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.     

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.     

Influence of the growth conditions on the hydrophobicity of Renibacterium salmoninarum evaluated by different methods

The influence of medium and salinity on the cell surface hydrophobicity of Renibacterium salmoninarum was investigated using three different methods: bacterial adherence to hydrocarbons (BATH), salt agglutination test (SAT), and binding to nitrocellulose filters (NCF). The possible relationship among hydrophobicity, haemagglutination and adherence to cell lines was also evaluated. R. salmoninarum showed to be highly hydrophobic regardless of the growth conditions or technique employed. Nevertheless, slight differences can be detected depending on the method used. In the SAT and NCF assays very uniform values were obtained within the strains. All the R. salmoninarum isolates agglutinated in (NH4)2SO4 in a range of 0.05-0.2 M and displayed a 77-100% of adherence to nitrocellulose filters. However, more variable results were observed in the BATH method depending on the hydrocarbon, buffer and strain employed. Although all of the isolates produced haemagglutinins for homeotherm erythrocytes, the majority of them failed to agglutinate poikilothermic red blood cells and were unable to adhere to fish cell lines. Therefore, a general correlation among hydrophobicity, agglutinating capacity for fish erythrocytes and adherence to fish cells can not be established for R. salmoninarum.     

A cohabitation challenge to compare the efficacies of vaccines for bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha

The relative efficacies of 1 commercial and 5 experimental vaccines for bacterial kidney disease (BKD) were compared through a cohabitation waterborne challenge. Groups of juvenile chinook salmon Oncorhynchus tshawytscha were vaccinated with one of the following: (1) killed Renibacterium salmoninarum ATCC 33209 (Rs 33209) cells; (2) killed Rs 33209 cells which had been heated to 37 degrees C for 48 h, a process that destroys the p57 protein; (3) killed R. salmoninarum MT239 (Rs MT239) cells; (4) heated Rs MT239 cells; (5) a recombinant version of the p57 protein (r-p57) emulsified in Freund's incomplete adjuvant (FIA); (6) the commercial BKD vaccine Renogen; (7) phosphate-buffered saline (PBS) emulsified with an equal volume of FIA; or (8) PBS alone. Following injection, each fish was marked with a subcutaneous fluorescent latex tag denoting its treatment group and the vaccinated fish were combined into sham and disease challenge tanks. Two weeks after these fish were vaccinated, separate groups of fish were injected with either PBS or live R. salmoninarum GL64 and were placed inside coated-wire mesh cylinders (liveboxes) in the sham and disease challenge tanks, respectively. Mortalities in both tanks were recorded for 285 d. Any mortalities among the livebox fish were replaced with an appropriate cohort (infected with R. salmoninarum or healthy) fish. None of the bacterins evaluated in this study induced protective immunity against the R. salmoninarum shed from the infected livebox fish. The percentage survival within the test groups in the R. salmoninarum challenge tank ranged from 59% (heated Rs MT239 bacterin) to 81% (PBS emulsified with FIA). There were no differences in the percentage survival among the PBS-, PBS/FIA-, r-p57- and Renogen-injected groups. There also were no differences in survival among the bacterin groups, regardless of whether the bacterial cells had been heated or left untreated prior to injection.     

First record of Renibacterium salmoninarum in the sea lamprey (Petromyzon marinus)

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a widespread problem with major implications for salmonid fish species. The mechanisms by which the bacterium has reached high levels of infection previously unrecorded in the Laurentian Great Lakes are presently unknown. Research involving reservoirs and mechanisms of R. salmoninarum transmission in fish is lacking because of the ecologic complexity of heterogeneous habitats and the lack of adequate funding. Herein, we report on the isolation of R. salmoninarum from the kidneys of the sea lamprey (Petromyzon marinus). The bacterium was cultured from kidneys of 16% and 4% of lampreys collected from two locations within the Lake Ontario watershed in 2003 and 2004, respectively. The identity of bacterial colonies was verified with the nested polymerase chain reaction and quantitative enzyme-linked immunosorbent assay.     

Characterization of Renibacterium salmoninarum with reduced susceptibility to macrolide antibiotics by a standardized antibiotic susceptibility test

Three cohorts of juvenile and subadult Chinook salmon Oncorhynchus tshawytscha received multiple treatments with macrolide antibiotics for bacterial kidney disease (BKD) during rearing in a captive broodstock program. A total of 77 mortalities among the cohorts were screened for Renibacterium salmoninarum, the etiologic agent of BKD, by agar culture from kidney, and isolates from 7 fish were suitable for growth testing in the presence of macrolide antibiotics. The minimum inhibitory concentration (MIC) of erythromycin and azithromycin was determined by a modification of the standardized broth assay using defined medium. The American Type Culture Collection (ATCC) type strain 33209 exhibited a MIC of 0.008 microg m(-1) to either erythromycin or azithromycin. Isolates from 3 fish displayed MICs identical to the MICs for the ATCC type strain 33209. In contrast, isolates from 4 fish exhibited higher MICs, ranging between 0.125 and 0.250 microg ml(-1) for erythromycin and between 0.016 and 0.031 microg ml(-1) for azithromycin. Sequence analysis of the mutational hotspots for macrolide resistance in the 23S rDNA gene and the open reading frames of ribosomal proteins L4 and L22 found identical sequences among all isolates, indicating that the phenotype was not due to mutations associated with the drug-binding site of 23S rRNA. These results are the first report of R. salmoninarum with reduced susceptibility to macrolide antibiotics isolated from fish receiving multiple antibiotic treatments.     

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.     

Efficacy of cellular vaccines and genetic adjuvants against bacterial kidney disease in chinook salmon (Oncorhynchus tshawytscha)

DNA adjuvants and whole bacterial cell vaccines against bacterial kidney disease (BKD) were tested in juvenile chinook salmon. Whole cell vaccines of either a nonpathogenic Arthrobacter spp. or an attenuated Renibacterium salmoninarum strain provided limited prophylactic protection against acute intraperitoneal challenge with virulent R. salmoninarum, and the addition of either synthetic oligodeoxynucleotides or purified R. salmoninarum genomic DNA as adjuvants did not increase protection. However, a combination of both whole cell vaccines significantly increased survival among fish naturally infected with R. salmoninarum, and the surviving fish treated with the combination vaccine exhibited reduced levels of bacterial antigens in the kidney. This is the first demonstration of a potential therapeutic effect of a whole cell vaccine against BKD.     

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 < or =10(5) cells per fish compared to isolates lacking the msa3 locus. In contrast, no difference in survival was observed at the highest dose, 10(6) 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.     

Accumulation and clearance of orally administered erythromycin and its derivative, azithromycin, in juvenile fall chinook salmon Oncorhynchus tshawytscha

Fall Chinook salmon Oncorhynchus tshawytscha were fed practical diets medicated with azithromycin (30 mg kg(-1) fish for 14 d) or erythromycin (100 mg kg(-1) fish for 28 d) either 1, 2, or 3 times beginning 14 d after initiation of exogenous feeding (February) and ending at smoltification (June). Average tissue concentrations of azithromycin increased from 19.0 microg g(-1) in fry to 44.9 microg g(-1) in smolts, and persisted in the tissues > 76 d after treatment ceased. Tissue concentrations of erythromycin were comparatively low, ranging from 0.2 microg g(-1) in fry to 10.4 microg g(-1) in smolts. Erythromycin was not detectable 21 d post-treatment. Neither antibiotic caused histopathologically significant lesions in the trunk kidney or other organ tissues. The high tissue concentrations and prolonged retention of azithromycin in Chinook may be factors that increase the efficacy of the antibiotic against Renibacterium salmoninarum, compared with erythromycin, particularly in early life history stages before covertly infected fish show clinical signs of disease.     

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.     

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.     

Chararacteristics of Vagococcus salmoninarum isolated from diseased salmonid fish

Isolates of the salmonid pathogen Vagococcus salmoninarum were recovered from Atlantic salmon, rainbow trout and brown trout with peritonitis. The phenotypes of these isolates and the type strain of Vag. salmoninarum NCFB 2777 were determined by morphological, biochemical and physiological tests and whole cell protein profiles by SDS-PAGE. There was a high level of phenetic similarity between the salmonid isolates and the type strain. The species forms short Gram-positive rods, hydrolyses L-pyrrolidonyl-β-naphthylamide, is α-haemolytic on sheep's blood agar, grows at pH 9·6 and 10°C but not at 40°C or in 6·5% NaCl and is catalase-negative; a Lancefield group N antigen is not present. Vagococcus salmoninarum can be distinguished phenetically from similar fish pathogens including Carnobacterium piscicola, Enterococcus seriolicida and Lactococcus piscium.     

Comparison and evaluation of Renibacterium salmoninarum quantitative PCR diagnostic assays using field samples of Chinook and coho salmon

Renibacterium salmoninarum is a Gram-positive bacterium causing bacterial kidney disease (BKD) in susceptible salmonid fishes. Several quantitative PCR (qPCR) assays to measure R. salmoninarum infection intensity have been reported, but comparison and evaluation of these assays has been limited. Here, we compared 3 qPCR primer/probe sets for detection of R. salmoninarum in field samples of naturally exposed Chinook and coho salmon first identified as positive by nested PCR (nPCR). Additional samples from a hatchery population of Chinook salmon with BKD were included to serve as strong positive controls. The 3 qPCR assays targeted either the multiple copy major soluble antigen (msa) genes or the single copy abc gene. The msa/non-fluorescent quencher (NFQ) assay amplified R. salmoninarum DNA in 53.2% of the nPCR positive samples, whereas the abc/NFQ assay amplified 21.8% of the samples and the abc/TAMRA assay 18.2%. The enzyme-linked immunosorbent assay (ELISA) successfully quantified only 16.4% of the nPCR positive samples. Although the msa/NFQ assay amplified a greater proportion of nPCR positive samples, the abc/NFQ assay better amplified those samples with medium and high ELISA values. A comparison of the geometric mean quantity ratios highlighted limitations of the assays, and the abc/NFQ assay strongly amplified some samples that were negative in other tests, in contrast to its performance among the sample group as a whole. These data demonstrate that both the msa/NFQ and abc/NFQ qPCR assays are specific and effective at higher infection levels and outperform the ELISA. However, most pathogen studies will continue to require multiple assays to both detect and quantify R. salmoninarum infection.     

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.     

Mortality and kidney histopathology of chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains

An isolate of Renibacterium salmoninarum (strain MT 239) exhibiting reduced virulence in rainbow trout Oncorhynchus mykiss was tested for its ability to cause bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha, a salmonid species more susceptible to BKD. Juvenile chinook salmon were exposed to either 33209, the American Type Culture Collection type strain of R. salmoninarum, or to MT 239, by an intraperitoneal injection of 1 x 10(3) or 1 x 10(6) bacteria fish(-1), or by a 24 h immersion in 1 x 10(5) or 1 x 10(7) bacteria ml(-1). For 22 wk fish were held in 12 degrees C water and monitored for mortality. Fish were sampled periodically for histological examination of kidney tissues. In contrast to fish exposed to the high dose of strain 33209 by either injection or immersion, none of the fish exposed to strain MT 239 by either route exhibited gross clinical signs or histopathological changes indicative of BKD. However, the MT 239 strain was detected by the direct fluorescent antibody technique in 4 fish that died up to 11 wk after the injection challenge and in 5 fish that died up to 20 wk after the immersion challenge. Viable MT 239 was isolated in culture from 3 fish that died up to 13 wk after the immersion challenge. Total mortality in groups injected with the high dose of strain MT 239 (12%) was also significantly lower (p < 0.05) than mortality in groups injected with strain 33209 (73 %). These data indicate that the attenuated virulence observed with MT 239 in rainbow trout also occurs in a salmonid species highly susceptible to BKD. The reasons for the attenuated virulence of MT 239 were not determined but may be related to the reduced levels of the putative virulence protein p57 associated with this strain.