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.     

Both msa genes in Renibacterium salmoninarum are needed for full virulence in bacterial kidney disease

Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression.     

Expression of duplicate msa genes in the salmonid pathogen Renibacterium salmoninarum

Renibacterium salmoninarum is a gram-positive bacterium responsible for bacterial kidney disease of salmon and trout. R. salmoninarum has two identical copies of the gene encoding major soluble antigen (MSA), an immunodominant, extracellular protein. To determine whether one or both copies of msa are expressed, reporter plasmids encoding a fusion of MSA and green fluorescent protein controlled by 0.6 kb of promoter region from msa1 or msa2 were constructed and introduced into R. salmoninarum. Single copies of the reporter plasmids integrated into the chromosome by homologous recombination. Expression of mRNA and protein from the integrated plasmids was detected, and transformed cells were fluorescent, demonstrating that both msa1 and msa2 are expressed under in vitro conditions. This is the first report of successful transformation and homologous recombination in R. salmoninarum.     

Both msa Genes in Renibacterium salmoninarum Are Needed for Full Virulence in Bacterial Kidney Disease

Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression.     

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.     

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.     

The relationship between auto-agglutination, cell surface hydrophobicity and virulence of the fish pathogen Renibacterium salmoninarum

Abstract The cell surface hydrophobicity of Renibacterium salmoninarum strains was examined using a salt aggregation method. Those strains which were virulent in the test animal were sticky, auto-agglutinating and possessed a hydrophobic cell surface. Those strains with a low virulence were non-sticky, non-agglutinating and failed to aggregate in a high molar salt. Strains could not be distinguished using biochemical tests. There was no change in hydrophobicity following re-isolation of the bacteria from experimentally infected rainbow trout, Salmo gairdneri .     

The cloning and expression of a gene encoding haemolytic activity from the fish pathogen Renibacterium salmoninarum

A gene encoding haemolytic activity from Renibacterium salmoninarum (strain PPD) was cloned into Escherichia coli using the cosmid vector pHC79, and subsequently subcloned on a 1.6 kbp SAlI fragment into pBR328. Southern blot hybridisation revealed that a homologous sequence is found in other strains of R. salmoninarum.     

Microevolution of Renibacterium salmoninarum: evidence for intercontinental dissemination associated with fish movements

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, a major pathogen of salmonid fish species worldwide. Very low levels of intra-species genetic diversity have hampered efforts to understand the transmission dynamics and recent evolutionary history of this Gram-positive bacterium. We exploited recent advances in the next-generation sequencing technology to generate genome-wide single-nucleotide polymorphism (SNP) data from 68 diverse R. salmoninarum isolates representing broad geographical and temporal ranges and different host species. Phylogenetic analysis robustly delineated two lineages (lineage 1 and lineage 2); futhermore, dating analysis estimated that the time to the most recent ancestor of all the isolates is 1239 years ago (95% credible interval (CI) 444–2720 years ago). Our data reveal the intercontinental spread of lineage 1 over the last century, concurrent with anthropogenic movement of live fish, feed and ova for aquaculture purposes and stocking of recreational fisheries, whilst lineage 2 appears to have been endemic in wild Eastern Atlantic salmonid stocks before commercial activity. The high resolution of the SNP-based analyses allowed us to separate closely related isolates linked to neighboring fish farms, indicating that they formed part of single outbreaks. We were able to demonstrate that the main lineage 1 subgroup of R. salmoninarum isolated from Norway and the UK likely represent an introduction to these areas ∼40 years ago. This study demonstrates the promise of this technology for analysis of micro and medium scale evolutionary relationships in veterinary and environmental microorganisms, as well as human pathogens.     

Comparison of serological techniques for the identification of Renibacterium salmoninarum

The optimum conditions for staphylococcal co-agglutination (CoA), latex agglutination (LA) and the fluorescent antibody test (FAT) for the identification of Renibacterium salmoninarum isolated in culture were determined. Their sensitivities were then compared with that of the enzyme-linked immunosorbent assay (ELISA). The ELISA was the most sensitive, followed by CoA, with the FAT and LA jointly the least sensitive. However, all four techniques were sufficiently sensitive for routine identification of R. salmoninarum. The agglutination techniques (CoA and LA) were preferred because of their speed, ease of use and low cost.     

Biochemical and immunochemical properties of the cell surface of Renibacterium salmoninarum.

The biochemical composition of the cell envelope of Renibacterium salmoninarum was investigated in a total of 13 strains isolated from different salmonid fish species at various geographical locations of the United States, Canada, and Europe. A marked similarity with the type strain R. salmoninarum ATCC 33209 was found both in the peptidoglycan and the cell wall polysaccharide. The primary structure of the peptidoglycan was found to be consistent with lysine in the third position of the peptide subunit, a glycyl-alanine interpeptide bridge between lysine and D-alanine of adjacent peptide subunits, and a D-alanine amide substituent at the alpha-carboxyl group of D-glutamic acid in position 2 of the peptide subunit. The cell wall polysaccharide contained galactose as the major sugar component which was accompanied by rhamnose, N-acetylglucosamine, and N-acetylfucosamine. The polysaccharide amounted to more than 60% of the dry weight of the cell walls. It was found to be covalently linked to the peptidoglycan and was released by hot formamide treatment. On gel filtration chromatography the extracted polysaccharide behaved like a homogeneous polymeric compound. The purified cell wall polysaccharide showed antigenic activity with antiserum obtained by immunization of rabbits with heat-inactivated trypsinized cells of R. salmoninarum. Immunoblotting experiments with nontrypsinized cell walls and antisera raised against R. salmoninarum cells revealed that antigenic proteins were attached to the cell walls.     

Nutrient Requirements of Renibacterium salmoninarum on Agar and in Broth Media

In well-aerated broth cultures, good growth of Renibacterium salmoninarum was obtained in a serum-free medium consisting of 1% peptone, 1% yeast extract, and 0.1% l-cysteine (PYC broth). In contrast, serum or charcoal is required for growth on agar medium. Charcoal treatment of broth media, either before bacterial inoculation or during growth, increased the growth of R. salmoninarum, whereas the surfactants Tween 20 and Tween 80 inhibited growth. l-Cysteine was essential for optimal growth. Other organic sulfur compounds, such as d-cysteine, l-methionine, homocysteine, homocysteine thiolactone, and reduced glutathione, supported only lower levels of growth, while cystine and dithiothreitol did not allow growth.     

Purification, and biochemical and structural characterization of a fimbrial haemagglutinin of Renibacterium salmoninarum

Renibacterium salmoninarum was shown to possess peritrichous fimbriae. Electron microscopy of strains FMV 84-01 and ATCC 33209T revealed short, flexible fimbriae less than 2 nm in diameter. These surface appendages were isolated from the bacteria by a procedure involving water extraction and urea solubilization. The fimbrin was purified to homogeneity by Fast Pressure Liquid Chromatography, and shown by SDS-PAGE to be a protein of 57 kDa. Isoelectric focusing under non-denaturing conditions indicated a pI of 4.8. The protein had an amino acid composition rich in glycine, Asx (aspartic acid and asparagine), valine and alanine; methionine was absent. Approximately 33% of the amino acid residues were hydrophobic. Immunoblotting using a polyclonal antiserum raised against whole cells showed that the 57 kDa protein was the immunodominant antigen on the cell surface. Immunogold labelling using polyclonal antibodies raised against the fimbrin revealed an alignment of gold particles along the fimbriae. Purified fimbriae caused agglutination of rabbit erythrocytes and antifimbrial serum inhibited this haemagglutination. Altogether the results indicate that the fimbriae on the surface of R. salmoninarum are responsible for the haemagglutinating activity.     

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.     

Epidemiology of Renibacterium salmoninarum in wild Arctic charr and brown trout in Iceland

Renibacterium salmoninarum (Rs) is common in wild Arctic charr Salvelinus alpinus and brown trout Salmo trutta in Iceland. Of 22 charr and nine trout populations none were free of Rs antigens. In two charr populations only one fish exceeded the Rs antigen detection limit and in one of these cases the ELISA value was within uncertainty limits of the infection criterion. Mean prevalence of infection was 46% for Arctic charr (range: 3–100%) and 35% for brown trout (range: 6–81%). No infected fish showed gross pathological signs of bacterial kidney disease (BKD). The ubiquity and high prevalences of infection indicated that the bacterium has been endemic for a long time, and is probably a normal, low density resident in the fish. A lack of correlation in mean intensity of Rs antigen and prevalence of infection between sympatricpopulations of Arctic charr and brown trout suggests that the dynamics of infection and internal proliferation of bacteria can be quite independent in the two species even if they live in the same lake. Rs intensity and its coefficient of variation decreased with age in older fish, suggesting a connection between Rs intensity and host mortality. However, this can be caused by other ecological factors that decrease survival, especially low food availability, which simultaneously increase the susceptibility to Rs infection and internal proliferation.     

Serine proteinase of Renibacterium salmoninarum digests a major autologous extracellular and cell-surface protein.

Renibacterium salmoninarum is a pathogen of salmonid fish that produces large amounts of extracellular protein (ECP) during growth. A proteolytic activity present in ECP at elevated temperatures digested the majority of the proteins in ECP. This digestion was also associated with the loss of ECP immunosuppressive function. In vitro activity of the proteinase in ECP was temperature dependent: it was not detected in an 18-h digest at 4 and 17 degrees C but became readily apparent at 37 degrees C. Proteinase activity was detected at bacterial physiological temperatures (17 degrees C) in reactions incubated for several days. Under these conditions, digestion of partially purified p57, a major constituent of ECP and a major cell-surface protein, yielded a spectrum of breakdown products similar in molecular weight and antigenicity to those in ECP. This pattern of digestion suggests that most of the immunologically related constituents of ECP are p57 and its breakdown products. The proteolytic activity was sensitive to phenylmethylsulfonyl fluoride, methanol, and ethanol and to 10-min incubation at temperatures above 65 degrees C. Electrophoretic analysis of the proteinase on polyacrylamide gels containing proteinase substrates indicated the native form to be 100 kDa or greater. The enzyme was active against selected unrelated substrates only when coincubated with a denaturant (0.1% lauryl sulfate) and (or) a reducing agent (20 mM dithiothreitol).     

A PCR-based assay for the identification of the fish pathogen Renibacterium salmoninarum

Abstract By means of a one-step one-tube extraction from less than 1 mg of tissue it is possible to identify, via the polymerase chain reaction, Renibacterium salmoninarum in salmon with bacterial kidney disease. A 149-bp DNA sequence unique to R salmoninarum was specifically amplified and its nature confirmed by Southern hybridization using a non-isotopically labelled probe. The sensitivity of the approach allowed the detection of 22 R. salmoninarum cells. The procedure was successfully applied in the identification of the causative agent of bacterial kidney disease in kidney tissue from infected fishes.     

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.     

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.