杀鲑气单胞菌杀日本鲑亚种胞外产物毒性及免疫原性分析

用玻璃纸平板法提取了杀鲑气单胞菌杀日本鲑亚种Aeromonassal monicida masoucida的胞外产物（ECP）。毒性试验证实,ECP对剑尾鱼Xiphophorus helleri具有致死性,其半致死量（LD50）为4.72μg蛋白/g体重。SDS-PAGE表明,ECP由13条蛋白带组成。利用大鼠抗ECP血清进行的Western-blot印迹显示,组成ECP的13条蛋白带中有7条具有免疫原性,能够引发机体的免疫应答反应产生抗体,其分子量分别为88、70、42、39、36、22和15kDa。     

Protein Hydrolysates and Ultrafiltration Fractions Obtained from Krill (Euphausia superba): Nutritional,Functional, Antioxidant,and ACE-Inhibitory Characterization

ABSTRACT

Krill (Euphausia superba) was hydrolyzed by proteolytic enzymes in order to produce multifunctional bioactive peptides, and their functional properties were evaluated. Krill protein hydrolysate (KPH) by pepsin with 4-h hydrolysis showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and angiotensin I converting enzyme (ACE) inhibitory activities. The solubility and foaming properties of KPH were higher than those of the unhydrolyzed krill protein at a wide range of pHs. KPH was further fractionated based on molecular weight. The 1- to 3-kDa peptide fraction exhibited the highest DPPH scavenging activity (IC₅₀ value of 0.5 mg/mL), oxygen radical absorbance capacity (497.39 ± 4.31 µM TE/mg fraction), 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid cation radical scavenging activity (48.41 ± 0.23 µM TE/mg fraction), and reducing power (110.40 ± 2.07 µM TE/mg fraction). However, the < 1-kDa peptide fraction exhibited a higher ACE inhibitory activity than that of other fractions. The 1- to 3- and < 1-kDa peptide fractions are rich in aromatic and hydrophobic amino acids, respectively.     

A study of the pathological effect of isolated Aeromonas salmonidda extracellular protease on Atlantic salmon, Salmo salar L.

Abstract. A comparison was made between the effects of Aeromonas salmonidda extracellular protease and total extracellular products (ECP) following intramuscular injection into juvenile Atlantic salmon, Salmo salar L. Thus, 20, 10, 5, 2.5 and 1.5 units of salt-free protease in 0.2 ml water were compared with ECP preparations with the same levels of proteolytic activity. The highest concentration of ECP produced a gross pathology with a large furuncular lesion 36 h after injection. The corresponding protease preparation had a lesser effect, although a furuncle was formed and tissue liquefaction was produced. These effects were less marked with reduced concentrations. At the lowest level studied, no significant effect was observed with protease alone but ECP (0.8 μg of protein) produced a small, characteristic lesion similar to that achieved with 5 units of isolated protease.     

Pathogenicity comparison of high- and low-virulence strains of Vibrio scophthalmi in olive flounder Paralichthys olivaceus

Vibrio scophthalmi, a bacterial pathogen of olive flounder Paralichthys olivaceus, exhibits strain-dependent virulence. No information is available on the comparative pathogenicity of different strains of V. scophthalmi toward olive flounder. In this study, high- and low-virulence strains (HVS and LVS, respectively) were compared in terms of their pathogenic characteristics, including adhesion and survival, superoxide dismutase (SOD) activity, and extracellular products (ECP) of bacterial cells. The cell-mediated defense of macrophages from olive flounder against V. scophthalmi infection in vitro was also investigated. The results demonstrated that the SOD activity of the HVS was higher than that of the LVS. The number of viable cells of the HVS in serum increased by two log units after 18 h, whereas that of the LVS decreased. The number of cells of the HVS in skin mucus increased significantly while that of the LVS remained constant. The LD₅₀ values of the HVS and LVS ECP toward olive flounder were 10.14 and 15.99 μg protein/g fish, respectively. The ECP were positive for naphthol-AS-BI-phosphohydrolase, lipase, gelatinase, and leucine arylamidase. The extracellular O₂ ^? overflow and intracellular O₂ ^? concentration of macrophages induced by the HVS were lower than those induced by the LVS. Significantly more nitric oxide was produced by the HVS than by the LVS.     

欧洲鳗血清免疫球蛋白纯化及部分特性分析

采用饱和硫酸铵分步盐析法结合柱层析提取，纯化欧洲鳗血清免疫球蛋白（Ｉg) ,实验证实欧洲鳗Ｉg主要分布在硫酸铵饱和度３０％－５０％的区间内，Ｓephacry1-S200进一步提纯的Ｉg主要存在于第一个蛋白峰，而Ｓepharose-4B柱层析提纯的Ｉg则存在于第二个蛋白峰，ＤＥＡE-52脑子交换柱层析可进一步纯化Ｓepharose-4B柱层析的产物，ＥＬＩＳＡ和Western-blot分别证实上述提取物具有抗体的免疫学活性，ＳＤＳ－ＰＡＥＧ分析纯化的Ｉg，显示了洲鳗Ｉg重链约为６８kD，轻链约为２６kD.     

Antigenicity of Streptococcus agalactiae extracellular products and vaccine efficacy

Streptococcus agalactiae is a major bacterial pathogen that is the cause of serious economic losses in many species of freshwater, marine and estuarine fish worldwide. A highly efficacious S. agalactiae vaccine was developed using extracellular products (ECP) and formalin-killed whole cells of S. agalactiae. The vaccine efficacy following storage of S. agalactiae ECP and formalin-killed S. agalactiae cells at 4 degrees C for 1 year was determined. The stored ECP containing S. agalactiae formalin-killed cells failed to prevent morbidity and mortality among the vaccinated fish, and the relative percentage survival was 29. Serum antibody responses of the stored ECP and freshly prepared ECP against soluble whole cell extract of S. agalactiae indicated that significantly less antibody was produced in fish immunized with stored ECP and S. agalactiae cells than in those fish immunized with freshly prepared ECP and S. agalactiae cells at day 31 post-vaccination. Silver staining of sodium dodecyl sulphate-polyacrylamide gels and immunostaining of Western blots with tilapia antiserum to S. agalactiae revealed that predominant 54 and 55 kDa bands were present in the freshly prepared ECP fraction. The 55 kDa band was absent from the stored ECP and new bands below 54 kDa appeared on the Western blot. The results of this study on S. agalactiae ECP provide evidence for a correlation between protection and antibody production to ECP and for the importance of the 55 kDa ECP antigen for vaccine efficacy.     

The non-specific activation of rainbow trout, Salmo gairdneri Richardson, complement by Aeromonas salmonicida extracellular products and the correlation of complement activity with the inactivation of lethal toxicity products

Abstract The possible mechanism of inactivation of the toxicity of Aeromonas salmonicida extracellular products (ECP) by normal rainbow trout serum was investigated using juvenile rainbow trout. ECP was prepared from culture supernatant by an acetone precipitation method. The ECP was incubated with normal rainbow trout serum at 20°C for 2 h, and the interrelationship between ECP proteolytic activity and immune complex-initiating, haemolytic complement activity (CH₅₀) of normal serum against antibody-sensitized goldfish red blood cells was evaluated. When normal serum was incubated with increasing concentrations of ECP, the CH₅₀ activity of serum decreased. The CH₅₀ activity was completely abolished in serum treated with undiluted ECP. ECP treated with serum was administered to trout intraperitoneally to determine mortality. All the fish receiving untreated ECP (0.05 ml = 0.5 mg protein) alone died within 24 h. When ECP was treated with serum at 1:1 to 4:1 (serum: ECP) in volume a similar high mortality was produced. These inocula possessed high protease activity and no or low CH₅₀ activity. However, mortality decreased and finally no mortality was recorded as ECP was treated with large volumes of serum (9:1 to 19:1). These inocula had lower protease activity and considerably higher CH₅₀ activity. Fish receiving ECP treated with heat-inactivated serum at 19:1 showed 100% mortality. A serum: ECP inoculum derived from fish which had been administered lipopolysaccharide from Salmonella enteritidis and which possessed a low CH₅₀ activity also gave a high mortality when used at 19:1. These results suggest that rainbow trout complement is implicated in the inactivation of toxicity of A. salmonicida ECP.     

高致病性维氏气单胞菌胞外产物对斑点鲖的致病性

为了研究维氏气单胞菌及其胞外产物的致病机制对预防和治疗斑点鲖维氏气单胞菌病的作用。通过对一株高致病性斑点鲖源维氏气单胞菌胞外产物(extracellular product,ECP)的提取并结合体内外实验研究其酶活性与致病性,以期为维氏气单胞菌的致病机制研究提供新思路。采用饱和硫酸铵沉淀法,对一株高致病性的斑点鲖源维氏气单胞菌的ECP进行了提取,将其进行浓度检测、SDS-PAGE分析和酶活测定;并将提取的ECP攻毒斑点鲖,通过病理学分析研究其致病机制。提取的ECP经考马斯亮蓝试剂盒确定其浓度为0.743 mg/m L;SDS-PAGE分析发现该ECP类型丰富,分子大小主要集中在20.0~33.0 ku。运用琼脂平板扩散法对ECP中主要毒力和代谢相关酶活性进行体外测定,发现ECP具有脂酶、蛋白酶、卵磷脂酶、DNA酶和溶血活性,不具有淀粉酶、明胶酶、脲酶活性;并对与毒力密切相关的溶血活性进行了进一步的溶血谱绘制,发现ECP对其他多种动物红细胞都有溶血活性,对鱼类红细胞溶血性较强,但对鸡、鸭红细胞无溶血活性。ECP攻毒健康斑点鲖后,发现其具有明显致病性,死亡率高达100%。病鱼大体病理变化为体表黏液增多,出现褪色斑以及不同程度的出血斑;眼球充血;肛门红肿外凸;剖检病变表现:鳃丝充血肿胀、肝脏肿大、散在少量出血点,脾脏肿大暗红,肠道扩张、肠壁变薄、肠腔内有大量黄色黏液。组织学病变主要表现为肝细胞变性、坏死;脾脏淋巴细胞减少,大量结缔组织增生;胃、肠道黏膜上皮坏死脱落。该株维氏气单胞菌的胞外产物具有多种酶活性,且对斑点鲖具有明显的致病性,推测该胞外产物在维氏气单胞菌入侵、感染甚至致死斑点鲖过程中都发挥了重要作用。     

Susceptibility of salmonid erythrocytes to lysis by bacterial lipopolysaccharides

Abstract. Bacterial lipopolysaccharide (LPS) is known to have binding affinity for mammalian erythrocyte membranes but does not cause haemolysis. In this study, LPS from eight different bacterial species all possessed haemolytic activity for salmonid erythrocytes. There were some differences in haemolytic activity among the different bacterial LPS after 2 h incubation. Lytic titres continued to increase with time for some LPS species reaching extremely high litres after 20 h incubation for the Salmonella minnesota LPS. The LPS extracted by the hot-phenol method from extracellular products (ECP) of Aeromonas salmonicida was more haemolytic than that extracted from the bacterial cell walls. Separated lipid A- and O-antigen fractions of A. salmonicida LPS had a similar low haemolytic activity. When A. salmonicida LPS, lipid A- and O-antigen were pre-incubated with bovine serum albumin, only LPS extracted from ECP retained any haemolytic activity. The LPS types used in this study were not haemolytic for rabbit erythrocytes.     

Instability of the major soluble antigen produced by Renibacterium salmoninarum

Abstract. Using Western blot to examine the nature of soluble antigens produced by Renibacterium salmoninarum , it was found that the major 57-kilodalton (kDa) antigen was unstable, SDS-PAGE of extracellular product (ECP) fractions showed that degradation of the 57-kDa protein increased with time and increased temperature. Several lower molecular mass pcptides accumulated temporarily from this degradation. Phenylmethylsulphonyl fluoride prevented breakdown of the 57-kDa protein suggesting a scrine protease present in the ECPs was responsible. The results indicated that most, if not all, immunoreactive bands in ECP fractions, other than the 57-kDa protein, arose as a result of degradation of this protein. Western blot analysis of two dimensional gels revealed that the presumptive proteolytic activity was associated with the 57-kDa antigen and several of the apparent degradation products. Many common peptide fragments appeared to be generated from heat-induced proteolysis of these protein moieties, confirming the familial relationship between much of the immunoreactive material in ECP fractions. The results suggested that the 57-kDa antigen is autolytic. Western blot analysis of tissue samples from Atlantic salmon, Satmo solar L., infected with R. salmoninarum suggested that this lability of the 57-kDa antigen also occurred in situ .     

Inclusion of size fractionated fish hydrolysate in high plant protein diets for Atlantic cod, Gadus morhua

Fish hydrolysate was evaluated as feed ingredient in high plant protein diets in an 89 days feed experiment with Atlantic cod (Gadus morhua). The fish hydrolysate was size fractionated by ultra- and nano-filtration and the various fractions were tested specifically as feed ingredients to trace any effect observed with the hydrolysate. All diets contained 68% of total protein as plant protein, added as a mixture of corn gluten, full-fat soy bean meal, soy protein concentrate and extracted soy bean meal. The diets were equal in protein, lipid and energy. The control diet contained 21.8% fish meal. Fish hydrolysate was tested in another diet where one third of the fish meal protein was exchanged with the fish hydrolysate. Retentate after ultra-filtration of fish hydrolysate and retentate and permeate after nano-filtration were used in three separate diets at dietary inclusion levels corresponding to the absolute dry matter level of the fractions in the hydrolysate. The cod tripled in weight during the experimental period. No significant differences were observed for growth or feed intake for any groups. The diets containing retentate from ultra- and nano-filtration showed lower feed efficiency than the control diet with fish meal or the diet containing fish hydrolysate or permeate after nano-filtration. In conclusion the results show that fish hydrolysate may successfully be used as a protein source in high plant protein diets for Atlantic cod in exchange of fish meal. Removal of small molecules from the fish hydrolysate by filtration reveals poorer feed utilization indicating that this marine fraction of small compounds is important for optimal growth of Atlantic cod. This may be important in the discussion of increased dietary utilization of plant protein sources in feed for fish.     

Putative virulence factors of atypical Aeromonas salmonicida isolated from Arctic charr,Salvelinus alpinus (L.), and European grayling,Thymallus thymallus (L.)

Atypical Aeromonas salmonicida (AAS) causes generalized lethal infections in farmed Arctic charr, Salvelinus alpinus (L.), and European grayling, Thymallus thymallus (L.), and is thus a serious threat for culture of these fish species. Virulence factors were studied among isolates of AAS from Arctic charr (n = 20), European grayling (n = 19) and other fish species (n = 20), of which 48 were of Finnish and 11 of Swedish origin. All isolates produced an A-layer. Extracellular products (ECP) of the AAS isolates did not produce detectable gelatinase and caseinase activity in test assays. Analysis of the same ECP preparations with substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed weak proteolytic activity, indicating the different sensitivity of the detection methods used. The ECP from AAS isolates showed low cytotoxic activity against cultured cells. However, the ECP did not induce mortality in challenged Arctic charr. The results suggest that toxic components, like ECP, secreted by the bacterium may not be the major virulence factor in AAS-infection in Arctic charr and European grayling, and hence the pathogenesis also differs from the pathogenesis of AAS-infection in Atlantic salmon, Salmo salar L.     

The effects of Vibrio anguillarum extracellular products on Japanese eels

To test the effect of Vibrio anguillarum extracellular products (ECP) on Japanese eels, test fish were injected intramuscularly with ECP at a dose of 1 mg protein/100 g body weight of fish. At 3, 6, 12, 24 and 36 h post-injection, blood samples were collected for haematocrit, haemoglobin, and serum protein determinations and tissues were fixed in Bouin's solution. Histopathological observations 24 h post-injection revealed that the ECP caused severe damage to muscle tissue, characterized by extensive muscle liquefaction and haemorrhaging. In addition, extensive haemosiderin deposits were observed in the spleen, with lesser deposits occurring in the kidney and liver. Haematocrit, haemoglobin, and serum protein values were lower in ECP-treated fish than in the untreated controls.     

羊栖菜和裙带菜中抗凝血活性物质的初步筛选

Components with blood-anticoagulant activity were fractionated from marine algae Sargassumfusiforrne and Undaria pinnatifida and the activity of the components was investigated by using the method of bioassay. It was found that the 80% ethanol soluble fraction of Sargassumfusiforme exhibited no blood-anticoagulant activity, while the components obtained from the 80 % ethanol insoluble fraction (hot water extract) gave obvious bloodanticoagulant activity. A further study suggests that the principal component in the hot water extract from Sargassumfusiforrne with high blood-anticoagulant activity should be sulfated polysaccharides, in which the activity was positively correlated with the content of total sugar and fucose in sulfated polysaccharides. As for Undaria pinnatifida, the n-butanol extract fractionated from the 80% ethanol soluble fraction had blood anticoagulant activity, while the petroleum ether, ether, ethyl acetate, or water extract showed no bloodanticoagulant activity.     

Size rank and growth potential in redclaw crayfish (Cherax quadricarinatus): are stunted juveniles suitable for grow‐out?

Considerable size variation emerges at an early stage among redclaw (Cherax quadricarinatus) juveniles. In order to explore whether juveniles from different size fractions of the same population differ in their growth potential, they were reared individually, in the absence of social effects. In the first experiment, post‐release siblings were reared for 5 weeks and then categorized into three size fractions: small, med‐sized and large. Subsequently, juveniles of each size fraction (n=24, 18, and 18, respectively) were reared in individual cells for 46 days and their moult increments and moult intervals were compared. In addition, the growth indices of the med‐ and large‐sized juveniles were compared with those of juveniles from other broods that were similar in age and size but belonged to a different size fraction, in order to separate the effect of absolute size from that of size rank. In a second experiment, juveniles from the small‐, med‐ and large‐size fractions (n=21) of a commercial nursery‐reared population were individually reared for 14 weeks and their growth indices were compared. Survival of individuals from the small‐size fraction was low, particularly in the first experiment, and increased with fraction size. However, small‐sized juveniles did not show reduced growth potential and even grew at a faster rate than large‐sized juveniles. The results indicate that social interactions plays an important role in the development of size variation, and that small juveniles have the potential to grow rapidly when reared in individual compartments. Overall, the size rank was a poor predictor of growth potential.     

Inhibitory Activities of Protein Hydrolysates from Spotted Babylon Snails on Tyrosinase and Melanogenesis

The optimal conditions for protein hydrolysates preparation from spotted babylon Babylonia areolata exhibiting tyrosinase inhibitory activity and antioxidant activity using alkaline protease (Protease G6) were undiluted Protease G6 (5.8 × 10⁵ DU/g) at 60 min of hydrolysis time and eightfold diluted Protease G6 (7.25 × 10⁴ DU/g) at 240 min of hydrolysis time. These two conditions were fractioned using molecular weight (MW) cutoff values of 10, 5, and 3 kDa membranes, and their anti-melanogenic and antioxidant properties were further analyzed. Among the fractions, the MW < 3 kDa fraction exhibited high levels of inhibitory activity toward the mono- and diphenolase activities of tyrosinase, with IC₅₀ values of 1.758 and 8.995 μg/mL, respectively. Kinetic studies revealed that this fraction behaved as an uncompetitive inhibitor. The results demonstrated that the MW < 3 kDa fraction suppressed melanin synthesis and decreased cellular tyrosinase activity with no cytotoxicity to B16F10 melanoma cells.     

Expression patterns of acidic and basic fibroblast growth factor in loach fish embryos

Heparin-binding fractions were taken from the heparin sepharose columns on which extracts of loach fish (Misgurnus anguillicaudatus) embryos from blastula, gastrula, 4–8 and 12–16 somites stages were applied. These heparin-binding fractions, except the fraction derived from 12–16 somite embryos, showed potent mitogenic activity on fibroblast-like cells derived from caudal fin blastema of goldfish. Western blot analysis of these heparin-binding fractions was carried out using monoclonal antibodies against human acidic and basic fibroblast growth factors (FGF-1 and-2). An immunoreactive FGF-1 band at 16 kD was detected in the heparin-binding fraction derived from embryos in each stage of blastula, gastrula and 4–8 somites. An immunoreactive FGF-2 band at 17 kDa was detected exclusively in the heparin-binding fraction derived from 4–8 somite embryos. In the heparin-binding fraction derived from 12–16 somite embryos neither immunoreactive FGF-1 nor-2 member band was detectable.     

Taste effects of oligopeptides in a Vietnamese fish sauce

ABSTRACT: A Vietnamese fish sauce Nuoc mam used in the present study was rich in oligopeptides of which nitrogen content accounted for 20% of total nitrogen. The high-molecular-weight peptide fractions fractionated by ion-exchange chromatographies and ultrafiltration enhanced sweetness and umami as well as sourness and bitterness of the fish sauce, and increased several flavor characteristics including continuity, first taste and aftertaste. Thus, it is apparent that large amounts of peptides produced during the long-term fermentation of fish sauce are responsible for the complicated taste of the sauce. From each fraction, 17 peptides in total were isolated and determined for their amino acid sequences. These di-, tri- and tetra-peptides synthesized by solid- or liquid-phase method gave any one of bitter, sour, umami taste, or practically no taste in the absence of salt. In the presence of 0.3% NaCl, however, almost all peptides showed sweet and umami tastes. Other than these 17 peptides, there existed many other kinds of peptides in the fish sauce. Thus, these peptides are thought to contribute to the overall taste of the fish sauce.     

Enhanced growth effects on shrimp (Litopenaeus vannamei) from inclusion of whole shrimp floc or floc fractions to a formulated diet

A growth trial was conducted to determine the effects of inclusion of whole shrimp floc or floc fractions to a control diet on growth and survival of shrimp (Litopenaeus vannamei). The floc sample was collected from marine shrimp culture tanks and partially fractionated by extraction with water, acetone and hexane. A series of diets was manufactured by inclusion of whole floc (intact or ground), each of the fractions or their combination to a control diet. These diets were fed to shrimp (approximately 1.0 g) in an indoor laboratory under flow‐through conditions for 8 weeks. It was found that addition of whole floc (200 g kg^?1) or floc fractions (24–200 g kg^?1) to the control diet improved (P < 0.05) shrimp growth rate without affecting (P > 0.05) shrimp survival (>81.3%). Although inclusion of whole floc reduced the crude protein and crude fat contents and gross energy of the control diet, shrimp fed the whole floc‐supplemented diets obtained the highest (P < 0.05) growth rates (1.01 and 1.03 g week^?1) among the shrimp fed the 11 tested diets including two control (0.81 and 0.85 g week^?1), two commercial (0.45 and 0.71 g week^?1) and five floc‐fraction‐added (0.91–1.00 g week^?1) diets. Many bioactive compounds in the floc that possibly affected shrimp growth were also analysed and quantified.     

The effect of feeding a novel multistrain yeast fraction on European seabass (Dicentrachus labrax) intestinal health and growth performance

Fish were fed a single‐strain yeast fraction (SsYF; 2 g/kg) or a multistrain yeast fraction (MsYF; 0.8 g/kg) for 10 weeks. The results demonstrated significant (p ≤ 0.03) elevations in weight gain, specific growth rate, protein efficiency ratio, and feed conversion ratio in fish fed the yeast fraction‐supplemented diets. In the distal intestine, a significant elevation in microvilli density was observed after 5 and 10 weeks of dietary supplementation with MsYF and SsYF, respectively, compared to control fed fish (p < 0.001). A significant elevation (p = 0.02) in the perimeter ratio was observed in fish fed diets supplemented with the yeast fractions. After 10 weeks of feeding on the experimental diets, Rt‐qPCR demonstrated a significant downregulation (p < 0.05) in the stress response genes, heat‐shock protein 70 (hsp70) and proliferating cell nuclear antigen (pcna), in fish fed diets supplemented with the yeast fractions. Significant (p < 0.05) elevations in interleukin 1‐beta (il1β) and interleukin‐10 (il10) gene expression were observed in fish fed diets supplemented with the MsYF compared to the other dietary groups. These findings suggest that feeding an MsYF specifically at a lower incorporation rate < 1 g/kg, compared to a commercial SsYF at 2 g/kg, is effective in improving the intestinal health status and growth performance of European seabass.