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.     

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.     

Seasonal variation of serum lysozyme levels in Atlantic halibut (Hippoglossus hippoglossus L.)

Serum lysozyme was measured in Atlantic halibut (Hippoglossus hippoglossus L.) kept in a range of different conditions that included ambient photoperiod and temperature and controlled photoperiod and temperature. There was no significant difference between animals held in ambient conditions of 6 degrees C and those held in controlled conditions of 12 degrees C. Similarly, there was no significant difference between animals maintained in a long day photoperiod and those in a short day photoperiod. However, there was a significant difference between summer and winter readings. Whilst this would indicate a link between season and the defence system, there appears to be no link with apparent entrainment to different photoperiods and serum lysozyme levels.     

Spontaneous haemolytic activity of Atlantic halibut (Hippoglossus hippoglossus L.) and sea bass (Dicentrarchus labrax) serum

The spontaneous haemolytic (SH) activity of sera was compared in groups of cultured halibut and sea bass. The optimum assay temperature was determined for each species and different red blood cell donors were tested. The effects of heat inactivation, storage temperature and of different agents like EDTA, EGTA, yeast cell components and bacterial LPS were compared. Halibut sera gave optimum lysis with sheep red blood cells (RBC) at 16 degrees C whereas sea bass sera showed optimum lysis with rabbit RBC at 37 degrees C. The haemolytic activity of halibut sera was inactivated at 45 degrees C while sea bass sera were inactivated at 56 degrees C. The haemolytic activity of halibut sera was significantly reduced during short-term storage at -80 degrees C, whereas the sea bass sera maintained fairly good activity after 1-year storage at -80 degrees C. EGTA and EDTA inhibited the spontaneous haemolytic activity of sera from both the species. Zymosan and MacroGard from yeast cells also inhibited the haemolytic activity of the sera of both species, whereas LPS had a very slight effect. Considerable variation in haemolytic activity was observed within both the halibut and sea bass groups studied.     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.     

The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of alpha-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 degrees C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.     

束缚应激大鼠血清淋巴细胞转化抑制因子的研究

为研究应激对淋巴细胞转化的影响,将 SD 大鼠四肢束缚于支架上,仰卧位,室温(20℃)下维持20h,对照组留置原饲养笼中,不予惊动。然后在乙醚轻麻下穿刺心脏取血,肝素化后密度梯度离心分离淋巴细胞,或待凝后分离血清。结果表明,应激大鼠外周血淋巴细胞对刀豆素(Con A)诱导的转化反应明显下降(p<0.01,n=8,ANOVA),而且应激大鼠血清可明显抑制正常小鼠淋巴细胞转化,这提示应激大鼠血清中可能存在某种具有抑制淋巴细胞转化的活性物质。进一步的分析实验表明,这种血清经加热56℃(30min),30%甲醇或透析(透析袋孔径阻滞分子量为6000)处理,抑制活性均不受影响;但经加热100℃(3min),80%甲醇或胰蛋白酶(64/μg/ml)处理,抑制活性丧失。提示这种抑制活性物质很可能是一类蛋白质。     

Short- and long-term effects of a dietary yeast beta-glucan (Macrogard) and alginic acid (Ergosan) preparation on immune response in sea bass (Dicentrarchus labrax)

The present study investigated the immunomodulatory activity of Ergosan, an algal extract containing alginic acid, and Macrogard, a yeast extract containing beta-glucans, on innate and specific immunity in sea bass (Dicentrarchus labrax). Four cycles of experimental feeding using normal fish feed formulation (control group) supplemented with Ergosan (0.5%) or Macrogard (0.1%) were performed at 60-day intervals (15 days of treatment+45 days of suspension). Serum complement, lysozyme, total proteins and heat shock protein (HSP) concentrations were measured at 15, 30 and 45 days from the end of the first 15-day feeding cycle (short term) and 45 days after the end of each feeding cycle over a 35-week period (long term). The percentage of B- and T-lymphocytes in peripheral blood leucocytes and gut were measured over long-term trial. Significant elevation (P < 0.05) in serum complement activity occurred in sea bass fed with alginic acid and glucans, at 15 days from the end of first cycle of treatment. Significant elevation (P < 0.05) in serum lysozyme, gill and liver HSP concentration were observed in the same experimental groups at 30 days from the end of treatment, whereas a significant increase (P < 0.05) of complement activity was only observed in fish that received an Ergosan diet. At 45 days from the end of treatment, complement, lysozyme and HSP concentration did not differ among groups. Over the long-term period, no significant differences were observed in innate and specific immune parameters, survival, growth performances and conversion index in treated and control fish. A dramatic decrease of both innate and acquired immune parameters was observed during the winter season in all groups, followed by a partial recovery when water temperature increased. Reduction in complement and lysozyme activities was significatively correlated (p < 0.01) to water temperature variation. The results suggested the potential of alginic acid and beta-glucans to activate some innate immune responses in sea bass, and particularly under conditions of immunodepression related to environmental stress.     

Pathogenicity of nodavirus strains from striped jack Pseudocaranx dentex and Atlantic halibut Hippoglossus hippoglossus, studied by waterborne challenge of yolk-sac larvae of both teleost species

The present study shows that differences in pathogenicity exist among fish nodavirus strains. In challenge trials, a Japanese strain (SJ93Nag) was highly virulent to larvae of the striped jack Pseudocaranx dentex but replication was not detected in larvae of Atlantic halibut Hippoglossus hippoglossus at 6 degrees C. Conversely, a Norwegian nodavirus strain (AH95NorA) that was highly virulent to the Atlantic halibut larvae did not replicate in striped jack larvae at 20 degrees C. Occurrence of the disease viral encephalopathy and retinopathy (VER) and cumulative mortality were significantly different in the 2 species when challenged with the 2 nodavirus strains. The presence of nodavirus in nervous tissue was monitored by immunohistochemical methods. Our results support the view that the genetic diversity among nodavirus strains reflects the existence of different viral phenotypes which may be adapted to infect different host species and/or for replicating at different temperatures. Fish nodaviruses represent surveyable pathogens well suited for studying the relation between viral genotypic and phenotypic properties such as host specificity, temperature optima, neuroinvasiveness and neurovirulence.     

Fluctuations in free or substrate-complexed lysozyme and a mutant of it detected on x-ray crystallography and comparison with those detected on NMR

A mutant lysozyme in which Arg14 and His15 were deleted together exhibited higher activity toward glycol chitin than the wild-type lysozyme. Moreover, the mutant lysozyme, which is less stable than the wild-type lysozyme by 7 degrees C, showed a shift of temperature dependence of activity to the low temperature side compared with the wild-type lysozyme [Protein Eng. 7, 743-748 (1994)]. In the free enzyme, the internal motion of the mutant lysozyme was similar to that of the wild-type. The internal motions of the wild-type and mutant lysozymes in the enzyme-substrate complex increased more than those in the free enzymes. Moreover, the increased internal motions of the substrate-complexed mutant lysozyme were greater than those of the substrate-complexed wild-type lysozyme in several residues [J. Mol. Biol. 286, 1547-1565 (1999)]. The structure of the mutant lysozyme was very similar to that of the wild-type lysozyme. Both structures were also alike in the complex of the trimer of N-acetyl-D-glucosamine. The mobility from B-factors agreed to some degree with that from order parameters in the regions showing great mobility of the protein, but this was not the case in the regions showing fast motion. However, we came to the same conclusion that the increased activity of the mutant lysozyme is due to the increase in the fluctuation of the lysozyme molecule. B-factor and order parameter do not always exhibit harmony because the time-scale of the analysis of mobility is different. However, they are not incompatible but complementary for detecting precise protein motions.     

Haematological studies in relation to environmental temperature and different periods of breeding cycle in an air breathing fish, Heteropneustes fossilis.

Investigations were made on different haematological parameters in H. fossilis at two environmental temperature (18 degrees C and 32.5 degrees C) and different periods of breeding cycles. The RBC counts 302.0-10(4)/mm3), WBC counts (10.6-10(4)/mm3), Hb concentration (19.0g%), haematocrit (36.0%), M.C.H.C. (57.7%), total blood volumes (0.966 ml) and per cent blood volumes (1.542%) gave significantly high values but M.C.V. (119.2 micrometer3) and erythrocyte surfaces (52.96 micrometer2) gave low values at higher temperature (32.5 +/- 1 degrees C) and during breeding period (G.S.I. = 10.4 +/- 0.52 g). The higher values obtained for different haematological parameters during breeding period at high temperature may be probably related to greater physical activity and higher metabolic activity at the time of reproduction.     

Bactericidal activity of C9-deficient human serum.

Escherichia coli B/SM, strain 1-1, was killed dose dependently by human hereditary C9-deficient serum (C9DHS), which was shown to contain no C9 Ag by an ELISA method. On the other hand, human hereditary C7-deficient serum did not kill the bacteria under similar conditions. The bactericidal activity of C9DHS was inhibited by rabbit anti-C5 antibody but not by murine anti-C9 mAb. The anti-C9 antibody decreased the bactericidal activity of normal human serum (NHS) to the level of that with C9DHS. Sheep anti-human lysozyme antibody did not affect the bactericidal activity of C9DHS or NHS even when added at more than twice the concentration required to block the serum lysozyme activity on Micrococcus luteus. After treatment with C9DHS and washing, surviving Escherichia coli were killed by C9, but not by lysozyme, transferrin, or both. Other strains of E. coli (K12 W3110, C600, and NIHJ) and Salmonella typhimurium (strain NCTC 74), all maintained in the laboratory, were also killed by C9DHS. However, pathogenic strains recently isolated from patients with traveler's diarrhea and some strains of S. typhimurium were resistant to both C9DHS and NHS, at least at the serum concentration tested. A concentration of 0.1 M Tris did not increase the susceptibility of serum-resistant strains of bacteria to C9DHS, but made one strain of S. typhimurium tested susceptible to NHS, but not to C9DHS. These results clearly showed that C9DHS kills bacteria that are sensitive to NHS through activation of C up to the step of C8 in the same way that C9-deficient C serum lyzed sensitized erythrocytes.     

beta-Galactosidase from Bacillus stearothermophilus.

Several strains of thermophilic aerobic spore-forming bacilli synthesize beta-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The beta-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 degrees C and the Arrhenius activation energy is about 24 kcal/mol below 47 degrees C and 16 kcal/mol above that temperature. At 55 degrees C the Km is 0.11 M for lactose and 9.8 X 10(-3) M for 9-nitrophenyl-beta-D-galactopyranoside. The enzyme is strongly product-inhibited by galactose (Ki equals 2.5 X 10(-3) M). It is relatively stable at 50 degrees C, losing only half of its activity after 20 days at this temperature. At 60 degrees C more than 60% of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein, and in the presence of 4 mg/ml of bovine serum albumin the enzyme is only 18% inactivated in 10 min at 60 degrees C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000.     

Disposition of 14C-flumequine in eel Anguilla anguilla, turbot Scophthalmus maximus and halibut Hippoglossus hippoglossus after oral and intravenous administration.

The absorption, distribution and elimination of 14C-labelled flumequine were studied using whole body autoradiography and liquid scintillation counting. Flumequine was administered to eel Anguilla anguilla, turbot Scophthalmus maximus and halibut Hippoglossus hippoglossus intravenously and orally as a single dose of 5 mg kg(-1), corresponding to 0.1 mCi kg(-1). The turbot and halibut studies were performed in salt water (salinity of 32%) at temperatures of 16 +/- 1 degrees C (turbot) and 9.5 +/- 0.5 degrees C (halibut). The eel study was conducted in fresh water at 23 +/- 1 degrees C. In the intravenously administered groups flumequine was rapidly distributed to all major tissues and organs. After oral administration flumequine also appeared to have rapid and extensive absorption and distribution in all 3 species. After the distribution phase, the level of flumequine was higher in most organs and tissues than in the blood, except in muscle and brain. The most noticeable difference between the species was the slow elimination of flumequine from eel compared to turbot and halibut. In orally administered eels, substantial amounts of flumequine remained in all major organs/tissues for 7 d. At 28 d significant levels of flumequine were present in liver, kidney and skin (with traces in muscle), and at the last sampling point (56 d) in eye, bone, bile and posterior intestine. In orally administered turbot significant levels of flumequine were observed over 96 h in bile, urine, bone, skin, intestine and eye, and traces were detected over 28 d in bone and eye in addition to a significant level in bile. In orally administered halibut, significant levels of flumequine were observed in bile, skin, intestine and eye over 96 h. Traces were present in skin and eye over 7 d. The maximal flumequine concentrations in blood were calculated to be 2.5 mg equivalents l(-1) (eel at 12 h), 0.8 mg l(-1) (turbot at 6 h) and 0.6 mg l(-1) (halibut at 6 h) after oral administration.     

Analysis of bacteriocinogenic properties of Yersinia enterocolitica strains

The aim of the study was the investigation of bacteriocinogenic properties of 102 Yersinia enterocolitica strains. The influence of selected factors on the production of bacteriocins by Y. enterocolitica and properties of jersiniacin 44JPSBKOH were also investigated. Bacteriocinogenic properties of Y. enterocolitica strains were tested by using the delayed cross-streaking method. It was found that the production of bacteriocins by Y. enterocolitica depended on the type of media on which the producer and indicator strains were grown. It turned out that some strains of Y. enterocolitica showed bacteriocinogenic properties at 25 degrees C, 30 degrees C and 37 degrees C irrespective of the presence of manganese ions in medium. In the presence of iron ions these strains showed bacteriocinogenic properties only at 25 degrees C. Y. enterocolitica strains which required Mn2+ or Mn7+ ions for bacteriocins production showed this activity only at 25 degrees C but in presence of Fe3+ ions they had no bacteriocinogenic properties. The partially purified jersiniacin 44JPSBKOH is a protein, its molecular weight was estimated to be 40 kDa. Yersiniacin 44JPSBKOH was active in the pH range of 3 to 9. Its bactericidal activity was rapidly lost when heated to 100 degrees C and treated with proteolytic enzymes. Yersiniacin 44JPSBKOH showed bactericidal activity against other Y. enterocolitica strains and some strains of Pseudomonas aeruginosa isolated from humans.     

Role of disulfide bonds in goose-type lysozyme

The role of the two disulfide bonds (Cys4-Cys60 and Cys18-Cys29) in the activity and stability of goose-type (G-type) lysozyme was investigated using ostrich egg-white lysozyme as a model. Each of the two disulfide bonds was deleted separately or simultaneously by substituting both Cys residues with either Ser or Ala. No remarkable differences in secondary structure or catalytic activity were observed between the wild-type and mutant proteins. However, thermal and guanidine hydrochloride unfolding experiments revealed that the stabilities of mutants lacking one or both of the disulfide bonds were significantly decreased relative to those of the wild-type. The destabilization energies of mutant proteins agreed well with those predicted from entropic effects in the denatured state. The effects of deleting each disulfide bond on protein stability were found to be approximately additive, indicating that the individual disulfide bonds contribute to the stability of G-type lysozyme in an independent manner. Under reducing conditions, the thermal stability of the wild-type was decreased to a level nearly equivalent to that of a Cys-free mutant (C4S/C18S/C29S/C60S) in which all Cys residues were replaced by Ser. Moreover, the optimum temperature of the catalytic activity for the Cys-free mutant was downshifted by about 20 degrees C as compared with that of the wild-type. These results indicate that the formation of the two disulfide bonds is not essential for the correct folding into the catalytically active conformation, but is crucial for the structural stability of G-type lysozyme.     

Inhibitory effect of combinations of heat treatment, pH, and sodium chloride on a growth from spores of nonproteolytic Clostridium botulinum at refrigeration temperature.

Nonproteolytic strains of Clostridium botulinum will grow at refrigeration temperatures and thus pose a potential hazard in minimally processed foods. Spores of types B, E, and F strains were used to inoculate an anaerobic meat medium. The effects of various combinations of pH, NaCl concentration, addition of lysozyme, heat treatment (85 to 95 degrees C), and incubation temperature (5 to 16 degrees C) on time until growth were determined. No growth occurred after spores were heated at 95 degrees C, but lysozyme improved recovery from spores heated at 85 and 90 degrees C.     

Temperature effect on the immune defense functions of Arctic charr Salvelinus alpinus

The Arctic charr Salvelinus alpinus is an endangered fish species in Finland, and thus farming is carried out mainly for stocking purposes. Farmed charr are susceptible to infection with atypical Aeromonas salmonicida (aAS). Losses of valuable brood stock will severely reduce the genetic diversity of stocked charr. No commercial vaccines are available to prevent aAS infection, and vaccines against furunculosis (caused by typical A. salmonicida, tAS) do not protect the charr against aAS infection. The effects of a metabolizable oil-adjuvanted, bivalent vaccine (containing killed aAS and A. salmonicida salmonicida bacteria) on the immune system of 1 yr old hatchery-reared charr originating from Lake Inari in Northern Finland were examined. Fish vaccination in Finland generally takes place either from October to November or from February to April, when the water temperature is low (1 to 3degrees C). The water temperature starts to increase in mid-May. Therefore, we also investigated whether post-vaccination (p.v.) temperature had an influence on the immune system of this cold-water fish species. The fish were immunized intraperitoneally at 2.9 degrees C at the end of April. After 52 d, during which the water temperature increased from 2.9 to 10.0 degrees C, the charr were exposed to 1 of 3 test temperatures: 10.3, 14.1 or 18.1 degrees C. Prior to vaccination, and 49, 75 and 103 d p.v., several immune parameters were measured in both unvaccinated and vaccinated charr. Vaccination induced a significant anti-aAS-specific antibody response, and increased plasma lysozyme activity at all p.v. temperatures. The haemolytic activity of the complement system was unaffected either by vaccination or p.v. temperatures. There was a slight positive correlation between p.v. temperature and lysozyme activity of the charr. The significant increase in lysozyme activity took place in vaccinated charr in the first 49 d p.v. as water temperatures increased from 2.9 to 10 degrees C. Furthermore, the highest activity of lysozyme in the plasma was observed 49 d p.v. Our results indicate that a rise in water temperature above 10 degrees C does not significantly enhance the vaccination response of charr. This could be one reason why farmed Arctic charr, which are well adapted to a cold climate, are highly susceptible to aAS infection in the summer.     

Use of gel electrophoresis for the study of enzymatic activities of cold-adapted bacteria

Glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH) activity of 13 cold-adapted strains, isolated from cold soils and showing GDH and/or LDH activity in spectrophotometric assays, were revealed by the use of electrophoresis on a nondenaturing acrylamide gel (zymogram). Psychrophilic strains were grown at 4 degrees C and 10 degrees C and the psychrotolerant strains at 4 degrees, 20 degrees and 28 degrees C. Incubation with the specific substrate and staining were done at 4, 28 or 37 degrees C. In the most cold-adapted strains, LDH and GDH production was high at 4 degrees C. In psychrotrophic strains, enzyme production and activity were greater at 20 or 28 degrees C than at lower temperatures. LDH remained active up to 37 degrees C while GDH activity was more thermolabile. GDH activity was NAD-dependent in some psychrophilic strains. In other strains, it was dependent on NAD(P) only or on both NAD and NAD(P). Two bands were seen for GDH or LDH activity in some strains. This method, which does not require a dialysis step, can be used to study the influence of temperature on enzyme production and activity, and the co-factor dependence. It detects phenotypic differences between isozymes, providing data for systematics.