Humoral immune parameters of cultured Atlantic halibut (Hippoglossus hippoglossus L.)

Several humoral immune factors were studied in a group of cultured halibut (Hippoglossus hippoglossus L.). The serum protein and IgM concentration was comparable to levels seen in other teleost species. A strong antibody activity against TNP-BSA was observed but not against other antigens tested. Lysozyme and anti-protease activity was detected and showed variable heat sensitivity. Unlike the anti-protease activity, the lysozyme activity of the sera was not sensitive to storage at -20 degrees C. No spontaneous haemolytic activity was observed and the sera had no bactericidal effect on any of the bacterial strains tested. Iron binding capacity of the sera was high. Individual variation was considerable in all the factors tested.     

Isolation and characterization of a fish F-type lectin from gilt head bream (Sparus aurata) serum

A novel fucose-binding lectin, designated SauFBP32, was purified by affinity chromatography on fucose-agarose, from the serum of the gilt head bream Sparus aurata. Electrophoretic mobility of the subunit revealed apparent molecular weights of 35 and 30 kDa under reducing and non-reducing conditions, respectively. Size exclusion analysis suggests that the native lectin is a monomer under the selected experimental conditions. Agglutinating activity towards rabbit erythrocytes was not significantly modified by addition of calcium or EDTA; activity was optimal at 37 degrees C, retained partial activity by treatment at 70 degrees C, and was fully inactivated at 90 degrees C. On western blot analysis, SauFBP showed intense cross-reactivity with antibodies specific for a sea bass (Dicentrarchus labrax) fucose-binding lectin. In addition, the similarity of the N-terminal sequence and a partial coding domain to teleost F-type lectins suggests that SauFBP32 is a member of this emerging family of lectins.     

Susceptibility of three different strains of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at two different temperatures to Vibrio anguillarum and temperature effect on antibody response

Three geographically distinct-reared strains (Canadian, Icelandic, Norwegian) of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at optimal and super-optimal growth temperatures (12 and 18 degrees C respectively), were challenged with a virulent isolate of Vibrio anguillarum by injection. The halibut were injected intraperitoneally with 100 microl of the bacterial suspension (1 x 10(6) cells per fish). After challenge, temperature and strain-related differences in survival were observed. Canadian and Icelandic halibut cultured at the super-optimal temperature of 18 degrees C were significantly more susceptible to infection than those strains cultured at 12 degrees C. Total mortality at 18 degrees C for the Canadian and Icelandic strains was 56.4 and 61.85% respectively, compared to 32 and 26.6% respectively at 12 degrees C. Norwegian halibut were significantly more resistant to infection with V. anguillarum at 18 degrees C compared to the other strains, with total mortality of 13.3%. There was no significant difference in total mortality of Norwegian halibut at 18 or 12 degrees C (13.3, 25% respectively). The specificity of the antibodies in sera from challenged halibut cultured at 18 degrees C was primarily to LPS. Immunoblots showed the presence of antibodies against O-side chain antigens. This reaction was strongest in sera from the Norwegian halibut strain compared with the Canadian and Icelandic halibut, which suggests that the difference in resistance to challenge may be ascribable to the presence of antibodies to LPS. Specific antibody levels, as measured by ELISA, increased with increasing temperature and strain differences were apparent, however these did not relate to disease resistance.     

Rearing temperature enhances hepatic glucokinase but not glucose-6-phosphatase activities in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) juveniles fed with the same level of glucose

The aim of this work was to elucidate if the previous results observed in hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) activities in European sea bass and gilthead sea bream are due to temperature per se or to differences in feed intake at different water temperatures. For that purpose triplicate groups of fish (30 g initial body weight) were kept at 18 degrees C or 25 degrees C during two weeks and fed a fixed daily ration of a glucose-free or 20% glucose diet. At the end of the experimental period, plasma glucose levels in both species were not influenced by water temperature but were higher in fish fed the glucose diet. Higher hepatic GK activity was observed in the two fish species fed the glucose diet than the glucose-free diet. In the glucose fed groups, GK activity was higher at 25 degrees C than at 18 degrees C. Glucose-6-phosphatase activities in both species were not influenced by water temperature. In European sea bass and in contrast to gilthead sea bream it was observed an effect of dietary composition on G6Pase activities with surprising higher activities recorded in fish fed the glucose diet than in fish fed the glucose-free diet. Overall, our data strongly suggest that European sea bass and gilthead sea bream are apparently capable to strongly regulate glucose uptake by the liver but not glucose synthesis, which is even enhanced by dietary glucose in European sea bass. Within limits, increasing water temperature enhances liver GK but not G6Pase activities, suggesting that both species are more able to use dietary carbohydrates at higher rearing temperatures.     

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.     

Production and partial characterization of high molecular weight extracellular alpha-amylase from Thermoactinomyces vulgaris isolated from Egyptian soil

Optimizing production of alpha-amylase production by Thermoactinomyces vulgaris isolated from Egyptian soil was studied. The optimum incubation period, temperature and initial pH of medium for organism growth and enzyme yield were around 24 h, 55 degrees C and 7.0, respectively. Maximum alpha-amylase activity was observed in a medium containing starch as carbon source. The other tested carbohydrates (cellulose, glucose, galactose, xylose, arabinose, lactose and maltose) inhibited the enzyme production. Adding tryptone as a nitrogen source exhibited a maximum activity of alpha-amylase. Bactopeptone and yeast extract gave also high activity comparing to the other nitrogen sources (NH4CI, NH4NO3, NaNO3, KNO3, CH3CO2NH4). Electrophoresis profile of the produced two alpha-amylase isozymes indicated that the same pattern at about 135-145 kDa under different conditions. The optimum pH and temperature of the enzyme activity were 8.0 and 60 degrees C, respectively and enzyme was stable at 50 degrees C over 6 hours. The enzyme was significantly inhibited by the addition of metal ions (Na+, Co2+ and Ca2+) whereas CI- seemed to act as activator. The enzyme was not affected by 0.1 mM EDTA while higher concentration (10 mM EDTA) totally inactivated the enzyme.     

Haemolytic and phospholipase C (PLC) activities of Rhodococcus equi

Rhodococcus equi, an intracellular organism causing pneumonia and lung abscesses in foals, is generally thought to be non-haemolytic. In the present study, however, 13 of 14 representative isolates were found to be haemolytic when tested on agar media containing washed red blood cells rather than whole blood. Red cells of rabbits, dogs, horses and man were more sensitive to lysis than were those of ruminants. Two new enzymatic activities of the species were defined: a lecithinase and a phosphatidylinositol-specific phospholipase C (PI-PLC). As judged from tests for trypsin, temperature and ethanol sensitivity, the haemolytic activity was primarily dependent on PI-PLC though the participation of lecithinase seemed probable. The haemolytic activity of growing strains, but not of cell-free preparations, was partially inhibited by lecithin but enhanced by cholesterol; however, cholesterol oxidase (CO) activity, known to mediate cooperative lysis of RBC sensitized with sphingomyelin-specific phospholipases C or D of some other species, did not contribute to the direct haemolysis caused by R. equi as demonstrated here.     

Identification of the cell wall receptor for bacteriophage E79 in Pseudomonas aeruginosa strain PAO.

Bacteriophage E79 was shown to interact with the lipopolysaccharide (LPS) of Pseudomonas aeruginosa strain PAO. LPS isolated from an E79-sensitive, smooth strain inactivated the phage, exhibiting a Phl50 value (concentration of LPS that caused a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) of 0.04 microgram/ml, whereas the LPS isolated from a rough mutant derived from the wild type showed no neutralizing activity towards E79. EDTA and sodium deoxycholate were demonstrated to abolish the neutralizing capacity of the smooth LPS. One E79 receptor site was shown to be equivalent to 10(-16) g of LPS.     

Evidence of complement-mediated killing of Discocotyle sagittata (Platyhelminthes, Monogenea) oncomiracidia

Discocotyle sagittata oncomiracidia were rapidly killed when incubated in na?ve plasma and immune sera from both rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta), the killing proceeding at a faster rate with blood material from the latter fish species. The lethal activity of na?ve plasma and immune sera was comparable. This was abolished after incubation at 45 degrees C for 30 min and by the addition of EDTA but not EGTA supplemented with Mg(2+), indicating that complement acting via the alternative pathway is responsible for the parasiticidal effect observed. Scanning electron micrographs showed varying degrees of surface disruption in larvae exposed to fish plasma, suggesting that complement acts by breaching the oncomiracidial tegument. Control (untreated) oncomiracidia showed no damage. Ultrastructural damage was more extensive in oncomiracidia exposed to brown trout plasma than to rainbow trout plasma for equal periods, suggesting that the complement cascade may be involved in mediating host susceptibility.     

Peroxide-triggered erythrocytes haemolysis as a model for the study of oxidative damage in marine fishes

The haemolysis of sea bass Dicentrarchus labrax red blood cells (RBC) was initiated by tert -butyl-hydroperoxide (t-BHP). The onset of the haemolytic process was accelerated by increasing t-BHP concentration. This process was preceded by a drop in the RBC glutathione content followed by the production of lipid peroxidation products. Also t-BHP induced DNA fragmentation in RBC nuclei as measured by COMET assay. The addition of the antioxidant Trolox C® dose-dependently delayed the onset of both lipid peroxidation and haemolysis, and protected GSH stores against t-BHP-induced depletion. DNA fragmentation was also pre-vented by Trolox C®. These results indicate that t-BHP induces haemolysis in sea bass RBC through the induction of oxidative stress. Such a simple model could prove useful for both fundamental and applied studies on marine fish antioxidant mechanisms.     

Alternative complement pathway-dependent ingestion of fluolite particles by human granulocytes

Fluorescent particles (Fluolite) with an average size of 0.1 micrometers were ingested by human granulocytes after incubation in fresh normal human serum (NHS). Ingestion was assessed by visual counting in a fluorescent microscope of cells containing particles. Ingestion required fresh normal serum and did not occur when serum was heated for 30 min at 50 degrees C or in the presence of ethylenediaminetetraacetic acid (EDTA). It did not occur in serum genetically deficient in C3b inactivator or in C3. Phagocytic activity was restored to C3-deficient serum by purified human C3 and to heat inactivated serum by purified factor B. Opsonic activity was present in NHS containing 5 mM Mg++ and 10 mM ethyleneglycoltetraacetic acid (EGTA) and in human serum genetically deficient in human C components C2 and C5. Agammaglobulinemic sera had normal opsonic activity. Opsonization of particles in this system is mediated through the alternative pathway of C activation, and its measurement serves as a simple quantitative functional assay for this system.     

荷斯坦种公牛血清替代补体溶血活性研究

本文将国外脊椎动物血清补体溶血活性标准测定方法,运用到荷斯坦种公牛研究中,首次建立了测定荷斯坦种公牛血清补体溶血ACH50的方法。种公牛血清经相应靶红细胞吸附后,可溶解悬浮在EGTAMgGVB缓冲液中的正常的兔血红细胞、人A,B,AB,O型红细胞,小鼠、大鼠、鸡红细胞,但对绵羊、山羊、猪红细胞溶血活性较低;对奶牛红细胞无溶血活性。且发现种公牛血清的溶血活性和靶红细胞的动物种类在系统发育上和种公牛的亲缘关系远近没有直接联系。种公牛血清在EGTAMgGVB缓冲液中对兔血红细胞发生溶血的最适条件是:温度是37℃,最适pH是7.3-7.4,最适Mg2 的浓度是4mmol/L,最适孵育时间为90min。溶血活性是二价离子依赖、热敏感(溶血活性热灭活温度是56℃)。种公牛血清对兔血红细胞的溶血活性在受到酵母聚糖、甲胺、肼、EDTA、鸡抗酵母聚糖牛血清结合物抗血清处理时,溶血活性可全部或部分消失,溶血活性抑制程度与补体抑制剂浓度相关。我们运用建立的标准溶血方法并以兔血红细胞作为指示细胞检测不同年龄的53头种公牛血清补体替代途径的溶血活性,溶血值在13.2-44.3u/ml之间,还发现不同年龄组公牛之间溶血活性有随年龄增加而逐步增大趋势,但差异不显著(P>0.05),在4-5岁公牛群中达到最大值。对种公牛血清补体系统溶血水平进行系统研究,一方面可以填补国内在此领域研究空白,另一方面也利于种公牛疾病监测、控制,此外也为兽医临床诊断试剂的研制提供新的技术手段。     

Proteolytic activity of a yeast cell wall lytic Arthrobacter species

AIMS: To investigate properties of the proteolytic activity of a yeast cell wall lytic soil bacterium identified as an Arthrobacter species. METHODS AND RESULTS: The organism was grown at pH 7.5 and 30 degrees C in shake flasks on media with different complex subtrates. Highest proteolytic activity assayed with azocaseine was detected in media with wheat gluten. In addition, l-leucine, l-alanine exopeptidase activity and esterase activity were found. The proteolytic activity showed stability up to pH 12, with a maximum at pH 11. The temperature optimum was at 55 degrees C, but there was a loss in enzyme activity of 50% within 2 h. The proteolytic activity was inhibited by 3,4-dichloroisocumarin, whereas there was little or no effect with EDTA, pepstatin A or E64. CONCLUSIONS: The proteolytic activity is highly alkaline stable. The formation of the enzyme can be induced by media with high protein content.     

Properties of a ribonuclease from Aedes aegypti larvae

1. The properties of a soluble ribonuclease from Aedes aegypti larvae have been compared with ribonuclease activity in adult female tissue. 2. In larval extracts ribonuclease activity was maximal at 40-45 degrees C whereas activity in tissue from adult females was highest at 50 degrees C. 3. Ribonuclease activity that was recovered in a 20-60% ammonium sulfate precipitate was further purified by batch elution from DEAE-Sephacel and from carboxymethylcellulose. 4. Ribonuclease activity in the partially purified fraction was sensitive to EDTA, stimulated by magnesium, had a pH optimum at 9.0 and a Mr of 45,000. 5. Agarose gels containing yeast RNA substrate were used to monitor partial purification of the larval ribonuclease.     

Complement-mediated lysis in vitro of newly excysted tapeworms: Hymenolepis diminuta, Hymenolepis microstoma, Hymenolepis nana and Hymenolepis citelli

Bøgh H., Christensen J.P.B. and Andreassen J. 1986. Complement-mediated lysis in vitro of newly excysted tapeworms: Hymenolepis diminuta, Hymenolepis microstoma, Hymenolepis nana and Hymenolepis citelii. International Journal for Parasitology16: 157–161. Newly excysted worms of Hymenolepis diminuta were lysed in 50% normal serum from all 13 animal species tested, including man. Since H. diminuta was neither lysed in complement inactivated serum—by heat or adding EDTA, LPS or CVF—nor in C5-deficient mouse serum, it is concluded that the lysis was associated with the complement cascade. It is shown that H. diminuta can activate the complement system via both the classical and alternative pathway. Furthermore, it is indicated that the lysis is independent of serum antibodies. Hymenolepis nana and H. citelli were also lysed in all normal sera tested, eight and six respectively, while newly excysted worms of H. microstoma were lysed in normal sera from 10 mammals and birds, but not in sera from its hosts, the mouse, rat and golden hamster. This indicates that the complement system of these three species differs from that of the other species tested in such a way that H. microstoma is able to avoid lysis in these sera.     

Magnesium chelation inactivates beta-galactosidase in -20 degrees C storage.

Mammalian cell lysate containing beta-galactosidase (beta Gal) derived from the transient expression of the bacterial lacZ gene driven by the human beta-actin promoter loses activity progressively over time in storage at -20 degrees C in the presence of EDTA. The simultaneous presence of NaCl with EDTA exacerbates such an inactivation, although NaCl by itself does not. However, EGTA, a chelating agent that preferentially binds Ca2+ over Mg2+, does not inactivate beta Gal. Addition of equal or higher molar concentration of Mg2+ (as MgCl2) or Ca2+ (as CaCl2), both effectively chelated by EDTA, to an EDTA-containing lysate prevents this cold-related inactivation, but does not reactivate the enzyme. Therefore, the chelation of Mg2+ by EDTA at -20 degrees C inactivates beta Gal. Storage of cell lysate at -70 degrees C completely prevents the EDTA-induced inactivation of beta Gal. It is recommended that when beta Gal activity is used as the reporter for gene expression 1) EDTA should not be used to prepare cell lysate and 2) the cell lysate should be stored in a -70 degrees C freezer to preserve full activity.     

Endotoxin inactivating activity of rat serum

The ability of rat serum to inactivate endotoxin (LPS) was assessed with the aid of the limulus amebocyte lysate assay. Following the addition of various amounts of endotoxin to normal serum the mixture was incubated for 1 hr at 37 degrees C and the residual endotoxin activity determined. One milliliter of rat serum inactivated between 5 and 10 micrograms Escherichia coli LPS per hour. Heating serum for 45 min at 56 degrees C resulted in loss of 80-90% of the LPS inhibitor (LPSI) activity. Serum from cobra venom factor (CVF)-treated rats inactivated between 0.5 and 2.5 micrograms LPS/ml serum. Serum from tolerant rats, even after heating for 45 min at 56 degrees C, inactivates between 10 and 15 micrograms LPS/ml serum/hr; decomplemented tolerant rat serum neutralizes between 5 and 10 micrograms LPS/ml serum/hr. Clearly, the tolerant rat has large quantities of LPSI activity, which does not appear to be complement. The inhibitor found in tolerant rat serum is not species specific since it inactivates Salmonella minnesota and Salmonella typhimurium endotoxins to the same degree and in the same amount as E. coli endotoxin, the agent used to induce tolerance. Both heating serum (56 degrees C) and lead acetate reduce LPSI activity.     

Stability of (2'-5')oligoriboadenylates in various sera

Avian and mammalian sera were found to contain an enzyme activity degrading 2-5A oligonucleotides. The most extensive degradation of the A2' p5' A was observed in chicken serum. Degradation of this compound is not affected by the presence of cAMP, dsRNA, Mg2+, but is significantly inhibited by EDTA. The enzyme activity described is not inactivated by heating to 56 degrees C for 30 min. The 5-mU3' p5' A has also been degraded in chicken serum.     

Isolation and characterization of a fish F-type lectin from gilt head bream (Sparus aurata) serum

A novel fucose-binding lectin, designated SauFBP32, was purified by affinity chromatography on fucose–agarose, from the serum of the gilt head bream Sparus aurata. Electrophoretic mobility of the subunit revealed apparent molecular weights of 35 and 30 kDa under reducing and non-reducing conditions, respectively. Size exclusion analysis suggests that the native lectin is a monomer under the selected experimental conditions. Agglutinating activity towards rabbit erythrocytes was not significantly modified by addition of calcium or EDTA; activity was optimal at 37 °C, retained partial activity by treatment at 70 °C, and was fully inactivated at 90 °C. On western blot analysis, SauFBP showed intense cross-reactivity with antibodies specific for a sea bass (Dicentrarchus labrax) fucose-binding lectin. In addition, the similarity of the N-terminal sequence and a partial coding domain to teleost F-type lectins suggests that SauFBP32 is a member of this emerging family of lectins.