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Notes: Abstract. A method for purification of the 70-KDa extracellular serine protease of Aeromonas salmonicida by hydrophobic chromatography and ion exchange is described. The purified protease, adsorbed onto mineral particles, was used for immunization of salmon. Other groups of salmon were immunized with particles coated with purified glycerophospholipid:cholesterol acyltransferase (GCAT) or with the acyltransferase complexed with LPS (GCAT-LPS). Humoral immune responses assayed after 6 weeks by ELISA, showed relatively good responses against GCAT-LPS, while titres of antisera against the purified protease and GCAT were scarcely above those of control sera. However, the antibody responses to each toxin were shown by Western blotting to be specific and qualitatively similar to responses seen in rabbits. The toxin preparations were also used (in combination with whole bacteria) for vaccination of salmon. On challenge 3 months later, only GCAT-LPS elicited significant immunological protection. However, a more convincing protection was seen when total extracellular product was present in the vaccine.

Notes: Abstract. Extracellular hacmolytic activities of Aeromonas salmonicida ssp. salmonicida to salmon red blood cells were shown to be due to different forms of the membrane-active enzyme glyccrophospholipidrcholcstcrol acyltransferase (GCAT). About 10% of the total haemolytic activity was due to a high molecular mass complex of LPS and GCAT (mol. mass 〉1000kDa), containing 35–50% neutral sugars and 1.5–2.0% protein. Some haemolytic activity (30–40% of total), corresponding to 50–70kDa by gel filtration, also contained GCAT-activity and may represent aggregated forms of GCAT. However, about 50% or more of the haemolytie activity was due to a protein of 26kDa free GCAT. Rabbit antibodies to GCAT neutralized the hacmolytic activity of both GCAT and GCAT-LPS. A transposon-produccd serinc protease negative mutant of the same A. salmonicida strain showed reduced haemolytic activity. The mutant produced a 38-kDa GCAT proform of low hacmolytic activity. The proform was processed by autogenous scrinc protease to a highly hacmolytic 26-kDa molecule with pl 6.3, similar to GCAT of the parent strain. The weakly haemolytic GCAT-LPS analogue of the mutant strain did not contain detectable amounts of the 26-kDa molecule and was not activated by proteases.

Notes: Abstract. Proteases produced by Vibrio anguillarum were isolated from culture supernatant by ultrafiltration, gel chromatography and ion exchange chromatography. The enzyme(s) were shown to be collagenolytic when assayed with native collagen substrates. In addition, the enzyme(s) hydrolysed azocasein, azocollagen, the collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg and the aminopeptidase substrate L-Leu-pNA effectively. Separation of the proteases by Mono Q ion exchange chromatography and native polyacrylamide gel electrophoresis revealed four distinct protein bands containing caseinase activity. However, only two of the bands showed aminopeptidase activity. The aminopeptidase activities could be separated from the caseinase activities by isoelectric focusing. Secreted proteases of different serotypcs of V. anguillarum showed a heterogeneous caseinolytic pattern. The molecular mass of the major enzyme was estimated at 35kDa as determined by its mobility on SDS-polyacrylamide gels. Serine protease inhibitors like PMSF, TPCK, TLCK and benzamidine had no inhibitory effects on the proteolytic activity when tested with azocasein as substrate. However, the enzyme was strongly inhibited by metal chelators like EDTA and 1, 10-phenanthroline. Also, normal salmon scrum and purified α2-macroglobulin from salmon serum strongly inhibited the caseinolytic activity of the enzyme.

Notes: Abstract. The predominant cell surface protein (A-protein) of Aeromonas salmonicida has been purified by a method utilizing a glycine/hydrochloridc extraction from whole cells and HPLC/ion exchanger (DEAE) columns. This procedure yielded two LPS-frec molecules (a 40- and a 50-kDa form) both shown to contain A-protein determinants. The former appears to be a digest product of the latter, as a serine protease produced by A. salmonicida was shown to process the 50-kDa form into a 40-kDa molecule in vitro. The A-layer protein was shown to contain one isoform, although multiple isoelectric forms appeared as preparative artifacts, probably due to deamidation. The A-layer protein and LPS arc the most significant surface antigens recognized by the Atlantic salmon B-lymphocytes or antibodies. Immunological studies of LPS-free and LPS-containing A-protein preparations were undertaken to test whether the two components behave like antigenie competitors or whether the LPS moiety could adjuvant the antibody response against the A-protein. The latter was shown to be the case.

Notes: Abstract. Vibrio anguillarum isolated from diseased cod, Gadus morhua L., were serotyped by use of a panel of monoclonal antibodies. Four serotypes could be distinguished, having different lipopolysaccharide determinants. These phenotypic differences were also reflected in the genetic map, as revealed by fingerprinting of bacterial DNA. Antisera were raised in cod after immunization with the V. anguillarum serotypes, and Western blot techniques demonstrated production of specific antibodies mainly to LPS-antigens. The immune system in cod discriminates to a eertain degree between the four serotypes as shown by crossreactions of the immune sera in elisa. Moreover, it was also shown that natural antibodies to bacterial antigens are present in non-immune sera, but these specificities are non-LPS in nature. As a consequence of the heterogeneity of the V. anguillarum strains, vaccination experiments were performed under laboratory conditions to compare the effectiveness of bacterins based on either single vaccines or polyvaccines. The results from these experiments were promising since challenge with one strain demonstrated 100% protection both in fish vaccinated with the homologous serotype as well as a mixture of all the four serotypes.

Notes: Lipopolysaccharide (LPS) and A-layer protein purified from Aeromonas salmonicida were administered intravenously in Atlantic salmon, Salmo salar L., either alone or in combination. Tissues from each organ were examined by immunohistochemical techniques, using a polyclonal antiserum against A-protein and a monoclonal antibody against LPS. When given simultaneously, the antigens seemed to be taken up by different cells in both the head and trunk kidney. The most striking finding was that A-protein was located in epithelial cells in renal proximal tubules, in contrast to LPS, which was not detected in this location. The amount of A-protein in the tissue increased with time until 2 h after injection. Autoradiography of SDS polyacrylamide gel electrophoresis of head kidney homogenates showed that in vivo processing of A-protein coupled to iodinated tyramine cellobiose (125I-TC-A-protein) was completed within 24 h, in contrast to LPS, which was maintained in tissues. The findings of the present study suggest that cells of the head kidney of Atlantic salmon are capable of taking up and processing the A-protein.

Notes: Τhe uptake and distribution of lipopolysaccharide (LPS), isolated from Aeromonas salmonicida, was investigated in Atlantic cod, Gadus morhua L., turbot, Scophthalmus maximus L., and Atlantic halibut, Hippoglossus hippoglossus L. LPS was radiolabelled by bromine oxidation and subsequent sodium borotritide reduction (3H-LPS), and fluorescence-labelled by introducing a fluorescein isothiocyanate derivative (FITC-LPS). After intravenous and intraperitoneal injections in cod, high amounts of radioactive LPS (3H-LPS) were present in heart, spleen and kidney throughout the experimental period (1–168 h). After peroral administration, a high amount of 3H-LPS was observed in intestinal tissues, whereas internal organs and tissues contained considerably lower amounts. Following intravenous administration of 3H-LPS in turbot, high contents of radioactivity were revealed in spleen, liver and kidney, whereas the content in heart was lower than in blood at the sampling times (1–24 h). The same pattern was observed after intraperitoneal administration. The spleen and liver contained high amounts of radioactivity when the turbots were intubated perorally with 3H-LPS. The spleen, kidney and heart were the main scavenging organs following intravenous administration of 3H-LPS in Atlantic halibut. A minor amount of radioactivity was present in the liver. The same pattern emerged after intraperitoneal injection in halibut. As observed for turbot, the spleen was the main accumulation site for 3H-LPS following peroral administration. Fluorescence microscopy of sections of organs and tissues from cod, intravenously and intraperitoneally injected with FITC-LPS, revealed that endocardial cells of both atrium and ventricle contained large amounts of the fluorochrome, whereas in turbot and halibut only atrial endothelial cells accumulated the substance. In all species, macrophages in kidney and spleen contained FITC-LPS and in the spleen the fluorochrome was trapped in the ellipsoidal walls. At later time points (e.g. 48 h) in the turbot spleen, FITC-LPS was located in cells adjacent to the ellipsoidal walls. Halibut endothelial cells that were located in the connective tissue of the intestine and gills also contained FITC-LPS. After peroral administration to the different fish species, specific fluorescence was found only in intestinal epithelial cells of halibut and in cells located in the lamina propria. Fluorescence was not detected in internal organs such as the kidney, spleen and liver after peroral administration of FITC-LPS. Gel chromatographic analysis of plasma samples from cod, turbot and halibut after intravenous and intraperitoneal injections showed that high molecular weight radioactivity was present. A minor amount of radioactivity that corresponded to low molecular weight substances was also observed. In conclusion, there is a high degree of variation with respect to the site of accumulation and some variation in the type of cells involved in the uptake of purified LPS in cod, turbot and halibut.

Notes: The antibacterial effects of synthetic cecropin B and cecropin P1 were tested against the fish-pathogenic bacteria Vibrio anguillarum, Vibrio salmonicida, Aeromonas salmonicida, Edwardsiella ictaluri and Yersinia ruckeri. Both cecropins were active against all bacteria tested, but the effect was strongly influenced by the growth media used. In brain heart infusion medium, the minimum inhibitory concentrations of cecropin B ranged from 0.3 to 1.3 μm and from 0.3 to 1.0 μm for cecropin P1, except for E. ictaluri, which was noticeably less sensitive to cecropin P1 (61 μm). The present authors have compared the bactericidal activity of these two peptides, showing that the killing rate for the selected bacteria was higher for cecropin B than for cecropin P1. V. anguillarum was the most sensitive to the cecropins, and in the present study, no colony forming units were detected after 4 and 8 min of treatment with cecropin B and P1, respectively. Electron microscopy was performed to document the effect of cecropin on the bacterial surface.