression of different trypsin isozymes in the pyloric caeca of Atlantic salmon. Hatching temperature of 10 °C induced the expression of the common isozyme TRP-2*100, and of 6 °C induced the variant TRP-2*92 (p$<$0.01). In contrast, start-feeding temperature of 12 °C significantly (p$<$0.0001) influenced the expression of the variant TRP-2*92, compared to 6 °C. The frequencies between different trypsin isozyme patterns were not changed at later stages under varied rearing temperatures.The frequency distribution between Atlantic salmon without and with trypsin variants was about 0.4:0.6. The trypsin isozyme TRP-2*92 was the major variant in Norwegian salmon, while the trypsin variant TRP-1*91 was dominant in Scottish salmon, at frequencies of 0.47 and 0.42, respectively. The presence of both the common and either variant trypsin isozymes were important for feed utilization and growth at varying rearing temperatures. Trypsin isozymes are functionally sensitive to different temperatures. The expression of the common trypsin isozyme TRP-2*100 is important when the water temperature is $>$ 8 °C, while it is important for the expression of the variant TRP-2*92 when the water temperature is $\leq$ 8 °C, especially below 6 °C. The variant TRP-1*91 was observed to perform effectively at a wider temperature range than the variant TRP-2*92, but not at temperature $\leq$ 6 °C.Genetic variation in trypsin isozyme pattern is a primary factor affecting food conversion efficiency and growth under different rearing temperatures.     

The influence of Gyrodactylus salaris Malmberg 1957 (Monogenea) on the epidermis of Atlantic salmon,Salmo salar L., and brook trout,Salvelinus fontinalis (Mitchill): experimental studies

The effect of Gyrodactylus salaris on the epidermal structure of hatchery-reared brook trout parr and Norwegian Atlantic salmon parr was evaluated. Both species were initially susceptible to this parasite, but whereas populations on salmon increased until the host died, brook trout responded to, and eventually eliminated, their infections. Salmon skin samples taken 14 days p.i. showed a reduced mucous cell concentration (less than 1500 cells mm^–2 on the head compared with 2000 cells mm^–2 in controls; the same trend was also seen in other sites) and the epidermis was thinner (48 μm compared with 60 μm on pectoral fins; similar trend seen in other sites) than in uninfected controls kept under identical conditions. Brook trout skin samples were taken 50 days p.i., when the hosts had responded to, and almost eliminated, their infections. No change was then observed in mucous cell density, while the number of epidermal cell layers and the epidermal thickness of brook trout had increased slightly following infection. These results are related to the nature of the host response, and the thinning of the epidermis and loss of mucous cells may in some way be related to the inability of Norwegian salmon parr to respond to the parasite.     

Effect of fish feed processing conditions on digestive protease activities, free amino acid pools, feed conversion efficiency and growth in Atlantic salmon (Salmo salar L.)

The responses of the digestive proteases trypsin and chymotrypsin and protein metabolism to differences in feed protein quality were investigated in Atlantic salmon (Salmo salar L.). Two sets of experimental feeds were produced. Each set of high and low quality feeds was provided to either 150 g or 2 kg salmon. Protein in the high quality feeds had significantly higher percentages of free (reactive) sulphydryl (SH) groups than the corresponding feeds based on low quality meals. After 90 days feeding, groups given high and low quality feeds did not differ in their specific growth rates (SGR) in either experiment. However, feed conversion efficiency (FCE) was significantly different between the high and low quality feed groups in 2 kg salmon, where the difference between the high and low feed protein qualities was larger, 10% versus 4% SH/[SH + (S–S)] in 150 g salmon. Higher FCE was preceded by significantly higher trypsin and chymotrypsin specific activities on day 60. SGR, in general, changed after the first month and was stable during the last 2 months in both experiments. Concurrently, both trypsin (T) and chymotrypsin (C) decreased with an increased activity ratio of trypsin to chymotrypsin (T/C ratio), and resulting in significantly lower T/C ratio on day 90 in salmon feeding on high quality feeds in both sizes of fish. Differences in FCE were associated with significant differences in levels of total free amino acids (TFAA) in the plasma and the white muscle, as well as in the ratio of essential to non‐essential free amino acids (EAA/NEAA ratio), free hydroxyproline, and RNA in the white muscle. Interestingly, after 3 days starvation (day 93), 5–7 h postprandial EAA/NEAA ratio in the plasma was significantly lower in the high quality diet groups in both experiments. Trypsin specific activity inversely correlated with muscle TFAA levels in 2 kg salmon, concurrent with higher muscle levels of RNA, lower free hydroxyproline and higher FCE in fish fed higher quality diets.     

Genetic differences in digestion and absorption of dietary protein in Atlantic salmon, Salmo salar L., with different trypsin isozyme patterns

Abstract. In the genetical presence of trypsin isozyme TRP -2(92), Atlantic salmon, Salmo salar L., of either 100g or 400g in weight showed similarity in significantly higher absorption of the dietary protein as their postprandial total free amino acids in the plasma were significantly higher than those in the salmon without the isozyme. Better digestion of the dietary protein was observed by the presence of significantly higher postprandial plasma lysine level of the TRP-2(92) salmon. Another indication of different protein metabolism between salmon with and without the isozyme was observed by differences in plasma alanine and hydroxyproline. Trypsin activity in the intestine showed a higher response to feeding than that in the pyloric     

Infectivity study of Streptococcus phocae to seven fish and mammalian cell lines by confocal microscopy

Streptococcus phocae is a beta-haemolytic bacterium that causes systemic infections in Atlantic salmon, Salmo salar L., cultured in southern Chile and also in seals. In this study, the host-pathogen interaction between S. phocae and seven types of cell lines (fish and mammalian) was examined using an indirect fluorescent antibody and confocal microscopy (CM). Chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), salmon head kidney (SHK-1) and Atlantic salmon kidney were used as the fish cell lines, while human cervix epithelial adenocarcinoma (HeLa), African green monkey kidney fibroblast (Cos-7) and mouse leukaemic monocyte macrophage (Raw 264.7) were included as mammalian cell lines. Streptococcus phocae type strain ATCC 51973(T) and isolates LM-08-Sp and P23 were selected as representatives from the salmon and seal host, respectively. For the CM examination, monolayers seeded on round coverslips were studied at 2- and 20-h post-inoculation (pi). The results showed that there is no common infectivity pattern between the three S. phocae strains at 2-h pi and the cell lines tested, regardless of the source of isolation (seal or salmon). All S. phocae strains could internalize and were found inside the fish and mammalian cell cytoplasm after 20-h pi. Regardless of the cells studied (fish or mammal) and incubation (2 and 20 h), S. phocae was never observed inside the nuclei. Seal and salmon isolates showed the highest number of bacteria entering into the primate cell lines (HeLa and Cos-7) from 2-h pi, while ATCC 51973(T) was not found outside or inside the HeLa and Cos-7 cells.     

Immunoreactive somatolactin cells in the pituitary of young,migrating, spawning and spent chinook salmon,Oncorhynchus tshawytscha

Immunocytochemical techniques using an antiserum to cod somatolactin (SL) demonstrated the presence of SL cells in the intermediate lobe of the pituitary in Oncorhynchus tshawytscha. The cells were small in yearling fish. Two groups of maturing fish were studied. In the spring run salmon collected in April and May during the upstream migration, the SL cells appeared stimulated. In September, during spawning, SL cell stimulation was maximal with indices of hypertrophy and degranulation often more marked in females than in males. In the other group, salmon of the fall run collected in the Pacific Ocean in August had well developed gonads, large gonadotropes and abundant SL cells. In spawning salmon (September) the SL cells were stimulated, mainly in females. However, the final stimulation was less intense than in spring run spawning fish. The SL cells were smaller, without evident granule release, but still abundant in spent salmon of the fall run caught at the end of November. Various factors (time spent in rivers before spawning, starvation, decalcification, stress, hypothalamic influences) were considered which might explain differences between spring and fall run salmon. These observations suggest that SL may play a role in the control of gonadal maturation in chinook salmon as it may also do in sockeye and chum salmon previously studied, and that SL cells may be sensitive to the ambient salinity.     

Cytotoxicity of surfactants to the FHM-sp cell line

Cytotoxicity of eight surfactants was determined by the neutral red assay with the fathead minnow (FHM)-sp cell line, a cell line in suspension culture from fish. The toxicity ranking of the surfactants was benzalkonium chloride > benzethonium chloride > sodium linear - dodecylbenzene–sulfonate (LAS) > potassium laurate > sodium dodecylsulfate > polyoxyethylene(20)sorbitan monolaurate > polyoxyethylene(20)sorbitan monooleate > betaine. The toxicity ranking of the surfactants classified into four groups based on the ion of the hydrophilic group was cationic surfactants > anionic surfactants > non-ionic surfactants > amphipathic surfactants. The FHM-sp cells, as well as the chinook salmon embryo (CHSE)-sp cells, could be inoculated directly to the microplate wells without dispersion by trypsin treatment of cell sheets at room temperature. Therefore, the cytotoxicity assay of the surfactants could be carried out quickly by using the FHM-sp cell line. The FHM-sp cell line had similar or higher sensitivity to sodium dodecylsulfate compared with several cell lines from mammals. The cytotoxicity assay could be shortened by the procedure exposing the surfactants to the FHM-sp cells before the cell monolayer formation in the microplate wells. To use the FHM-sp cell line as a screening tool prior to in vivo testing, studies on the correlation between in vivo data and in vitro data on the toxicity of surfactants are necessary.     

Antioxidant Activity of Hydrolysates and Peptide Fractions of Nemipterus japonicus and Exocoetus volitans Muscle

The focus of the study was to investigate the antioxidant activity of hydrolyzed muscle protein of Nemipterus japonicus and Exocoetus volitans. The trypsin protein hydrolysates of both fish showed maximum free radical scavenging potential and lipid peroxidation inhibition. Furthermore, it was purified by chromatographic methods followed by the lipid peroxidation inhibition; free radical scavenging assay was performed before and after purification. The purified peptide fractions of N. japonicus and E. volitans exhibited higher activity against polyunsaturated fatty acids (PUFA) peroxidation which was similar to natural antioxidants like α-tocopherol. Free radical scavenging potencies were measured by electron spin resonance technique (ESR). The purified peptide of E. volitans quenched free radicals (DPPH, hydroxyl, and superoxide) slightly more than N. japonicus. The amino acid composition of both fish protein hydrolysates showed variations in their ratio. The purified peptides were tested for cell cytotoxicity for Vero (kidney epithelial cells of the African Green Monkey) and Hep G₂ (human hepatocellular liver carcinoma) cell lines. It was found that peptides did not show any cytotoxic effect for Vero cell lines and exerted a significant antiproliferative effect on Hep G₂ cell lines.     

Heart and skeletal muscle inflammation in Atlantic salmon, Salmo salar L: a new infectious disease

Heart and skeletal muscle inflammation (HSMI) is a disease syndrome of unknown aetiology first observed in farmed Atlantic salmon, Salmo salar, in 1999. In the present study we have demonstrated for the first time that HSMI is an infectious disease. It was induced in Atlantic salmon post-smolts after injection with tissue homogenate from farmed Atlantic salmon previously diagnosed with HSMI. The lesions were also induced in cohabitating salmon given a corresponding injection without tissue homogenate. Six weeks post-challenge the fish that had been injected with tissue homogenate developed a serious epicarditis and myocarditis with mononuclear cell infiltrations in compact and spongy layers of the heart. Similar lesions were found in cohabitants after 10 weeks. The lesions were consistent with samples from field outbreaks of HSMI. No lesions were found in control fish. A viral aetiology is strongly suggested, as no difference in disease induction between an inoculum containing antibiotics and a non-treated inoculum was found. Further investigations are required in order to make conclusions regarding the cause and pathogenesis of HSMI.     

饲料中添加重组鱼生长激素对罗非鱼鱼种的促生长作用研究

采用基因工程菌ＥｃｏｌｉＤＨ５ａ（ｐＢＶＧＨ１８）生产重组鲑鱼生长激素。经初步纯化后作为添加剂投喂罗非鱼鱼种。实验结果表明,应用基因工程生产的重组鲑鱼生长激素具有生物学活性,能够通过口服经肠道被鱼体吸收,具有很强的促进鱼体生长作用。通过改进工艺,降低成本可望能作为饲料添加剂应用于水产养殖业,特别是名贵品种的养殖     

Isolation,Purification, and Biochemical Characterization of Trypsin from Indian Mackerel (Rastralliger kanagurta)

Trypsin from viscera of Indian mackerel (Rastralliger kanagurta) was purified by ammonium sulphate precipitation and chromatographic techniques such as size exclusion, ion exchange, and affinity chromatography, with a 14.4-fold increase in specific activity and 18.7% recovery. The molecular weight of the trypsin was estimated to be approximately 26 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified trypsin showed amidase-specific activity which was determined using benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for isolated trypsin activity were 9.0 and 50°C, respectively. The purified trypsin was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK). Purified trypsin showed almost 40% recovery at high NaCl concentration (30%). The N-terminal amino acid sequence of the first 10 amino acids of purified trypsin was IVGGYESQPH. The Michaelis-Menten constant (K_m) and catalytic constant (K_cat) of purified trypsin were 0.430 mM and 0.77 s^?1, respectively, determined using BAPNA as a substrate. Purified trypsin showed digestion of casein similar to bovine trypsin by the fluorometric method.     

Inactivated Piscine orthoreovirus vaccine protects against heart and skeletal muscle inflammation in Atlantic salmon

Heart‐ and skeletal muscle inflammation (HSMI) caused by infection with Piscine orthoreovirus (PRV) is one of the most common viral diseases in farmed Atlantic salmon (Salmo salar) in Norway, and disease outbreaks have been reported in most countries with large‐scale Atlantic salmon aquaculture. Currently there is no vaccine available for protection against HSMI, partly due to the lack of a cell line for efficient virus propagation. Erythrocytes are the primary target cells for PRV in vivo and a potential source for isolation of PRV particles. In this study, PRV was purified from infected erythrocytes, inactivated and used in a vaccination trial against HSMI. A single immunization with adjuvanted, inactivated PRV induced protection against HSMI in Atlantic salmon infected by virus injection 6 weeks later, while a moderate protection was obtained in fish infected through natural transmission, i.e. cohabitation. The PRV vaccine significantly reduced PRV loads and histopathological lesions typical for HSMI compared to the unvaccinated control group. This is the first demonstration of protective vaccination against PRV, and promising for future control of HSMI in Atlantic salmon aquaculture.     

Rapid temperature‐dependent wound closure following adipose fin clipping of Atlantic salmon Salmo salar L

Three groups of Atlantic salmon were kept at a constant temperature of 4, 10 and 14 °C. The adipose fins were removed; six fish/group were sampled at 11 subsequent time points post‐clipping. Samples were prepared for histopathological examination to study the course of re‐epithelization. A score sheet was developed to assess the regeneration of epidermal and dermal cell types. Wounds were covered by a thin epidermal layer between 4 and 6 h post‐clipping at 10 and 14 °C. In contrast, wound closure was completed between 6 and 12 h in fish held at a constant temperature of 4 °C. By 18 h post‐clipping, superficial cells, cuboidal cells, prismatic basal cells and mucous cells were discernible in all temperature groups, rapidly progressing towards normal epidermal structure and thickness. Within the observation period, only minor regeneration was found in the dermal layers. A positive correlation between water temperature and healing rates was established for the epidermis. The rapid wound closure rate, epidermal normalization and the absence of inflammatory reaction signs suggest that adipose fin clipping under anaesthesia constitutes a minimally invasive method that may be used to mark large numbers of salmon presmolts without compromising fish welfare.     

Purification and characterization of two anionic trypsins from the hepatopancreas of carp

SUMMARY: Two trypsins, designated as trypsin A and trypsin B, have been purified from the hepatopancreas of carp. The purification procedures consisted of ammonium sulfate fractionation, and chromatographies on DEAE-Sephacel, Ultrogel AcA54 and Q-Sepharose. Trypsin A was purified to homogeneity with the molecular mass of approximately 28 kDa, while trypsin B gave two close bands of 28.5 kDa and 28 kDa on sodium dodecylsulfate polyacrylamide gel electrophoresis both under reducing and non-reducing conditions. On native-PAGE, both trypsin A and trypsin B showed a single band. Trypsin A and trypsin B revealed optimum temperature of 40°C and 45°C, respectively, and shared the same optimum pH 9.0 using Boc-Phe-Ser-Arg-MCA as substrate. Both enzymes were effectively inhibited by trypsin inhibitors and their susceptibilities were similar. The NH₂-terminal amino acid sequences of trypsin A and trypsin B were determined to 37th and 40th amino acid residue, respectively. Their sequences were very homologous, but not identical to that of a trypsin-type serine proteinase from carp muscle and these of other trypsins. Immunoblotting test using the antibody raised against trypsin A cross-reacted with trypsin B positively.     

Effects of diet supplementation of soya‐saponins,isoflavones and phytosterols on Atlantic salmon (Salmo salar,L) fry fed from start‐feeding

A 14‐week trial was conducted to investigate the effects of antinutritional factors (ANFs) commonly present in soybean ingredients, singly and in combination, on Atlantic salmon (Salmo salar L.) fed from start‐feeding. The experimental diets consisted of a negative control fish meal diet (FM), and a positive control diet with 167 g kg^?1 soybean meal inclusion (SBM) and four diets based on the FM diet supplemented with 2 g kg^?1 soya‐saponins (SAP), 1.5 g kg^?1 isoflavones (IFL), 0.3 g kg^?1 phytosterols (PHS) or a mixture of these (MIX). Fish fed the SAP diet showed significantly higher growth performance than those fed FM, while the IFL treatment significantly decreased growth performance of salmon fry. Fish fed the IFL diet had significantly lower maltase activity and higher trypsin activity in proximal intestine than fish fed the FM diet. Histological differences were observed in the liver of fish fed the IFL diet, characterized by reduced size of the hepatocytes. Fish fed the PHS and IFL diets showed the highest frequencies of skeletal deformities among the six treatments. In conclusion, the results indicate that purified isoflavones may negatively affect growth performance, intestinal function, liver metabolism and bone formation of salmon fry.     

Isolation and characterization of a surface layer antigen from Vibrio salmonicida

Abstract. A cell surface product (VS-P1) of Vibrio salmonicida has been purified from culture supernatant by a combination of extensive dialysis, filtration and centrifugation, as well as by salt precipitation and hydrophobic chromatography. SDS-PAGE analysis showed that the monomeric form of the antigen is a single polypeptide with an apparent molecular weight of 40000. Size exclusion HPLC of purified VS-P1 as well as VS-Pl-containing fish serum revealed, however, oligomeric forms in the range from 300000 to more than 700000 daltons. The antigen contained 6% carbohydrate and several isolectric forms were distinguishable when analysed on an analytical isoelectric focusing electrophoresis system. A 'sandwich' ELISA, utilizing polyclonal antibodies, was developed for screening sera from both healthy and moribund Atlantic salmon for the presence of the VS-P1 antigen.     

Surface properties of Streptococcus phocae strains isolated from diseased Atlantic salmon, Salmo salar L

Streptococcus phocae is an emerging pathogen for Chilean Atlantic salmon, Salmo salar, but the factors determining its virulence are not yet elucidated. In this work, cell surface–related properties such as hydrophobicity and haemagglutination, adhesion to mucus and cell lines, capsule detection, survival and biofilm formation in skin mucus and serum resistance of the isolates responsible for outbreaks in Atlantic salmon and seals were examined. Adhesion to hydrocarbons and the results of salt aggregation tests indicated most of the S. phocae were strongly hydrophobic. All isolates exhibited a similar ability to attach to the Chinook salmon embryo (CHSE) cells line, but were not able to enter CHSE cells. Haemagglutination was not detected. Our data clearly indicate that S. phocae can resist the killing activity of mucus and serum and proliferate in them, which could be associated with the presence of a capsular layer around the cells. Pathogenicity studies using seal and fish isolates demonstrated mortality or pathological signs in fish injected only with the Atlantic salmon isolate. No mortalities or histopathological alterations were observed in fish injected with extracellular products.