Characterization of the Renibacterium salmoninarum haemagglutinin

Water-extracted proteins from nine geographically diverse strains of Renibacterium salmoninarum, all of which agglutinated rabbit erythrocytes and rainbow trout spermatozoa, were compared by SDS-PAGE. Extracts from eight strains, including the type strain, ATCC 33209, were similar, containing a major protein of 57 kDa and a minor protein of 58 kDa. The SDS-PAGE protein profile of the Char strain did not contain the 58 kDa protein. A non-agglutinating strain, MT-239, which was also non-hydrophobic, did not produce any water-extractable protein. Immunoblot reactions with rabbit antiserum prepared against whole cells of the type strain demonstrated that the water-extracted haemagglutinins from the various strains were antigenically related. When purified by polyacrylamide gel zone electrophoresis, the haemagglutinin from R. salmoninarum ATCC 33209 formed a doublet band with molecular masses of 57 and 58 kDa, similar to the previously described F antigen. The water-extracted haemagglutinin agglutinated salmonid spermatozoa, was degraded by protease K and trypsin, and was shown to self-assemble onto the cell surface.     

Further characterization of Renibacterium salmoninarum extracellular products.

Renibacterium salmoninarum, the agent of bacterial kidney disease in salmonids, releases high concentrations of extracellular protein in tissues of infected fish. The extracellular protein consists almost entirely of a 57-kDa protein and derivatives of degradation and aggregation of the same molecule. The 57-kDa protein and its derivatives were fractionated into defined ranges of molecular mass. Separated fractions continued to produce degradation and aggregation products. One-dimensional electrophoretic separation of extracellular protein revealed a number of proteolytically active bands from > 100 to approximately 18 kDa associated with various 57-kDa protein derivatives in the different molecular mass fractions. Two-dimensional separation of extracellular protein showed that continued degradation and aggregation, similar both in location and behavior to some of the 57-kDa protein derivatives, was also displayed by the proteolytically active bands after their separation. Effects of reducing agents and sulfhydryl group proteinase inhibitors indicated a common mechanism for the proteolytically active polypeptides characteristic of a thiol proteinase. The results suggested that the 57-kDa protein and some of its derivatives undergo autolytic cleavage, releasing a proteolytically active polypeptide(s) of at least 18 kDa. Soluble polysaccharide-like material also was detected in extracellular products and tissue from infected fish. Antiserum to the polysaccharide-like material cross-reacted with O-polysaccharide of the fish pathogen Aeromonas salmonicida, suggesting some structural similarity between these polysaccharides. The polysaccharide and the proteolytic activity associated with the 57-kDa protein derivatives should be investigated with respect to the pathogenesis of R. salmoninarum infections.     

Cloning, functional expression and partial characterization of the glucose kinase from Renibacterium salmoninarum

In recent years, different molecular techniques have led to an important progress in the characterisation of Colletotrichum species, but there are no available methods which permit the easy identification of Colletotrichum strains and their assignation to classical species. In the present work, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene, were used to identify a total of 80 strains of Colletotrichum, the majority of them isolated from strawberry. One of the most interesting results derived from this study was the easy and reliable distinction, using the endonuclease MvnI, between Colletotrichum fragariae and Colletotrichum gloeosporoides, both responsible of anthracnose on strawberry and phenotypically indistinguishable. Moreover, we propose the restriction fragments generated by the endonucleases MvnI, PvuII and ScrFI as a rapid method to differentiate species of the Colletotrichum genus.     

Uniformity in the biochemical properties of Renibacterium salmoninarum isolates obtained from several sources

Abstract A marked similarity in the biochemical properties of 26 Renibacterium salmoninarum cultures was found. Properties not previously recorded for this bacterium include haemolytic and proteolytic activity, DNase and the presence of glycogen in cells, and negative reactions in tests for nitrates, Tween-80 hydrolysis, amylase, bile solubility, arginine hydrolysis and phosphatase. Selected properties enabled bacteria morphologically similar to R. salmoninarum to be identified.     

Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum.

Renibacterium salmoninarum, the causative agent of bacterial kidney disease, produces large quantities of a 57-58 kDa protein (p57) during growth in broth culture and during infection of salmonid fish. Biological activities of secreted p57 include agglutination of salmonid leucocytes and rabbit erythrocytes. We define the location of epitopes on p57 recognized by agglutination-blocking monoclonal antibodies (MAbs) 4C11, 4H8 and 4D3, and demonstrate that the majority of secreted p57 is a monomer that retains salmonid leucocyte agglutinating activity. The 3 MAbs bound a recombinant, amino-terminal fragment of p57 (211 aa) but not a carboxy-terminal fragment (315 aa) demonstrating that the neutralizing epitopes are located within the amino-terminal portion of p57. When combinations of the MAbs were used in an antigen capture ELISA, the epitopes recognized by the 3 MAbs were shown to be sterically separate. However, when the same MAb was used as both the coating and detection MAb, binding of the biotinylated detection MAb was not observed. These data indicate that the epitopes recognized by the 3 agglutination-blocking antibodies are functionally available only once per molecule and that native p57 exists as a monomer. Similar ELISA results were obtained when kidney tissues from 3 naturally infected chinook salmon were assayed. Finally, a p57 monomer was purified using anion exchange and size exclusion chromatography that retained in vitro agglutinating activity. A model in which p57 is released from R. salmoninarum as a biologically active monomer during infection of salmonid fish is proposed.     

Molecular cloning of Renibacterium salmoninarum DNA fragments

A Renibacterium salmoninarum enriched recombinant DNA library was constructed to isolate DNA fragments which could be used as probes to detect gene sequences specific for the causative agent of bacterial kidney disease in salmonid fish. One fragment of 149 base pairs was isolated and its specificity and sequence determined. This probe may prove useful in the design of diagnostic tests for the disease in asymptomatic fish and ova.     

Purification and characterization of a novel haemagglutinin from Chlorella pyrenoidosa

We previously identified a strong haemagglutination activity in the freshwater unicellular green alga, Chlorella pyrenoidosa. Here, we sought to purify and characterize the haemagglutinin associated with this activity. Ammonium sulfate precipitation, gel filtration on sephacryl S-200 and DEAE-Sepharose ion-exchange chromatography were used to purify the haemagglutinin, which was designated CPH (Chlorella pyrenoidosa haemagglutinin). The molecular weight of CPH was estimated as 58 kDa by SDS-PAGE and 60 kDa by gel filtration of the native protein, indicating that this haemagglutinin exists as a monomer. The haemagglutinin activity of CPH was inhibited by glycoproteins, especially yeast mannan, but not by monosaccharides or disaccharides, indicating that CPH is carbohydrate-specific. In addition to the composition of CPH shown to be rich in glycine and acidic amino acids, heamagglutinating activity of CPH was insensitive to variations in pH or the presence of divalent cations, and atomic force microscopy revealed that the protein is rod-shaped. These results indicate that the characteristics of CPH are consistent with its identification as a haemagglutinin, and suggest that CPH may be a viable candidate for applications in a variety of biomedical fields.     

Description and characterization of IS994, a putative IS3 family insertion sequence from the salmon pathogen, Renibacterium salmoninarum

The motor properties of myosin reside in the globular S1 region of the myosin heavy chain (MHC) subunit. All vertebrates express a family of MHC isoforms in skeletal muscle that have a major influence on the mechanical properties of the various fiber types. Differences in molecular composition of S1 among MHC isoforms within a species have not been studied to any great detail. Presently, we have isolated, cloned and sequenced the S1 subunit of four MHC isoforms from skeletal muscle in Rana pipiens that are specifically expressed in four mechanically divergent fiber types. Paired analysis showed that the overall amino acid identity was higher between the three S1 isoforms expressed in twitch fibers than between the twitch and tonic isoforms. Relatedness in amino acid composition was evaluated in regions reported to govern cross-bridge kinetics. Surface loops 1 and 2, thought to influence motor velocity and ATPase, respectively, were both highly divergent between isoforms. However, the divergence in the loops was roughly equal to that of the amino-terminal region, a domain considered less important for motor function. We tested the hypothesis that the loops are more conserved in pairs of isoforms with more similar kinetics. Comparisons including other vertebrate species showed no tendency for loops from pairs with similar kinetics to be more conserved. These data suggest that the overall structure of loops 1 and 2 is not critical in regulating the kinetic properties of R. pipiens S1 isoforms. Cloning of this family of frog S1 isoforms will facilitate future structure/function studies of the molecular basis of variability in myosin cross-bridge kinetics.     

Evaluation of a whole cell, p57- vaccine against Renibacterium salmoninarum.

A whole cell Renibacterium salmoninarum vaccine was developed using 37 degrees C heat treated cells that were subsequently formalin fixed; this treatment reduced bacterial hydrophobicity and cell associated p57. Coho salmon Oncorhynchus kisutch were immunized with the p57- vaccine by either a combination of intraperitoneal (i.p.) and intramuscular (i.m.) injections or per os. In the first experiment, i.p./i.m. vaccination of coho salmon with p57- cells in Freund's Incomplete Adjuvant (FIA) conferred a statistically significant increase in mean time to death after the salmon were i.p. challenged with 4.1 x 10(6) colony forming units (cfu) of R. salmoninarum. There was no significant difference in response between fish immunized with R. salmoninarum cell surface extract in FIA and those immunized with extracellular protein (ECP) concentrated from culture supernatant in FIA. The i.p. challenge dose resulted in complete mortality of all fish by Day 43. In a second experiment, fish were orally vaccinated with p57- R. salmoninarum cells encased in a pH protected, enteric-coated antigen microsphere (ECAM). Fish were bath challenged with 4.2 x 10(6) cfu ml-1 on Day 0 and sampled at time points of 0 (pre-challenge), 50, 90, or 150 d immersion challenge. Vaccine efficacy was determined by monitoring the elaboration of p57 in the kidneys of vaccinated and control fish. Fish vaccinated orally demonstrated a significantly lower concentration of p57 (p < 0.01) at Day 150 post challenge compared to fish receiving ECAMs alone. Fish receiving p57 cells without ECAM coating also showed a significantly lower p57 level (p < 0.03) versus control. In contrast, fish injected intraperitoneally with the p57- cells or fish fed p57+ R. salmoninarum cells in ECAMs demonstrated no significant difference (p > 0.05) versus controls. In summary, these studies suggest the preliminary efficacy of 37 degrees C treatment of R. salmoninarum cells as an oral bacterial kidney disease vaccine.     

Identification of Renibacterium salmoninarum surface proteins by radioiodination

Abstract Treatment of Clostridium perfringens alpha toxin with aminopeptidase resulted in no effect on various activities of the toxin. Aminopeptidase did not hydrolyze the native toxin or the toxin treated with urea in the presence of EDTA. Treatment with carboxypeptidase for 30 min resulted in a 75% decrease in these activities. Incubation of the native toxin with carboxypeptidase for 30 min released approximately 15 amino acids from the C-terminus of the toxin. The biological activities of a mutant toxin lacking 20 C-terminal residues of the toxin (AT_1–350) showed about 59–87% of the activity of native toxin. The mutant toxin showed partial antigenic identity with the native toxin. These data suggest that the C-terminal domain contributes to maintaining the active form of the toxin.     

Purification,biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262

Fibrinolytic proteases are enzymes that degrade fibrin. They provide a promising alternative to existing drugs for thrombolytic therapy. A protease isolated from the filamentous fungus Mucor subtilissimus UCP 1262 was purified in three steps by ammonium sulfate fractionation, ion exchange, and molecular exclusion chromatographies, and characterized biochemically and structurally. The purified protease exhibited a molecular mass of 20 kDa, an apparent isoelectric point of 4.94 and a secondary structure composed mainly of α-helices. Selectivity for N-succinyl-Ala–Ala–Pro–Phe-p-nitroanilide as substrate suggests that this enzyme is a chymotrypsin-like serine protease, whose activity was enhanced by the addition of Cu²⁺, Mg²⁺, and Fe²⁺. The enzyme showed a fibrinolytic activity of 22.53 U/mL at 40 °C and its contact with polyethylene glycol did not lead to any significant alteration of its secondary structure. This protein represents an important example of a novel fibrinolytic enzyme with potential use in the treatment of thromboembolic disorders such as strokes, pulmonary emboli, and deep vein thrombosis.

     

Renibacterium salmoninarum: effect of hypochlorite treatment, and survival in water

The effect of different concentrations of sodium hypochlorite on Renibacterium salmoninarum and the survival of the bacterium in autoclaved river water and groundwater were examined. The disinfection trial was performed using R. salmoninarum ATCC 33209. The concentrations of free chlorine were 10, 50, 100 and 200 mg 1(-1), the contact times were 5, 15, and 30 min and 24 h, and the test suspensions were subcultured both on Kidney disease medium (KDM2) agar and in 3 parallel KDM2 broths, which were then subcultured on KDM2 and selective KDM (SKDM) agar. The survival of the bacterium in river water and groundwater was studied using 4 isolates of R. salmoninarum including ATCC 33209. Treatment with sodium hypochlorite effectively reduced the number of culturable cells of R. salmoninarum, but use of the recovery broth showed that small numbers of cells remained viable at all concentrations of free chlorine. The numbers of R. salmoninarum decreased to an undetectable level after 4 wk incubation in the survival trials, but low numbers of colonies were again found in the subculture after 5 wk incubation. Viable cells of R. salmoninarum were still detected in subcultures of all strains after 20 wk of incubation in river water.     

Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations

Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences.     

Charcoal agar, a new growth medium for the fish disease bacterium Renibacterium salmoninarum

Charcoal is an effective replacement for serum in media for the isolation and culture of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish. The medium, KDM-C, contains 10 g of peptone, 0.5 g of yeast extract, 1 g of L-cysteine hydrochloride, 1 g of activated charcoal, and 15 g of agar per liter and is adjusted to pH 6.8 with NaOH before autoclaving. Eight strains of R. salmoninarum grew from dilute inocula as well on KDM-C as on a standard serum-containing medium (KDM-2). The medium was effective for both primary isolations from fish and repeated transfers and has potential value for antigen preparation and physiological studies.     

Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation

The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population is described. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R. salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When the concentration of R. salmoninarum was 8.65 x 10(6) bacteria ml(-1) and the bacteria exposed to chlorine at 1 mg l(-1) for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescence intensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p < 0.0001). When the concentration of R. salmoninarum was reduced to 1.76 x 10(6) bacteria ml(-1) and exposed to 0.8 mg l(-1) free chlorine level for periods from 20 s to 5 min (reduced-Rs assessment), the mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when the R. salmoninarum was exposed to chlorine for at least 1 min (p < or = 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivation derived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r2 < or = 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 > or = 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation estimates correlated with bacteriological culture (r2 < or = 0.22). In both assessments, there was a correlation between the estimates of inactivation based upon HRFI and CS analyses (r2 > 0.99). These results suggest that flow cytometry can be used as a supplementary or alternative method to bacteriological culture for monitoring the inactivation of R. salmoninarum.     

Murein type and polysaccharide composition of cell walls from Renibacterium salmoninarum

Abstract The primary structure of the murein of Renibacterium salmoninarum ATCC33209 was determined. It contained lysine in the third position of the peptide subunit, a glycyl-alanine interpeptide bridge between lysine and the d -alanine of adjacent peptide subunits and a d -alanine-amide substitution at the α-carboxyl group of d -glutamic acid in position 2 of the peptide side chain.
Cell walls contained a considerable amount of polysaccharide with galactose as major sugar component. In addition N -acetyl-glucosamine, rhamnose and N -acetyl-fucosamine were detected.     

Identification of a Third msa Gene in Renibacterium salmoninarum and the Associated Virulence Phenotype

Renibacterium salmoninarum, a gram-positive diplococcobacillus, causes bacterial kidney disease, a condition that can result in extensive morbidity and mortality among stocks of fish. An immunodominant extracellular protein, called major soluble antigen (MSA), is encoded by two identical genes, msa1 and msa2. We found evidence for a third msa gene, msa3, which appears to be a duplication of msa1. Unlike msa1 and msa2, msa3 is not present in all isolates of R. salmoninarum. The presence of the msa3 locus does not affect total MSA production in culture conditions. In a challenge study, isolates possessing the msa3 locus reduced median survival in juvenile chinook salmon (Oncorhynchus tshawytscha) by an average of 34% at doses of ≤10⁵ cells per fish compared to isolates lacking the msa3 locus. In contrast, no difference in survival was observed at the highest dose, 10⁶ cells per fish. The phenotype associated with the msa3 locus and its nonuniform distribution may contribute to observed differences in virulence among R. salmoninarum isolates.     

Shedding of Renibacterium salmoninarum by infected chinook salmon Oncorhynchus tschawytscha

Laboratory studies of the transmission and pathogenesis of Renibacterium salmoninarum may describe more accurately what is occurring in the natural environment if test fish are infected by waterborne R. salmoninarum shed from infected fish. To quantify bacterial shedding by chinook salmon Oncorhynchus tschawytscha at 13 degrees C in freshwater, groups of fish were injected intraperitoneally with R. salmoninarum at either 1.3 x 10(6) colony forming units (CFU) fish (-1) (high-dose injection group) or 1.5 x 10(3) CFU fish (-1) (low-dose injection group). R. salmoninarum infection levels were measured in the exposed fish by the enzyme-linked immunosorbent assay (BKD-ELISA). At regular intervals for 30 d, the numbers of R. salmoninarum shed by the injected fish were calculated on the basis of testing water samples by the membrane filtration-fluorescent antibody test (MF-FAT) and bacteriological culture. Mean BKD-ELISA optical densities (ODs) for fish in the low-dose injection group were not different from those control fish (p > 0.05), and no R. salmoninarum were detected in water samples taken up to 30 d after injection of fish in the low-dose group. By 12 d after injection a proportion of the fish from the high-dose infection group had high (BKD-ELISA OD > or = 1.000) to severe (BKD-ELISA OD > or = 2.000) R. salmoninarum infection levels, and bacteria were detected in the water by both tests. However, measurable levels of R. salmoninarum were not consistently detected in the water until a proportion of the fish maintained high to severe infection levels for an additional 8 d. The concentrations of R. salmoninarum in the water samples ranged from undetectable up to 994 cells ml(-1) on the basis of the MF-FAT, and up to 1850 CFU ml(-1) on the basis of bacteriological culture. The results suggest that chinook salmon infected with R. salmoninarum by injection of approximately 1 x 10(6) CFU fish (-1) can be used as the source of infection in cohabitation challenges beginning 20 d after injection.