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Sample records for renibacterium salmoninarum distinguishes

  1. Bacterial kidney disease (Renibacterium salmoninarum)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a prevalent disease of salmonid fish that impacts sustainable production for consumption and species conservation efforts. The disease is chronic in nature and mortality most often occurs in juvenile salmonids and prespawning a...

  2. Variation in the Spacer Regions Separating tRNA Genes in Renibacterium salmoninarum Distinguishes Recent Clinical Isolates from the Same Location

    PubMed Central

    Alexander, Sarah M.; Grayson, T. Hilton; Chambers, Edel M.; Cooper, Lynne F.; Barker, Gavin A.; Gilpin, Martyn L.

    2001-01-01

    A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS994. Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS994 profiles. PMID:11136759

  3. Variation in the spacer regions separating tRNA genes in Renibacterium salmoninarum distinguishes recent clinical isolates from the same location.

    PubMed

    Alexander, S M; Grayson, T H; Chambers, E M; Cooper, L F; Barker, G A; Gilpin, M L

    2001-01-01

    A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS994. Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS994 profiles. PMID:11136759

  4. Molecular cloning of Renibacterium salmoninarum DNA fragments.

    PubMed

    Etchegaray, J P; Martínez, M A; Krauskopf, M; León, G

    1991-03-15

    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. PMID:2044941

  5. Further characterization of Renibacterium salmoninarum extracellular products.

    PubMed

    Barton, T A; Bannister, L A; Griffiths, S G; Lynch, W H

    1997-10-01

    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. PMID:9480644

  6. Further characterization of Renibacterium salmoninarum extracellular products.

    PubMed Central

    Barton, T A; Bannister, L A; Griffiths, S G; Lynch, W H

    1997-01-01

    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. PMID:9480644

  7. The fish pathogen Renibacterium salmoninarum: growth in a microaerophilic atmosphere.

    PubMed

    Hirvelä-Koski, Varpu

    2008-02-01

    Renibacterium salmoninarum is the etiologic agent of bacterial kidney disease (BKD) occurring worldwide in salmonid fish. This bacterium has previously been regarded as a strict aerobic species. However, in this study it is shown that R. salmoninarum grows well in microaerophilic atmosphere, the colony size being larger and the colonies being more mucoid than in aerobic conditions. Microaerophilic cultivation might be one possibility to increase the sensitivity of the cultivation method for the detection of this slowly growing pathogen. PMID:17884309

  8. Virulence of Renibacterium salmoninarum to salmonids

    USGS Publications Warehouse

    Starliper, C.E.; Smith, D.R.; Shatzer, T.

    1997-01-01

    Virulence of Renibacterium salmoninarum isolates representing five origins was evaluated in eight salmonid hosts; four origins were of Lake Michigan and the fifth was of the Pacific Northwest. The species type strain, ATCC (American Type Culture Collection) 33209, was also included. Each isolate was grown in a kidney disease medium (KDM2) supplemented with 1 % ATCC 33209 culture metabolite; serial 10-fold dilutions were prepared, and groups of fish were challenged by intraperitoneal injection with 0.1 mL of each dilution. A 70-d observation period followed, and bacterial kidney disease (BKD) was diagnosed by the fluorescent antibody technique. Virulence of isolates was quantified as a dose lethal to 50% of fish (LD50) for each host–isolate challenge. In the first set of experiments, 23 isolates were used to challenge groups of brook trout Salvelinus fontinalis. The mean LD50 was 1.087 x 106 colony-forming units per milliliter (cfu/mL; SD = 2.022 x 106), and the LD50 values ranged from 8.457 x 106 to 2.227 x 104 cfu/mL. Analysis of variance to evaluate the effect of isolate origin on virulence in brook trout revealed no significant difference (F = 1.502; P = 0.243). Susceptibilities of the other salmonid hosts were evaluated by challenge with six isolates of R. salmoninarum representing each origin and the species type strain. For many of the host–isolate challenge combinations, time to death was highly dependent on the dilution (number of bacteria) injected. In general, the isolates MCO4M, B26, and A34 (all of Lake Michigan origin) tended to be more virulent. Also, LD50 values were dispersed throughout a wider range among the more susceptible hosts. Lake trout Salvelinus namaycush, rainbow trout Oncorhynchus mykiss, and brook trout were relatively resistant to challenge with the strains, whereas coho salmon O. kisutch, domestic Atlantic salmon Saltno salar, and chinook salmon O. tshawytscha were relatively susceptible. Another challenge evaluated the effect of

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

    PubMed

    Hirvelä-Koski, Varpu

    2004-04-21

    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. PMID:15212289

  10. Shedding of Renibacterium salmoninarum by infected chinook salmon Oncorhynchus tschawytscha

    USGS Publications Warehouse

    McKibben, C.L.; Pascho, R.J.

    1999-01-01

    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??C in freshwater, groups of fish were injected intraperitoneally with R. salmoninarum at either 1.3 x 106 colony forming units (CFU) fish-1 (high-dose injection group) or 1.5 x 103 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 of 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 ??? 1.000) to severe (BKD-ELISA OD ??? 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 106 CFU fish-1 can be used as the source of infection in cohabitation challenges beginning 20 darter injection.

  11. Shedding of Renibacterium salmoninarum by infected chinook salmon Oncorhynchus tschawytscha.

    PubMed

    McKibben, C L; Pascho, R J

    1999-10-11

    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

  12. Rhamnolipid biosurfactants produced by Renibacterium salmoninarum 27BN during growth on n-hexadecane.

    PubMed

    Christova, Nelly; Tuleva, Borjana; Lalchev, Zdravko; Jordanova, Albena; Jordanov, Bojidar

    2004-01-01

    A new strain Renibacterium salmoninarum 27BN was isolated for its capacity to utilize n-hexadecane as sole substrate. Growth on n-hexadecane was accompanied with the production of glycolipid surface active substances detected by surface pressure lowering and emulsifying activity. Glycolipid detection by thin layer chromatography and infrared spectra analyses showed for the first time that Renibacterium salmoninarum 27BN secretes the two rhamnolipids RLL and RRLL typical for Pseudomonas aeruginosa. Growth of Renibacterium salmoninarum 27BN on n-hexadecane depended on the bioavailability of the substrate and the secreted rhamnolipids appeared to be efficient in increasing hexadecane availability for the cells. PMID:15018056

  13. MICs and MBCs of chemotherapeutic agents against Renibacterium salmoninarum.

    PubMed Central

    Bandín, I; Santos, Y; Toranzo, A E; Barja, J L

    1991-01-01

    The efficacies of 21 chemotherapeutic agents for controlling bacterial kidney disease were evaluated. The bactericidal and/or bacteriostatic effects of these drugs were tested against 11 Renibacterium salmoninarum strains with different origins. The most effective compounds displaying both bacteriostatic and bactericidal activity for all the isolates were tetracycline and erythromycin, with MICs ranging from less than 0.62 to 10.95 micrograms/ml for tetracycline and from less than 0.62 to 5.47 micrograms/ml for erythromycin. Whereas tetracycline showed identical MICs and MBCs, erythromycin showed bactericidal effects at concentrations of 5.47 to 21.87 micrograms/ml. Similarly, cefazolin and tiamulin proved to be very effective bactericidal compounds against the majority of R. salmoninarum isolates, with MBCs for 90% of the strains tested of 21.87 and 10.95 micrograms/ml, respectively. Neither nitrofuranes, quinolones, nor sulfonamides showed inhibitory effects on the growth of the strains. PMID:1854157

  14. MICs and MBCs of chemotherapeutic agents against Renibacterium salmoninarum.

    PubMed

    Bandín, I; Santos, Y; Toranzo, A E; Barja, J L

    1991-05-01

    The efficacies of 21 chemotherapeutic agents for controlling bacterial kidney disease were evaluated. The bactericidal and/or bacteriostatic effects of these drugs were tested against 11 Renibacterium salmoninarum strains with different origins. The most effective compounds displaying both bacteriostatic and bactericidal activity for all the isolates were tetracycline and erythromycin, with MICs ranging from less than 0.62 to 10.95 micrograms/ml for tetracycline and from less than 0.62 to 5.47 micrograms/ml for erythromycin. Whereas tetracycline showed identical MICs and MBCs, erythromycin showed bactericidal effects at concentrations of 5.47 to 21.87 micrograms/ml. Similarly, cefazolin and tiamulin proved to be very effective bactericidal compounds against the majority of R. salmoninarum isolates, with MBCs for 90% of the strains tested of 21.87 and 10.95 micrograms/ml, respectively. Neither nitrofuranes, quinolones, nor sulfonamides showed inhibitory effects on the growth of the strains. PMID:1854157

  15. The detection of two antigenic groups among Renibacterium salmoninarum isolates.

    PubMed

    Bandín, I; Santos, Y; Magariños, B; Barja, J L; Toranzo, A E

    1992-07-01

    The analysis of the membrane proteins and their antigenic properties in a group of 14 geographically diverse strains of Renibacterium salmoninarum revealed the existence of antigenic diversity within this species. Eleven isolates, including the type strain ATCC 33209, shared a similar protein profile with a major component of 57 kDa whereas three strains showed a common pattern with a major protein of 30 kDa. The quantitative agglutination tests and Western blotting assays seem to indicate the existence of serological heterogeneity, with two distinct groups being detected. PMID:1521757

  16. Identification of Renibacterium salmoninarum surface proteins by radioiodination.

    PubMed

    Fredriksen, A; Bakken, V

    1994-09-01

    Surface exposed proteins of Renibacterium salmoninarum were identified by radiolabelling whole bacterial cells with 125I, followed by SDS-PAGE and autoradiography. The most prominent bands had molecular masses of approximately 57 kDa and 22 kDa; in addition, some less intensively labelled bands were detected. Polyclonal sera raised against the 22 kDa protein did not react with the 57 kDa protein. N-terminal amino acid sequence analysis of the purified 22 kDa protein showed no similarity with the sequence of the 57 kDa protein. PMID:7926685

  17. Growth of the fish pathogen Renibacterium salmoninarum on different media.

    PubMed

    Bandín, I; Santos, Y; Barja, J I; Toranzo, A E

    1996-09-01

    In the present study, the ability of a group of Renibacterium salmoninarum strains to grow in the presence or absence of the amino acid cysteine and other mineral and organic sources of sulfur and nitrogen has been evaluated. Most of the isolates tested were able to grow on a mineral media supplemented with L-cysteine-HCl or other organic compounds, such as the vitamin thiamine and a casein hydrolysate (Bacto Casamino Acids, Difco). Bacterial growth was also recorded on commercial and specific media not supplemented with L-cysteine-HCl, or in which this amino acid was replaced by the compounds cited above. PMID:8897425

  18. Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations.

    PubMed

    Grayson, T H; Cooper, L F; Atienzar, F A; Knowles, M R; Gilpin, M L

    1999-03-01

    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. PMID:10049848

  19. Atypical growth of Renibacterium salmoninarum in subclinical infections.

    PubMed

    Hirvelä-Koski, V; Pohjanvirta, T; Koski, P; Sukura, A

    2006-01-01

    Two growth types of Renibacterium salmoninarum were isolated from subclinically infected rainbow trout, one producing the smooth colonies typical of R. salmoninarum and the other forming a thin film on the surface of the agar with no separate colonies. The atypical growth was present on kidney disease medium agar in primary cultures of the kidney but not on selective kidney disease medium (SKDM). Fluorescent antibody staining of the fresh isolate and polymerase chain reaction amplification were the most reliable techniques to identify the atypical growth of R. salmoninarum. The condition was reversible, with growth reverting from atypical to the smooth colony form in experimentally infected rainbow trout and under laboratory conditions. There was no mortality, or any clinical signs of bacterial kidney disease (BKD) in the fish challenged with the atypical growth, although small numbers of smooth colonies of R. salmoninarum were isolated from 8% of these fish. The atypical growth reported here may explain some of the failures of culture, when SKDM agar alone is used for the detection of BKD in subclinically infected fish. PMID:16351695

  20. Molecular Differentiation of Renibacterium salmoninarum Isolates from Worldwide Locations

    PubMed Central

    Grayson, Thomas H.; Cooper, Lynne F.; Atienzar, Franck A.; Knowles, Mark R.; Gilpin, Martyn L.

    1999-01-01

    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. PMID:10049848

  1. Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation

    USGS Publications Warehouse

    Pascho, R.J.; Ongerth, J.E.

    2000-01-01

    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 106 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 106 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 ??? 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 ??? 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 ??? 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

  2. Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation.

    PubMed

    Pascho, R J; Ongerth, J E

    2000-07-14

    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

  3. Characterization of attenuated Renibacterium salmoninarum strains and their use as live vaccines.

    PubMed

    Daly, J G; Griffiths, S G; Kew, A K; Moore, A R; Olivier, G

    2001-03-01

    Two nutritionally mutant strains of Renibacterium salmoninarum (Rs) were isolated that grew on tryticase soy agar (Rs TSA1) or brain heart infusion agar (Rs BHI1). These 2 strains could be continuously cultured on these media, whereas typical R. salmoninarum would only grow on KDM-2 agar. We determined no other phenotypic difference that could be used to distinguish them from wild-type R. salmoninarum. Both strains were found to be avirulent when 5 x 10(6) bacteria were intraperitoneally (i.p.) injected into Atlantic salmon. Rs TSA1, Rs BHI1, and Rs MT-239 (a R. salmoninarum strain previously shown to be attenuated) were tested as live vaccines in 2 separate trials. The best protection was seen with Rs TSA1. Vaccinated Atlantic salmon had relative percent survival (RPS) of 50 at 74 d post-challenge in Trial 1 and 76 at 60 d post-challenge in Trial 2. In both trials, 100% of the control salmon died from bacterial kidney disease (BKD) (within 40 d for Trial 1 and 50 d for Trial 2) after i.p. challenge with 5 x 10(6) live cells of the virulent isolate Rs Margaree. PMID:11324812

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

    PubMed

    Evenden, A J; Gilpin, M L; Munn, C B

    1990-09-01

    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. PMID:2276613

  5. Monoclonal antibody characterization of a leukoagglutinin produced by Renibacterium salmoninarum.

    PubMed

    Wiens, G D; Kaattari, S L

    1991-02-01

    Renibacterium salmoninarum causes a chronic disease of salmonid fish known as bacterial kidney disease. High concentrations of bacterially produced extracellular protein (ECP) are present in plasma, kidney, and spleen tissue of naturally and experimentally infected fish. ECP agglutinated salmonid leukocytes in vitro at concentrations which correspond to levels found in highly infected fish. Association of biological activity with the structure of the major protein constituent of ECP, p57, was accomplished by monoclonal antibody (MAb) analysis. Location of the antigenic binding sites recognized by the MAbs was determined by two-dimensional electrophoresis and Western immunoblotting of the proteolytic breakdown fragments of p57. Eight MAbs have been classified into three groups on the basis of their differential recognition of these proteolytic breakdown products. Group I MAbs bound a region proximal to the amino terminus of the protein. Two of these MAbs were also able to block leukoagglutinating activity. Group III MAbs bound to a region associated with the bacterial cell surface, while group II MAbs bound a region between group I and group III. These analyses have allowed the identification of potential structural and functional regions of p57. PMID:1987079

  6. Monoclonal antibody characterization of a leukoagglutinin produced by Renibacterium salmoninarum.

    PubMed Central

    Wiens, G D; Kaattari, S L

    1991-01-01

    Renibacterium salmoninarum causes a chronic disease of salmonid fish known as bacterial kidney disease. High concentrations of bacterially produced extracellular protein (ECP) are present in plasma, kidney, and spleen tissue of naturally and experimentally infected fish. ECP agglutinated salmonid leukocytes in vitro at concentrations which correspond to levels found in highly infected fish. Association of biological activity with the structure of the major protein constituent of ECP, p57, was accomplished by monoclonal antibody (MAb) analysis. Location of the antigenic binding sites recognized by the MAbs was determined by two-dimensional electrophoresis and Western immunoblotting of the proteolytic breakdown fragments of p57. Eight MAbs have been classified into three groups on the basis of their differential recognition of these proteolytic breakdown products. Group I MAbs bound a region proximal to the amino terminus of the protein. Two of these MAbs were also able to block leukoagglutinating activity. Group III MAbs bound to a region associated with the bacterial cell surface, while group II MAbs bound a region between group I and group III. These analyses have allowed the identification of potential structural and functional regions of p57. Images PMID:1987079

  7. Detection of Renibacterium salmoninarum antigen in migrating adult chum salmon (Oncorhynchus keta) in Japan.

    PubMed

    Sakai, M; Atsuta, S; Kobayashi, M

    1992-01-01

    Renibacterium salmoninarum antigen was detected in the kidney of migrating chum salmon (Oncorhynchus keta) using the indirect dot blot assay and indirect fluorescent antibody test. The adult chum salmon had migrated into a bay in which cultured coho salmon infected with R. salmoninarum were present. Antigen was detected in 5% of the chum salmon although they did not have clinical signs of bacterial kidney disease (BKD). This report describes the first case of R. salmoninarum antigen detection among wild chum salmon populations in eastern Asia. PMID:1548789

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

    PubMed

    León, G; Maulén, N; Figueroa, J; Villanueva, J; Rodríguez, C; Vera, M I; Krauskopf, M

    1994-01-15

    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. PMID:8138127

  9. Evaluation by enzyme-linked immunosorbent assay (ELISA) of Renibacterium salmoninarum bacterins affected by persistence of bacterial antigens

    USGS Publications Warehouse

    Pascho, R.J.; Goodrich, T.D.; McKibben, C.L.

    1997-01-01

    Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with a bacterin containing killed Renibacterium salmoninarum cells delivered alone or in an oil-based adjuvant. We evaluated the relative abilities of the batterins to prevent the initiation or progression of infection in fish challenged by waterborne exposure to R. salmoninarum. Sixty-one days after vaccination, fish were held for 24 h in water containing either no bacteria or approximately 1.7 x 103, 1.7 x 105, or 5.3 x 106 live R. salmoninarum cells/mL. An enzyme-linked immunosorbent assay (ELISA) was used to monitor changes in the levels of R. salmoninarum antigen in live fish before and after the immersion challenges. High levels of R. salmoninarum antigens were detected by ELISA in kidney-spleen tissue homogenates from vaccinated fish immediately before the challenges. Levels of those antigens remained high in the tissues of unchallenged fish throughout the study. We found that the ELISA used in this study may be unsuitable for evaluating the efficacy of batterins because it did not distinguish antigens produced by the challenge bacteria during an infection from those of the bacterins. Groups of control and vaccinated fish also were injected with either 1.7 x 104 or 1.7 x 106 R. salmoninarum cells and served as R. salmoninarum virulence controls. Relative survival among the various subgroups in the injection challenge suggests that adverse effects might have been associated with the adjuvant used in this study. The lowest survival at both injection challenge levels was among fish vaccinated with bacteria in adjuvant.

  10. Testing of candidate non-lethal sampling methods for detection of Renibacterium salmoninarum in juvenile Chinook salmon Oncorhynchus tshawytscha.

    PubMed

    Elliott, Diane G; McKibben, Constance L; Conway, Carla M; Purcell, Maureen K; Chase, Dorothy M; Applegate, LynnMarie J

    2015-05-11

    Non-lethal pathogen testing can be a useful tool for fish disease research and management. Our research objectives were to determine if (1) fin clips, gill snips, surface mucus scrapings, blood draws, or kidney biopsies could be obtained non-lethally from 3 to 15 g Chinook salmon Oncorhynchus tshawytscha, (2) non-lethal samples could accurately discriminate between fish exposed to the bacterial kidney disease agent Renibacterium salmoninarum and non-exposed fish, and (3) non-lethal samples could serve as proxies for lethal kidney samples to assess infection intensity. Blood draws and kidney biopsies caused ≥5% post-sampling mortality (Objective 1) and may be appropriate only for larger fish, but the other sample types were non-lethal. Sampling was performed over 21 wk following R. salmoninarum immersion challenge of fish from 2 stocks (Objectives 2 and 3), and nested PCR (nPCR) and real-time quantitative PCR (qPCR) results from candidate non-lethal samples were compared with kidney tissue analysis by nPCR, qPCR, bacteriological culture, enzyme-linked immunosorbent assay (ELISA), fluorescent antibody test (FAT) and histopathology/immunohistochemistry. R. salmoninarum was detected by PCR in >50% of fin, gill, and mucus samples from challenged fish. Mucus qPCR was the only non-lethal assay exhibiting both diagnostic sensitivity and specificity estimates>90% for distinguishing between R. salmoninarum-exposed and non-exposed fish and was the best candidate for use as an alternative to lethal kidney sample testing. Mucus qPCR R. salmoninarum quantity estimates reflected changes in kidney bacterial load estimates, as evidenced by significant positive correlations with kidney R. salmoninarum infection intensity scores at all sample times and in both fish stocks, and were not significantly impacted by environmental R. salmoninarum concentrations. PMID:25958804

  11. Rapid identification of Renibacterium salmoninarum using an oligonucleotide probe complementary to 16S rRNA.

    PubMed

    Mattsson, J G; Gersdorf, H; Jansson, E; Hongslo, T; Göbel, U B; Johansson, K E

    1993-02-01

    Bacterial kidney disease in salmonid fish is caused by the slow-growing Gram-positive rod, Renibacterium salmoninarum. The partial sequence of 16S rRNA from R. salmoninarum was determined and compared with published bacterial 16S rRNA sequences. From this sequence information, a 30-bases-long oligonucleotide was designed and used as a specific probe for identification of R. salmoninarum in filter hybridization experiments. Strong specific hybridization signals were observed for all strains of R. salmoninarum tested. Furthermore, no cross-hybridization could be seen against 22 other bacterial species, among them other salmonid fish pathogens. The detection limit for the probe in direct filter hybridization by the dot-blot technique was 2.5 x 10(4) bacteria. It was also possible to detect R. salmoninarum in clinical samples by direct filter hybridization. PMID:8455640

  12. DETECTION OF RENIBACTERIUM SALMONINARUM IN CHINOOK SALMON ONCORHYNCHUS TSHAWYTSCHA USING QUANTITATIVE PCR.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed a quantitative PCR assay to detect varying levels of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). This assay allows for the direct enumeration of bacterial DNA or RNA copy number within tissues and body fluids. The assay can be applied nonletha...

  13. Renibacterium salmoninarum p57 antigenic variation is restricted in geographic distribution and correlated with genomic markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 57 kDa protein (p57) is an important diagnostic antigen that is implicated in the pathogenesis of salmonid bacterial kidney disease. Little is known about the nature and extent of antigenic variation in p57. Previously, we reported that p57 produced by Renibacterium salmoninarum Strain 684 conta...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 b...

  15. Multilocus variable-number tandem-repeat genotyping of Renibacterium salmoninarum, a bacterium causing bacterial kidney disease in salmonid fish

    PubMed Central

    2013-01-01

    Background Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a bacterial disease of fish, which is both geographically widespread and difficult to control. Previously, application of various molecular typing methods has failed to reliably discriminate between R. salmoninarum isolates originating from different host species and geographic areas. The current study aimed to utilize multilocus variable number tandem repeats (VNTR) to investigate inter-strain variation of R. salmoninarum to establish whether host-specific populations exist in Atlantic salmon and rainbow trout respectively. Such information would be valuable in risk assessment of transmission of R. salmoninarum in a multispecies aquaculture environment. Results The present analysis utilizing sixteen VNTRs distinguished 17 different haplotypes amongst 41 R. salmoninarum isolates originating from Atlantic salmon and rainbow trout in Scotland, Norway and the US. The VNTR typing system revealed two well supported groups of R. salmoninarum haplotypes. The first group included R. salmoninarum isolates originating from both Atlantic salmon and rainbow trout circulating in Scottish and Norwegian aquaculture, in addition to the type strain ATCC33209T originating from Chinook salmon in North America. The second group comprised isolates found exclusively in Atlantic salmon, of mainly wild origin, including isolates NCIB1114 and NCIB1116 associated with the original Dee disease in Scotland. Conclusions The present study confirmed that VNTR analysis can be successfully applied to discriminate R. salmoninarum strains. There was no clear distinction between isolates originating from Atlantic salmon and rainbow trout as several haplotypes in group 1 clustered together R. salmoninarum isolates from both species. These findings indicate a potential exchange of pathogens between Atlantic salmon and rainbow trout in Scottish and Norwegian aquaculture during the last 20 years. In a scenario of

  16. Recovery of Renibacterium salmoninarum from naturally infected salmonine stocks in Michigan using a modified culture protocol

    USGS Publications Warehouse

    Faisal, M.; Eissa, A.E.; Starliper, C.E.

    2010-01-01

    Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), is a fastidious and slow-growing bacterium that is extremely difficult to grow in vitro. Herein, we describe a modified primary culture protocol that encompasses a modified bacteriological culture medium and a tissue processing procedure. In order to facilitate the release of R. salmoninarum from granulomatous tissues, kidneys of infected fish were homogenized in a high speed stomacher. The kidney disease medium (KDM2), routinely used for primary culture of R. salmoninarum was modified by the addition of antibiotics and metabolites. When a relatively large inoculum of diluted kidney homogenate was streak-plate inoculated onto the modified KDM2, colonial growth of R. salmoninarum was achieved within 5-7. days, compared to the standard of two weeks or more. The modified procedure was then used to determine the prevalence of R. salmoninarum among representative captive and feral salmonid stocks in Michigan. Prevalence and clinical manifestations varied among species, strains of fish, and locations; however, R. salmoninarum isolates were biochemically homogenous. The improved primary culture procedure described in this study enabled selective and quick isolation of R. salmoninarum. Also, the isolates retrieved in this study constitute a unique biological resource for future studies of R. salmoninarum in the Laurentian Great Lakes. ?? 2009 University of Cairo.

  17. Testing of candidate non-lethal sampling methods for detection of Renibacterium salmoninarum in juvenile Chinook salmon Oncorhynchus tshawytscha

    USGS Publications Warehouse

    Elliott, Diane G.; McKibben, Constance L.; Conway, Carla M.; Purcell, Maureen K.; Chase, Dorothy M.; Applegate, Lynn M.

    2015-01-01

    Non-lethal pathogen testing can be a useful tool for fish disease research and management. Our research objectives were to determine if (1) fin clips, gill snips, surface mucus scrapings, blood draws, or kidney biopsies could be obtained non-lethally from 3 to 15 g Chinook salmon Oncorhynchus tshawytscha, (2) non-lethal samples could accurately discriminate between fish exposed to the bacterial kidney disease agent Renibacterium salmoninarum and non-exposed fish, and (3) non-lethal samples could serve as proxies for lethal kidney samples to assess infection intensity. Blood draws and kidney biopsies caused ≥5% post-sampling mortality (Objective 1) and may be appropriate only for larger fish, but the other sample types were non-lethal. Sampling was performed over 21 wk following R. salmoninarum immersion challenge of fish from 2 stocks (Objectives 2 and 3), and nested PCR (nPCR) and real-time quantitative PCR (qPCR) results from candidate non-lethal samples were compared with kidney tissue analysis by nPCR, qPCR, bacteriological culture, enzyme-linked immunosorbent assay (ELISA), fluorescent antibody test (FAT) and histopathology/immunohistochemistry. R. salmoninarum was detected by PCR in >50% of fin, gill, and mucus samples from challenged fish. Mucus qPCR was the only non-lethal assay exhibiting both diagnostic sensitivity and specificity estimates >90% for distinguishing between R. salmoninarum-exposed and non-exposed fish and was the best candidate for use as an alternative to lethal kidney sample testing. Mucus qPCR R. salmoninarum quantity estimates reflected changes in kidney bacterial load estimates, as evidenced by significant positive correlations with kidney R. salmoninaruminfection intensity scores at all sample times and in both fish stocks, and were not significantly impacted by environmentalR. salmoninarum concentrations.

  18. Expression of duplicate msa genes in the salmonid pathogen Renibacterium salmoninarum.

    PubMed

    Rhodes, Linda D; Coady, Alison M; Strom, Mark S

    2002-11-01

    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. PMID:12406741

  19. Expression of Duplicate msa Genes in the Salmonid Pathogen Renibacterium salmoninarum

    PubMed Central

    Rhodes, Linda D.; Coady, Alison M.; Strom, Mark S.

    2002-01-01

    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. PMID:12406741

  20. Identification of a Renibacterium salmoninarum DNA fragment associated with bacterial internalization into CHSE-cultured cells.

    PubMed

    Maulén, N P; Morales, P J; Aruti, D; Figueroa, J E; Concha, M I; Krauskopf, M; León, G

    1996-01-01

    We report here the isolation of a Renibacterium salmoninarum DNA sequence capable of transforming a non-invasive Escherichia coli strain into a microorganism able to enter the fish cell line, CHSE-214. Immunofluorescence and electron microscopy techniques were used to assess the acquired invasive phenotype by HB101 E. coli cells, upon transformation with pPMV-189. This plasmid carries a 2282-bp R. salmoninarum DNA segment. The invasive phenotype is conserved upon deletion of approximately 1000 bp at the 3' end of the insert. The remaining segment contains an ORF region encoding a putative protein of about 30 kDa. PMID:8598275

  1. Effects of temperature on Renibacterium salmoninarum infection and transmission potential in Chinook salmon, Oncorhynchus tshawytscha (Walbaum).

    PubMed

    Purcell, M K; McKibben, C L; Pearman-Gillman, S; Elliott, D G; Winton, J R

    2016-07-01

    Renibacterium salmoninarum is a significant pathogen of salmonids and the causative agent of bacterial kidney disease (BKD). Water temperature affects the replication rate of pathogens and the function of the fish immune system to influence the progression of disease. In addition, rapid shifts in temperature may serve as stressors that reduce host resistance. This study evaluated the effect of shifts in water temperature on established R. salmoninarum infections. We challenged Chinook salmon with R. salmoninarum at 12 °C for 2 weeks and then divided the fish into three temperature groups (8, 12 and 15 °C). Fish in the 8 °C group had significantly higher R. salmoninarum-specific mortality, kidney R. salmoninarum loads and bacterial shedding rates relative to the fish held at 12 or 15 °C. There was a trend towards suppressed bacterial load and shedding in the 15 °C group, but the results were not significant. Bacterial load was a significant predictor of shedding for the 8 and 12 °C groups but not for the 15 °C group. Overall, our results showed little effect of temperature stress on the progress of infection, but do support the conclusion that cooler water temperatures contribute to infection progression and increased transmission potential in Chinook salmon infected with R. salmoninarum. PMID:26449619

  2. Effects of temperature on Renibacterium salmoninarum infection and transmission potential in Chinook salmon, Oncorhynchus tshawytscha (Walbaum)

    USGS Publications Warehouse

    Purcell, Maureen K.; McKibben, Constance L.; Pearman-Gillman, Schuyler; Elliott, Diane G.; Winton, James R.

    2016-01-01

    Renibacterium salmoninarum is a significant pathogen of salmonids and the causative agent of bacterial kidney disease (BKD). Water temperature affects the replication rate of pathogens and the function of the fish immune system to influence the progression of disease. In addition, rapid shifts in temperature may serve as stressors that reduce host resistance. This study evaluated the effect of shifts in water temperature on established R. salmoninarum infections. We challenged Chinook salmon with R. salmoninarum at 12°C for 2 weeks and then divided the fish into three temperature groups (8, 12 and 15°C). Fish in the 8°C group had significantly higher R. salmoninarum-specific mortality, kidney R. salmoninarum loads and bacterial shedding rates relative to the fish held at 12 or 15°C. There was a trend towards suppressed bacterial load and shedding in the 15°C group, but the results were not significant. Bacterial load was a significant predictor of shedding for the 8 and 12°C groups but not for the 15°C group. Overall, our results showed little effect of temperature stress on the progress of infection, but do support the conclusion that cooler water temperatures contribute to infection progression and increased transmission potential in Chinook salmon infected with R. salmoninarum.

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

    PubMed

    Eissa, A E; Elsayed, E E; McDonald, R; Faisal, M

    2006-07-01

    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. PMID:17092886

  4. Production of putative virulence factors by Renibacterium salmoninarum grown in cell culture.

    PubMed

    McIntosh, D; Flaño, E; Grayson, T H; Gilpin, M L; Austin, B; Villena, A J

    1997-10-01

    A cell culture system, employing the fish cell line Epithelioma papillosum cyprini (EPC), was developed to study the synthesis of intracellular antigen and the expression of putative virulence factors by Renibacterium salmoninarum. EPC cultures infected with R. salmoninarum could be maintained for 7 weeks, during which the pathogen multiplied intracellularly. Immunohistochemical examination of infected cultures revealed the production of the p57 antigen, haemolysin and cytolysin. The intracellular nature of the infection was confirmed by transmission electron microscopic examination of EPC monolayers. A comparison of the relative virulence of bacterial cells cultured in EPC cells and on agar plates revealed that the former were markedly more virulent in challenge experiments with juvenile rainbow trout (Oncorhynchus mykiss Walbaum). The EPC cell culture model provided a system for the study of R. salmoninarum under more natural conditions than those achieved with plate culture techniques. PMID:9353936

  5. Sortase inhibitor phenyl vinyl sulfone inhibits Renibacterium salmoninarum adherence and invasion of host cells.

    PubMed

    Sudheesh, Ponnerassery S; Crane, Samuel; Cain, Kenneth D; Strom, Mark S

    2007-12-13

    Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fishes, is a Gram-positive diplococcobacillus belonging to the family Micrococcaceae. Analysis of the genome sequence of the bacterium demonstrated the presence of a sortase homolog (srtD), a gene specifying an enzyme found in Gram-positive bacteria and required for covalent anchoring of cell surface proteins. Interference of sortase activity is being examined as a target for therapeutic prevention of infection by several pathogenic Gram-positive bacterial species. In silico analysis identified 8 open reading frames containing sortase recognition motifs, suggesting these proteins are translocated to the bacterial cell wall. The sortase and potential sortase substrate genes are transcribed in R. salmoninarum, suggesting they encode functional proteins. Treatment of R. salmoninarum with phenyl vinyl sulfone (PVS) significantly reduced bacterial adherence to Chinook salmon fibronectin. In addition, the ability of the PVS-treated bacteria to adhere to Chinook salmon embryo cells (CHSE-214) in vitro was dramatically reduced compared to that of untreated bacteria. More importantly, PVS-treated bacteria were unable to invade and replicate within CHSE-214 cells (demonstrated by an intracellular growth assay and by light microscopy). When treated with PVS, R. salmoninarum was not cytopathic to CHSE-214 cells, whereas untreated bacteria produced cytopathology within a few days. These findings clearly show that PVS, a small molecule drug and a known sortase inhibitor, can interfere with the ability of R. salmoninarum to adhere and colonize fish cells, with a corresponding decrease in virulence. PMID:18286808

  6. Zygosity at the major histocompatibility class IIB locus predicts susceptibility to Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.).

    PubMed

    Turner, S M; Faisal, M; DeWoody, J A

    2007-10-01

    Major histocompatibility (MH) class II genes play an important role in the vertebrate immune response. Here, we investigate the relationship between Atlantic salmon (Salmo salar) MH class IIB zygosity and susceptibility to Renibacterium salmoninarum, the causal agent of bacterial kidney disease. By combining DNA sequences from the salmon MH class IIB gene with quantitative ELISA data on R. salmoninarum antigen levels, we found that MH class IIB homozygotes were significantly more susceptible to R. salmoninarum than heterozygotes. These findings are discussed in the context of current evolutionary theory. PMID:17627802

  7. Comparison of five techniques for the detection of Renibacterium salmoninarum in adult coho salmon.

    USGS Publications Warehouse

    Pascho, R.J.; Elliott, D.G.; Mallett, R.W.; Mulcahy, D.

    1987-01-01

    Samples of kidney, spleen, coelomic fluid, and blood from 56 sexually mature coho salmon Oncorhynchus kisutch were examined for infection by Renibacterium salmoninarum by five methods. The overall prevalence (all sample types combined) of R. salmoninarum in the fish was 100% by the enzyme-linked immunosorbent assay, 86% by the combined results of the direct fluorescent antibody and the direct filtration-fluorescent antibody techniques, 39% by culture, 11% by counterimmunoelectrophoresis, and 5% by agarose gel immunodiffusion. There was a significant positive correlation (P < 0.001) between the enzyme-linked immunosorbent assay absorbance levels and the counts by fluorescent antibody techniques for kidney, spleen, and coelomic fluid, and significant positive correlations (P < 0.001) in enzyme-linked immunosorbent assay absorbance levels for all four of the sample types.

  8. Specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum.

    PubMed

    León, G; Martinez, M A; Etchegaray, J P; Vera, M I; Figueroa, J; Krauskopf, M

    1994-03-01

    To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova. PMID:24420936

  9. Nutrient Requirements of Renibacterium salmoninarum on Agar and in Broth Media.

    PubMed

    Daly, J G; Stevenson, R M

    1993-07-01

    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. PMID:16348993

  10. Nutrient Requirements of Renibacterium salmoninarum on Agar and in Broth Media

    PubMed Central

    Daly, J. G.; Stevenson, R. M. W.

    1993-01-01

    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. PMID:16348993

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

    PubMed Central

    Daly, J G; Stevenson, R M

    1985-01-01

    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. Images PMID:4083882

  12. Evidence that coded-wire-tagging procedures can enhance transmission of Renibacterium salmoninarum in chinook salmon

    USGS Publications Warehouse

    Elliott, D.G.; Pascho, R.J.

    2001-01-01

    Binary coded wire tags (CWTs) are used extensively for identification and management of anadromous salmonid populations. A study of bacterial kidney disease (BKD) in two brood year groups of hatchery-reared spring chinook salmon Oncorhynchus tshawytscha provided strong evidence that horizontal transmission of Renibacterium salmoninarum, the causative agent of BKD, might be enhanced by CWT-marking procedures. About 4 months after CWTs were implanted in the snouts of juvenile fish, 14-16 different tissues were sampled from each of 60 fish per brood year group for histological analysis. Of the fish that were positive for R. salmoninarum by histological examination, 41% (7 of 17) of the 1988 brood year fish and 24% (10 of 42) of the 1989 brood year fish had BKD lesions confined to the head near the site of tag implantation. These lesions often resulted in the destruction of tissues of one or both olfactory organs. No focal snout infections were observed in fish that had not been marked with CWTs. Further data obtained from tissue analyses by use of an enzyme-linked immunosorbent assay and a fluorescent antibody test for detection of R. salmoninarum supported the hypothesis that infections of R. salmoninarum can be initiated in the snout tissues of CWT-marked fish and then spread to other organs. The tagging procedures might promote transmission of the pathogen among fish via contaminated tagging needles, by facilitating the entry of pathogens through the injection wound, or both. Limited evidence from this study suggested that implantation of passive integrated transponder tags in the peritoneal cavities of fish might also promote the transmission of R. salmoninarum or exacerbate existing infections. The results indicated a need for strict sanitary procedures during the tagging of fish in populations positive for R. salmoninarum to reduce the probability of enhanced horizontal transmission of the pathogen.

  13. Bactericidal activity of juvenile chinook salmon macrophages against Aeromonas salmonicida after exposure to live or heat-killed Renibacterium salmoninarum or to soluble proteins produced by R. salmoninarum

    USGS Publications Warehouse

    Siegel, D.C.; Congleton, J.L.

    1997-01-01

    Macrophages isolated from the anterior kidney of juvenile chinook salmon Oncorhynchus tshawytscha in 96-well microtiter plates were exposed for 72 h to 0, 105, or 106 live or heat-killed Renibacterium salmoninarum cells per well or to 0, 0.1, 1.0, or 10 ??g/mL of R. salmoninarum soluble proteins. After treatment, the bactericidal activity of the macrophages against Aerornonas salmonicida was determined by a colorimetric assay based on the reduction of the tetrazolium dye MTT to formazan by viable bacteria. The MTT assay was modified to allow estimation of the percentage of bacteria killed by reference to a standard curve relating the number of bacteria added to microtiter wells to absorbance by formazan at 600 nm. The live and heat-killed R. salmoninarum treatments significantly (P < 0.001) increased killing of A. salmonicida by chinook salmon macrophages. In each of the five trials, significantly (P < 0.05) greater increases in killing occurred after exposure to 105 R. salmoninarum cells than to 106 R. salmoninarum cells per well. In contrast, treatment of macrophages with 10 ??g/mL R. salmoninarum soluble proteins significantly (P < 0.001) decreased killing of A. salmonicida, but treatment with lower doses did not. These results show that the bactericidal activity of chinook salmon macrophages is stimulated by exposure to R. salmoninarum cells at lower dose levels but inhibited by exposure to R. salmoninarum cells or soluble proteins at higher dose levels.

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

    USGS Publications Warehouse

    Coady, A.M.; Murray, A.L.; Elliott, D.G.; Rhodes, L.D.

    2006-01-01

    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. Copyright ?? 2006, American Society for Microbiology. All Rights Reserved.

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

    PubMed

    Coady, Alison M; Murray, Anthony L; Elliott, Diane G; Rhodes, Linda D

    2006-04-01

    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. PMID:16597972

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

    PubMed Central

    Coady, Alison M.; Murray, Anthony L.; Elliott, Diane G.; Rhodes, Linda D.

    2006-01-01

    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. PMID:16597972

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

    PubMed Central

    Fiedler, F; Draxl, R

    1986-01-01

    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. Images PMID:3782026

  18. Development of a specific biotinylated DNA probe for the detection of Renibacterium salmoninarum.

    PubMed

    Hariharan, H; Qian, B; Despres, B; Kibenge, F S; Heaney, S B; Rainnie, D J

    1995-10-01

    A specific DNA probe for the identification of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was developed from one of 3 clones pRS47, pRS49, and pRS26 of 5.1 kb, 5.3 kb, and 11.3 kb, respectively. The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA extracted from 3 strains of R. salmoninarum and from 1 strain each of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. In a dot blot assay, this probe hybridized only with the DNA from the R. salmoninarum strains. When used on kidney samples from fish challenged with R. salmoninarum, the dot blot hybridization assay with the probe was found to be as sensitive as culture. In a fluorescent antibody test, samples that were negative in culture and dot blot hybridization showed no more than one fluorescing cell in 50 microscopic fields examined. This DNA probe, therefore, has the potential for use in the diagnosis of BKD of fish. PMID:8548693

  19. Development of a specific biotinylated DNA probe for the detection of Renibacterium salmoninarum.

    PubMed Central

    Hariharan, H; Qian, B; Despres, B; Kibenge, F S; Heaney, S B; Rainnie, D J

    1995-01-01

    A specific DNA probe for the identification of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was developed from one of 3 clones pRS47, pRS49, and pRS26 of 5.1 kb, 5.3 kb, and 11.3 kb, respectively. The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA extracted from 3 strains of R. salmoninarum and from 1 strain each of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. In a dot blot assay, this probe hybridized only with the DNA from the R. salmoninarum strains. When used on kidney samples from fish challenged with R. salmoninarum, the dot blot hybridization assay with the probe was found to be as sensitive as culture. In a fluorescent antibody test, samples that were negative in culture and dot blot hybridization showed no more than one fluorescing cell in 50 microscopic fields examined. This DNA probe, therefore, has the potential for use in the diagnosis of BKD of fish. Images Fig. 2. Fig. 3. PMID:8548693

  20. Diagnostic testing patterns of Renibacterium salmoninarum in spawning salmonid stocks in Michigan.

    PubMed

    Faisal, M; Eissa, A E

    2009-04-01

    Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a slowly progressing disease that threatens salmon conservation and restoration programs in North America. The purpose of this study was to track naturally occurring R. salmoninarum infection in representative, Michigan, USA, salmonid stocks using nested polymerase chain reaction (nPCR), quantitative enzyme-linked immunosorbent assay (Q-ELISA), and culture. The Q-ELISA test detected 67.6% infection prevalence, which is lower than culture (77.2%) or nPCR (94.2%), yet it provided semiquantitative data on infection intensity. The disagreement in results among the three assays may reflect the different phases of R. salmoninarum infection at the time of sampling. The testing results demonstrated the presence of six patterns, with each of the patterns representing a probable stage along the course of natural R. salmoninarum infection. Findings also suggest that fish stocks tested in this study were not uniform in the distribution of the diagnostic patterns and that, from studying such patterns, one can determine the course of BKD infection in a particular population. PMID:19395754

  1. Genome sequence of the fish pathogen Renibacterium salmoninarum suggests reductive evolution away from an environmental Arthrobacter ancestor.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Renibacterium salmoninarum (Rs) is the causative agent of bacterial kidney disease and a significant threat to the healthy and sustainable production of salmonid fish worldwide. The pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative...

  2. A new value for mol percent guanine + cytosine of DNA for the salmonid fish pathogen Renibacterium salmoninarum.

    PubMed

    Banner, C R; Rohovec, J S; Fryer, J L

    1991-03-15

    The mol% G + C of DNA extracted from seven different isolates of Renibacterium salmoninarum was determined. Organisms studied were from selected geographical areas (U.S.A., Canada, England and France) and were isolated from five different species of salmonid fish. The mol% G + C was determined to be 55.5, higher than the currently reported value of 53. PMID:2044940

  3. Genes associated with an effective host response by Chinook salmon to Renibacterium salmoninarum.

    PubMed

    Rhodes, Linda D; Wallis, Steviebrooke; Demlow, S Ellen

    2009-02-01

    An effective host response to Renibacterium salmoninarum, the etiologic agent of bacterial kidney disease, is poorly characterized. Using suppression subtractive hybridization, we exploited the difference in early host response in the pronephros of fish challenged by an attenuated strain (MT239) or a virulent strain (ATCC 33209) of R. salmoninarum. Among the 132 expressed sequence tag (EST) clones that were sequenced, 20 were selected for expression analysis at 24 and 72h after challenge. ESTs matching two interferon inducible genes (IFN-inducible GBP and VLIG1), the ligand GAS6, and the kinase VRK2 were upregulated in fish exposed to MT239, but downregulated or unchanged in fish exposed to 33209. A second group of ESTs matching genes involved in apoptosis (caspase 8) and immune function (IkappaBalpha, p47(phoX), EMR/CD97) were more slowly upregulated in fish exposed to 33209 compared to fish exposed to MT239. The ESTs displaying elevated expression in MT239-exposed fish may represent important cellular processes to bacterial challenge, and may be useful indicators of an effective host response to R. salmoninarum infection. PMID:18793667

  4. Identification of a third msa gene in Renibacterium salmoninarum and the associated virulence phenotype.

    PubMed

    Rhodes, Linda D; Coady, Alison M; Deinhard, Rebecca K

    2004-11-01

    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. PMID:15528510

  5. Pathological and immunological responses associated with differential survival of Chinook salmon following Renibacterium salmoninarum challenge

    USGS Publications Warehouse

    Purcell, Maureen K.; Elliott, Diane G.; Metzger, C. David; Wargo, Andrew; Park, K. Linda

    2010-01-01

    Chinook salmon Oncorhynchus tshawytscha are highly susceptible to Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). Previously we demonstrated that introduced Chinook salmon from Lake Michigan, Wisconsin (WI), USA, have higher survival following R. salmoninarum challenge relative to the progenitor stock from Green River, Washington, USA. In the present study, we investigated the pathological and immunological responses that are associated with differential survival in the 2 Chinook salmon stocks following intra-peritoneal R. salmoninarum challenge of 2 different cohort years (2003 and 2005). Histological evaluation revealed delayed appearance of severe granulomatous lesions in the kidney and lower overall prevalence of membranous glomerulopathy in the higher surviving WI stock. The higher survival WI stock had a lower bacterial load at 28 d post-infection, as measured by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). However, at all other time points, bacterial load levels were similar despite higher mortality in the more susceptible Green River stock, suggesting the possibility that the stocks may differ in their tolerance to infection by the bacterium. Interferon-y, inducible nitric oxide synthase (iNOS), Mx-1, and transferrin gene expression were up-regulated in both stocks following challenge. A trend of higher iNOS gene expression at later time points (≥28 d post-infection) was observed in the lower surviving Green River stock, suggesting the possibility that higher iNOS expression may contribute to greater pathology in that stock.

  6. Pathological and immunological responses associated with differential survival of Chinook salmon following Renibacterium salmoninarum challenge.

    PubMed

    Metzger, David C; Elliott, Diane G; Wargo, Andrew; Park, Linda K; Purcell, Maureen K

    2010-05-18

    Chinook salmon Oncorhynchus tshawytscha are highly susceptible to Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). Previously we demonstrated that introduced Chinook salmon from Lake Michigan, Wisconsin (WI), USA, have higher survival following R. salmoninarum challenge relative to the progenitor stock from Green River, Washington, USA. In the present study, we investigated the pathological and immunological responses that are associated with differential survival in the 2 Chinook salmon stocks following intra-peritoneal R. salmoninarum challenge of 2 different cohort years (2003 and 2005). Histological evaluation revealed delayed appearance of severe granulomatous lesions in the kidney and lower overall prevalence of membranous glomerulopathy in the higher surviving WI stock. The higher survival WI stock had a lower bacterial load at 28 d post-infection, as measured by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). However, at all other time points, bacterial load levels were similar despite higher mortality in the more susceptible Green River stock, suggesting the possibility that the stocks may differ in their tolerance to infection by the bacterium. Interferon-gamma, inducible nitric oxide synthase (iNOS), Mx-1, and transferrin gene expression were up-regulated in both stocks following challenge. A trend of higher iNOS gene expression at later time points (> or = 28 d post-infection) was observed in the lower surviving Green River stock, suggesting the possibility that higher iNOS expression may contribute to greater pathology in that stock. PMID:20597428

  7. Detection of Renibacterium salmoninarum in chinook salmon, Oncorhynchus tshawytscha (Walbaum), using quantitative PCR.

    PubMed

    Powell, M; Overturf, K; Hogge, C; Johnson, K

    2005-10-01

    A quantitative polymerase chain reaction (QPCR) assay has been developed to detect varying levels of Renibacterium salmoninarum, the causative agent of bacterial kidney disease. This assay allows for the direct enumeration of bacterial DNA or RNA copy number within tissues and body fluids. The assay can be applied non-lethally and can be used to determine whether R. salmoninarum is transcriptionally active. The presence of R. salmoninarum in kidney tissues from 430 chinook salmon collected from five Idaho Fish and Game operated hatcheries was initially evaluated using the widely employed enzyme-linked immunosorbent assay (ELISA) with two sets of Kirkegaard and Perry Laboratories polyclonal antibodies, 'mother batches' 1 and 2. The same tissue samples were then analysed using the novel QPCR assay and the results compared. At moderate to high levels of infection [optical density (OD > 0.5)], ELISA values and estimated DNA copy number were highly correlated (r(2) > 0.80), although correlation to specific antibody batches varied. However, lower ELISA values (OD < 0.5) observed with either antibody batch did not correlate well with the QPCR assay (R(2)

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

    PubMed Central

    Rhodes, Linda D.; Coady, Alison M.; Deinhard, Rebecca K.

    2004-01-01

    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. PMID:15528510

  9. Detection of Renibacterium salmoninarum, the Causative Agent of Bacterial Kidney Disease in Salmonid Fish, from Pen-Cultured Coho Salmon.

    PubMed

    Sakai, M; Kobayashi, M

    1992-03-01

    The detection of Renibacterium salmoninarum antigen from pen-cultured coho salmon was attempted. Flounder (Limanda herzensteini) (n = 24), greenling (Hexagrammos otakii) (n = 5), Japanese sculpin (Cottus japonicus) (n = 1), and flathead (Platycephalus indicus) (n = 22) captured by fishing around coho salmon net pens were examined for the presence of R. salmoninarum antigen by an indirect dot blot assay and by an indirect fluorescent-antibody technique using polyclonal and monoclonal antibodies. R. salmoninarum antigen was detected from kidney samples of one greenling and six flathead. Moreover, 86 scallops (Patinopecten yessoensis) were hung from the edge of the net pen for 50 days, and R. salmoninarum antigen was demonstrated in 31 samples by the indirect dot blot assay and the indirect fluorescent-antibody technique. PMID:16348666

  10. Detection of Renibacterium salmoninarum, the Causative Agent of Bacterial Kidney Disease in Salmonid Fish, from Pen-Cultured Coho Salmon

    PubMed Central

    Sakai, Masahiro; Kobayashi, Masanori

    1992-01-01

    The detection of Renibacterium salmoninarum antigen from pen-cultured coho salmon was attempted. Flounder (Limanda herzensteini) (n = 24), greenling (Hexagrammos otakii) (n = 5), Japanese sculpin (Cottus japonicus) (n = 1), and flathead (Platycephalus indicus) (n = 22) captured by fishing around coho salmon net pens were examined for the presence of R. salmoninarum antigen by an indirect dot blot assay and by an indirect fluorescent-antibody technique using polyclonal and monoclonal antibodies. R. salmoninarum antigen was detected from kidney samples of one greenling and six flathead. Moreover, 86 scallops (Patinopecten yessoensis) were hung from the edge of the net pen for 50 days, and R. salmoninarum antigen was demonstrated in 31 samples by the indirect dot blot assay and the indirect fluorescent-antibody technique. PMID:16348666

  11. Impact of stressors on transmission potential of Renibacterium salmoninarum in Chinook salmon

    USGS Publications Warehouse

    Purcell, Maureen K.; Winton, James R.

    2014-01-01

    Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD) affecting several species of Pacific salmon.  The severity of BKD can range from a chronic infection to overt disease with high mortality as in the case of large losses of adult Chinook salmon (Oncorhynchus tshawytscha) in the Great Lakes during late 1980s. The goal of this study was to empirically evaluate how environmental stressors relevant to the Great Lakes impact R. salmoninarum disease progression and bacterial shedding, the latter parameter being a proxy of horizontal transmission. In the first study (Aim 1), we focused on how endogenous host thiamine levels and dietary fatty acids impacted resistance of Chinook salmon to R. salmoninarum. Juvenile fish were fed one of four experimental diets, including a (1) thiamine replete diet formulated with fish oil, (2) thiamine deplete diet formulated with fish oil, (3) thiamine replete diet formulated with soybean oil, and (4) thiamine deplete diet formulated with soybean oil, before being challenged with buffer or R. salmoninarum. We observed significantly higher mortality in the R. salmoninarum infected groups relative to the corresponding mock controls in only the thiamine replete diet groups. We also observed a significant effect of time and diet on kidney bacterial load and bacterial shedding, with a significant trend towards higher shedding and bacterial load in the fish oil – thiamine replete diet group. However, during the course of the study, unexpected mortality occurred in all groups attributed to the myxozoan parasite Ceratomyxa shasta. Since the fish were dually-infected with C. shasta, we evaluated parasite DNA levels (parasitic load) in the kidney of sampled fish. We found that parasite load varied across time points but there was no significant effect of diet. However, parasite load did differ significantly between the mock and R. salmoninarum challenge groups with a trend towards longer persistence of C. shasta

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

    PubMed

    Piganelli, J D; Wiens, G D; Zhang, J A; Christensen, J M; Kaattari, S L

    1999-04-15

    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. PMID

  13. Molecular diversity of Renibacterium salmoninarum isolates determined by randomly amplified polymorphic DNA analysis.

    PubMed

    Grayson, T H; Atienzar, F A; Alexander, S M; Cooper, L F; Gilpin, M L

    2000-01-01

    The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture. PMID:10618262

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

    PubMed

    Concha, M I; León, G

    2000-05-01

    The complete glcK gene from the fish pathogen Renibacterium salmoninarum, encoding a glucose kinase, was analyzed and expressed. The partial characterization of the recombinant enzyme confirmed that it belongs to a group of glucose kinases involved in carbon catabolite repression. Multiple sequence alignments were used to deduce a new consensus sequence for this family of bacterial proteins, characterized by several conserved Cys residues. This sequence was more specific and allowed the detection of the first eukaryotic protein of this family. The recombinant enzyme was inhibited by N-ethylmaleimide and the substrates protected the enzyme from this inhibition, suggesting the presence of Cys residues in or close to the active site. PMID:10779719

  15. Detection of a vascular permeability factor in the extracellular products of Renibacterium salmoninarum.

    PubMed

    Bandín, I; Santos, Y; Toranzo, A E; Barja, J L

    1992-09-01

    The presence of vascular permeability factors in the extracellular products (ECP) of 10 strains of Renibacterium salmoninarum with different geographical origin and serological characteristics are reported. All the ECP produced haemorrhagic and/or oedematous zones at the injection site with a diameter ranging from 10-30 mm. However, the ECP samples did not display toxic effect in fish at the same dose as inoculated in rabbit (180-400 micrograms protein/0.1 ml). No differences were observed in the production of this dermatotoxic factor between the two antigenic groups found in this microorganism. Whereas heating (80 and 100 degrees C/15 min) the ECP samples resulted in a complete loss of their proteolytic activity, only a decrease (but not total inactivation) of the dermatotoxic effects was detected. Therefore, although proteases could be implicated in the permeability factor, they are not totally responsible for this activity. PMID:1291845

  16. Structural studies of the major polysaccharide in the cell wall of Renibacterium salmoninarum.

    PubMed

    Sørum, U; Robertsen, B; Kenne, L

    1998-01-01

    The galactose-rich polysaccharide (GPS) in the cell wall of the Gram-positive bacterium Renibacterium salmoninarum, the causative agent in of bacterial kidney disease (BKD) of salmonids, has been studied by sugar and methylation analysis, partial acid hydrolysis, Smith degradation, FABMS, and 1H and 13C NMR spectroscopy. The data show that the GPS has a heptasaccharide repeating unit with the following structure: alpha-D-Rhap-(1-->3)-alpha-L-FucpNAc-(1-->)-beta-D-GlcpNAc 1 decreases 2 -->3)-beta-D-Galf-(1-->6)-beta-D-Galf-(1-->3)-beta-D-Galf -(1-->6) -beta-D-Galf-(1-->. PMID:9691455

  17. Molecular Diversity of Renibacterium salmoninarum Isolates Determined by Randomly Amplified Polymorphic DNA Analysis

    PubMed Central

    Grayson, T. Hilton; Atienzar, Franck A.; Alexander, Sarah M.; Cooper, Lynne F.; Gilpin, Martyn L.

    2000-01-01

    The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture. PMID:10618262

  18. Renibacterium salmoninarum bar forms: characterization, occurrence, and evidence of a host response to a R. salmoninarum infection.

    PubMed

    Cvitanich, J D

    2004-04-01

    Unique-staining Renibacterium salmoninarum (Rs) cells, termed bar forms, first observed in a coho salmon, Oncorhynchus kisutch (Walbaum), in 1983, could not be cultured, making their characterization difficult and significance obscure. They can be detected only by the fluorescent-antibody technique (FAT) and their numbers estimated only by a quantitative FAT (QFAT). Data collected over a 10-year period showed that bar forms were observed only in vivo and appeared associated with a host response. Bar forms were observed in 10 salmonid species from five countries and in fish from < 1 g to spawning adults. They were observed in 50.1% of kidney smears prepared from 10,061 Rs positive chinook, O. tshawytscha (Walbaum), coho, and Atlantic salmon, Salmo salar L. Bar forms were shown to be Rs cells based on absorption studies, their reaction with an Rs-specific FAT and enzyme-linked immunosorbent assay, and a transition from 'typical' Rs cells to bar forms in naturally and experimentally infected fish. Bar forms were determined to be non-virulent, damaged or dead Rs cells, based on fluorescence and electron microscopy observations, the inability to culture them, and mortality data. Bar forms appeared to represent visual markers of recovery from an Rs infection. PMID:15049888

  19. A gene from Renibacterium salmoninarum encoding a product which shows homology to bacterial zinc-metalloproteases.

    PubMed

    Grayson, T H; Evenden, A J; Gilpin, M L; Martin, K L; Munn, C B

    1995-06-01

    A genomic library constructed from Renibacterium salmoninarum isolate MT444 DNA in the plasmid vector pBR328 was screened using Escherichia coli host strain DH1 for the expression of genes encoding putative virulence factors. A single haemolytic clone was isolated at 22 degrees C and found to contain a 3.1 kb HindIII fragment of inserted DNA. This fragment was present in seven isolates of R. salmoninarum which were examined. Western blots of extracts from clones exhibiting haemolytic activity were performed with antisera raised against either cellular or extracellular components of R. salmoninarum and failed to identify any additional proteins compared to control E. coli containing pBR328. However, minicell analysis revealed that a polypeptide with an apparent molecular mass of 65 kDa was associated with a haemolytic activity distinct from that previously described for R. salmoninarum. The nucleotide sequence of the gene encoding this product was determined and the amino acid sequence deduced. The product was 548 amino acids with a predicted molecular mass of 66757 Da and a pl of 5.57. The deduced amino acid sequence of the gene possessed strong similarities to those of a range of secreted bacterial zinc-metalloproteases and was tentatively designed hly. Neither protease nor lecithinase activities were detectable in E. coli recombinants expressing gene hly. Haemolytic activity was observed from 6 degrees C to 37 degrees C for erythrocytes from a number of mammalian species and also from fish. Gene hly was expressed in E. coli as a fusion protein consisting of maltose-binding protein at the N-terminus linked to all but the first 24 amino acids, largely constituting the putative signal peptide, of the N-terminus of Hly. The soluble fusion protein was produced and purified by affinity chromatography. Antiserum raised against the purified fusion protein was used to probe Western blots of cell lysates and extracellular products from seven isolates of R. salmoninarum

  20. Performance of serum-free broth media for growth of Renibacterium salmoninarum

    USGS Publications Warehouse

    Starliper, C.E.; Schill, W.B.; Mathias, J.

    1998-01-01

    Growth of Renibacterium salmoninarum was compared in 14 different broth media; 13 serum-free, and 1 that contained newborn calf serum, KDM2+M. Supplementation with 1% v/v R. salmoninarum MCO4M metabolite was evaluated for 6 of the media that do not utilize it as part of their ingredients. Viable cells were enumerated on Days 10, 20, and 30 post inoculation to evaluate performance. The experiment was repeated 3 times using high, low, and medium (trials 1 to 3, respectively) cell concentrations as inoculum. In general there was no optimal medium and all performed well. The choice of which to employ depends on the ease of preparation and presence of certain ingredients that might affect subsequent assays. In trials 2 and 3, the pH was estimated using test papers at the same time as cells were counted. Maximum pH increase occurred with KDM2+M and those media containing charcoal. For most media, a simple pH determination could be used as a means to check that growth has occurred in a culture, particularly if charcoal was added directly to the media and a visual inspection could not be made to detect growth.

  1. Performance of serum-free broth media for growth of Renibacterium salmoninarum.

    PubMed

    Starliper, C E; Schill, W B; Mathias, J

    1998-09-11

    Growth of Renibacterium salmoninarum was compared in 14 different broth media; 13 serum-free, and 1 that contained newborn calf serum, KDM2+M. Supplementation with 1% v/v R. salmoninarum MCO4M metabolite was evaluated for 6 of the media that do not utilize it as part of their ingredients. Viable cells were enumerated on Days 10, 20, and 30 post inoculation to evaluate performance. The experiment was repeated 3 times using high, low, and medium (trials 1 to 3, respectively) cell concentrations as inoculum. In general there was no optimal medium and all performed well. The choice of which to employ depends on the ease of preparation and presence of certain ingredients that might affect subsequent assays. In trials 2 and 3, the pH was estimated using test papers at the same time as cells were counted. Maximum pH increase occurred with KDM2+M and those media containing charcoal. For most media, a simple pH determination could be used as a means to check that growth has occurred in a culture, particularly if charcoal was added directly to the media and a visual inspection could not be made to detect growth. PMID:9789976

  2. Use of Arthrobacter davidanieli as a live vaccine against Renibacterium salmoninarum and Piscirickettsia salmonis in salmonids.

    PubMed

    Salonius, K; Siderakis, C; MacKinnon, A M; Griffiths, S G

    2005-01-01

    Arthrobacter davidanieli (proposed species nomenclature) is a non-pathogenic Gram-variable bacterium related to, but taxonomically distinct from, Renibacterium salmoninarum, the aetiological agent of bacterial kidney disease (BKD). We have demonstrated that vaccination with live A. davidanieli is effective against BKD in Atlantic salmon (Salmo salar) showing above 80 relative percent survival in experimental challenge trials. Good protection was also demonstrated in long-term field trials where Atlantic salmon were naturally exposed to R. salmoninarum challenge until 23 months after vaccination. The same vaccine, which is licensed in Canada against BKD has also proved effective in reducing mortality from experimental challenge of coho salmon (Oncorhynchus kisutch) with Piscirickettsia salmonis, the causative agent of piscirickettsiosis. Under field conditions in Chile, use of the vaccine led to a significant reduction in piscirickettsiosis mortality in coho salmon over 10 months following sea transfer. The vaccine strain is unique in that it is the first live organism to be licensed as a vaccine for use in aquaculture. Potential mechanisms of protection against the two taxonomically disparate pathogens are discussed. PMID:15962482

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

    PubMed

    Rhodes, Linda D; Nguyen, Oanh T; Deinhard, Rebecca K; White, Teresa M; Harrell, Lee W; Roberts, Marilyn C

    2008-08-01

    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. PMID:18814542

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

    PubMed

    Dubreuil, J D; Jacques, M; Graham, L; Lallier, R

    1990-12-01

    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. PMID:1981894

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

    PubMed Central

    Brynildsrud, Ola; Feil, Edward J; Bohlin, Jon; Castillo-Ramirez, Santiago; Colquhoun, Duncan; McCarthy, Una; Matejusova, Iveta M; Rhodes, Linda D; Wiens, Gregory D; Verner-Jeffreys, David W

    2014-01-01

    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. PMID:24173459

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

    PubMed

    Brynildsrud, Ola; Feil, Edward J; Bohlin, Jon; Castillo-Ramirez, Santiago; Colquhoun, Duncan; McCarthy, Una; Matejusova, Iveta M; Rhodes, Linda D; Wiens, Gregory D; Verner-Jeffreys, David W

    2014-04-01

    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. PMID:24173459

  7. Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis

    USGS Publications Warehouse

    Chase, D.M.; Elliott, D.G.; Pascho, R.J.

    2006-01-01

    Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

  8. Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis.

    PubMed

    Chase, Dorothy M; Elliott, Diane G; Pascho, Ronald J

    2006-07-01

    Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids. PMID:16921877

  9. In vitro effects of the extracellular protein of Renibacterium salmoninarum on phagocyte function in brook trout (Salvelinus fontinalis).

    PubMed

    Densmore, C L; Smith, S A; Holladay, S D

    1998-04-30

    Renibacterium salmoninarum is a facultative intracellular pathogen often found in host phagocytes where it appears to successfully avoid the host fish's immunological defenses. The objective of this investigation was to determine if soluble extracellular protein (ECP) produced by R. salmoninarum may contribute to the immunomodulation in bacterial kidney disease (BKD) via inhibition of phagocyte respiratory burst and/or phagocytosis mechanisms. Splenic cells from adult brook trout (Salvelinus fontinalis) were incubated with two different concentrations of ECP (0.1 mg/ml and 1.0 mg/ml) and viable R. salmoninarum. Splenic cell cultures were evaluated for respiratory burst activity via flow cytometry with the dichlorofluorescin diacetate (DCF-DA) assay and for phagocytosis via light microscopic assessment of microsphere engulfment. Respiratory burst activity was significantly inhibited in all treatment groups as compared to untreated fish, while no differences were noted in phagocytic activity. PMID:9646439

  10. Epidemiological investigation of Renibacterium salmoninarum in three Oncorhynchus spp. in Michigan from 2001 to 2010.

    PubMed

    Faisal, Mohamed; Schulz, Carolyn; Eissa, Alaa; Brenden, Travis; Winters, Andrew; Whelan, Gary; Wolgamood, Martha; Eisch, Edward; VanAmberg, Jan

    2012-12-01

    Bacterial kidney disease (BKD) has caused mortalities and chronic infections in wild and farm-raised salmonids throughout the world. In the Laurentian Great Lakes of North America, BKD was associated with several large-scale mortality events of Oncorhynchus spp. throughout the 1980s and 1990s. In response to these mortality events, the state of Michigan implemented several enhanced biosecurity measures to limit the occurrence of BKD in state-operated hatcheries and gamete-collection weirs. The objectives of this study were to assess if infection levels (prevalence and intensity) of Renibacterium salmoninarum, the causative agent of BKD, have changed in broodstock and pre-stocking fingerlings of three feral Oncorhynchus spp. (Chinook salmon (O. tshawytscha), coho salmon (O. kisutch), and steelhead (O. mykiss)) over a decade, following the implementation of the enhanced biosecurity measures. Between 2001 and 2010, a total of 3,530 broodstock salmonids collected from lakes Huron and Michigan tributaries during spawning runs and 4,294 propagated pre-stocking salmonid fingerlings collected from three state of Michigan fish hatcheries were tested for the presence of R. salmoninarum antigens using the enzyme-linked immunosorbent assay. Substantial declines in the overall prevalence of the bacterium were detected in each of the examined broodstocks. Most propagated pre-stocking fingerlings also exhibited substantial declines in R. salmoninarum prevalence. Prevalence was typically higher in Chinook salmon from Lake Michigan than from Lake Huron; prevalence was also generally higher in the Hinchenbrooke strain of coho salmon than in the Michigan-adapted strain. For most strains and stocks examined, intensity of R. salmoninarum infection was found to have declined. Although there were declines in the potential for shedding the bacteria for both male and female Chinook and coho salmon, overall shedding rates were generally low (<15%) except for Hinchenbrooke coho salmon strain

  11. Real-time PCR for quantification of viable Renibacterium salmoninarum in chum salmon Oncorhynchus keta.

    PubMed

    Suzuki, Kunio; Sakai, D K

    2007-03-13

    Quantification of msa gene mRNA of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was investigated using reverse transcription followed by real-time PCR assay on R. salmoninarum in culture, and in experimentally challenged chum salmon Oncorhynchus keta fry kidney tissues (total of 70 samples) after intraperitoneal (i.p.) injection and bath infection. Correlations of msa gene mRNA concentrations with culturable cell concentrations (as colony forming units [CFU]), determined by drop-plate culture method on selective kidney disease medium (SKDM) agar through a 12 wk incubation time, and msa gene DNA concentrations by real-time PCR assay were examined. Furthermore, ovarian fluid samples from wild chum salmon adults with no clinical signs of disease were collected from 8 rivers and from clinically infected kokanee 0. nerka and masu salmon O. masou that were reared in 1 and 2 hatcheries, respectively (total of 414 samples). All samples were examined by nested PCR assay. Then, positive samples were examined by real-time PCR assays for mRNA and DNA; mRNA was detectable at 8 log units (5.0 x 101 to 5.0 x 10(9) copies p11(-1)) with high correlation (R2 = 0.999). The mRNA concentration correlated with CFU in kidney tissue from fish infected by i.p. injection (R2 = 0.924), by bath infection (R2 = 0.502) and in culture (R2 = 0.888). R. salmoninarum was detected and quantified by real-time PCR assay for mRNA in ovarian fluid samples in both subclinically infected chum salmon adults and clinically infected kokanee and masu salmon adults; detection rates ranged from 0 to 44.4% and concentrations ranged from 9.7 x 10(2) to 5.6 x 10(5) copies pl(-1). These results indicate that real-time PCR assay for the mRNA is a rapid, sensitive and reliable method to detect and quantify the viability of R. salmoninarum in kidney and ovarian fluid samples of salmonid fishes with both clinical and subclinical infection of the pathogen. PMID:17465306

  12. Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum

    USGS Publications Warehouse

    Weins, G.; Chien, M.S.; Winton, J.R.; Kaatari, S.L.

    1999-01-01

    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 salrnonid leucocytes and rabbit erythrocytes. We define the location of epitopes on p57 recognized by agglutination-blocking monoclonal antibodies (MAbs) 4Cl1, 4H8 and 4D3, and demonstrate that the majority of secreted p57 is a nlonomer that retains salrnonid leucocyte agglutinat~ng 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.

  13. Renibacterium salmoninarum p57 antigenic variation is restricted in geographic distribution and correlated with genomic markers.

    PubMed

    Wiens, Gregory D; Dale, Ole Bendik

    2009-02-12

    The 57 kDa protein (p57) is an important diagnostic antigen that is implicated in the pathogenesis of salmonid bacterial kidney disease. Little is known about the nature and extent of antigenic variation in p57. Previously, we reported that p57 produced by Renibacterium salmoninarum Strain 684 contains a mutation that disrupts monoclonal antibody (MAb) 4C11 binding. In the present study, we examined MAb binding to a panel of 23 additional R. salmoninarum isolates obtained from diverse geographic locations to examine the prevalence of this variant and whether additional variability exists within other p57 epitopes. Six p57-specific MAbs (4C11, 4D3, 3H1, 4H8, 4D10 and 1A1) were used to probe dot and western blots to determine the relative expression, size and cellular association of p57. Full-length p57 was produced by all isolates, and for each isolate, the protein was associated with the bacterial cell surface. The epitopes recognized by 4 MAbs, 4D3, 4H8, 3H1 and 1A1, were conserved among all strains tested. The 4C11 epitope was absent in 5 of 8 strains originating from Norway, while the 4D10 epitope was partially disrupted in one isolate from British Columbia, Canada. The 5 Norwegian antigenic-variant strains appeared to be clonally related as they shared the following characteristics: one tandem repeat in the ETRA locus, a Sequovar-4 16-23S rRNA intervening DNA sequence, a larger XhoI fragment in the msa1 5' region, and absent msa3 gene. These results indicate that limited antigenic and genomic variation exists between strains and this variation appears geographically restricted in distribution. PMID:19326793

  14. Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum.

    PubMed

    Wiens, G D; Chien, M S; Winton, J R; Kaattari, S L

    1999-06-23

    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. PMID:10439902

  15. Modeling fish health to inform research and management: Renibacterium salmoninarum dynamics in Lake Michigan.

    PubMed

    Fenichel, Eli P; Tsao, Jean I; Jones, Michael L

    2009-04-01

    Little is known about the interaction between fish pathogens and managed freshwater fish populations. We develop a model of chinook salmon (Oncorhynchus tschawytscha)-Renibacterium salmoninarum (Rs) dynamics based on free-swimming Lake Michigan fish by synthesizing population and epidemiological theory. Using the model, we expose critical uncertainties about the system, identify opportunities for efficient and insightful data collection, and pose testable hypotheses. Our simulation results suggest that hatcheries potentially play an important role in Lake Michigan Rs dynamics, and understanding vertical transmission will be critical for quantifying this role. Our results also show that disease-mediated responses to chinook salmon density need to be considered when evaluating management actions. Related to this, a better understanding of the stock-recruitment relationship and natural mortality rates for wild-spawned fish and the impact of hatchery stocking on recruitment is required. Finally, to further develop models capable of assisting fishery management, fish health surveys ought to be integrated with stock assessment. This is the first time a host-pathogen modeling framework has been applied to managed, freshwater ecosystems, and we suggest that such an approach should be used more frequently to inform other emerging and chronic fish health issues. PMID:19425436

  16. Diagnosis of bacterial kidney disease by detection of Renibacterium salmoninarum by real-time PCR.

    PubMed

    Jansson, E; Lindberg, L; Säker, E; Aspán, A

    2008-10-01

    Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), is a serious threat to salmon in aquaculture as well as to wild populations. We have developed a real-time polymerase chain reaction (PCR) for detection of Rs in kidney samples. The PCR is based on detection of unique parts of the 16S rRNA gene of Rs and DNA equivalent to 1-10 Rs genomes was detected per reaction. No cross-reactivity with other fish pathogenic or related bacteria could be demonstrated. Analysis of individual kidney samples collected from BKD classified populations identified 39.9% of the fish as positive by real-time PCR compared with 28.0% by polyclonal enzyme-linked immunosorbent assay (ELISA). The real-time PCR assay was found to be well suited for complementary use with ELISA for diagnosis of BKD, with the ability to detect clinical as well as covert Rs infections. The infection level determined by the polyclonal ELISA and by real-time PCR was significantly correlated. PMID:18681904

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

    PubMed

    Rockey, D D; Turaga, P S; Wiens, G D; Cook, B A; Kaattari, S L

    1991-10-01

    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). PMID:1777853

  18. A phylogenetic comparison of the 16S rRNA sequence of the fish pathogen, Renibacterium salmoninarum, to gram-positive bacteria.

    PubMed

    Gutenberger, S K; Giovannoni, S J; Field, K G; Fryer, J L; Rohovec, J S

    1991-01-15

    The 16S rRNA of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonids, was sequenced by reverse transcriptase to produce a nearly complete sequence (97%) of 1475 nucleotides. Phylogenetic comparisons to seventeen genera and signature sequence analysis indicated that R. salmoninarum was a member of the high G + C Gram-positive eubacterial subdivision although the reported G + C value is only 53%. A phylogenetic tree details the relationship of R. salmoninarum to ten actinomycetes from diverse environments. PMID:1709893

  19. A simplified PCR-based method for the detection of Renibacterium salmoninarum utilizing preparations of rainbow trout (Oncorhynchus mykiss, Walbaum) lymphocytes.

    PubMed

    McIntosh, D; Meaden, P G; Austin, B

    1996-11-01

    A method for the detection of Renibacterium salmoninarum by PCR is described. A rapid, reliable procedure was developed for the extraction of DNA, which could be applied to infected kidney homogenates and head kidney lymphocyte preparations. The target for DNA amplification was a 376-bp region of the gene encoding the 57-kDa major surface antigen (MSA). The PCR was specific for R. salmoninarum and allowed the detection of 10 to 100 cells of the pathogen. Use of the PCR for the examination of experimentally infected rainbow trout showed it to be as reliable as plate culture methods for the detection of R. salmoninarum in infected kidneys. PMID:8899978

  20. A simplified PCR-based method for the detection of Renibacterium salmoninarum utilizing preparations of rainbow trout (Oncorhynchus mykiss, Walbaum) lymphocytes.

    PubMed Central

    McIntosh, D; Meaden, P G; Austin, B

    1996-01-01

    A method for the detection of Renibacterium salmoninarum by PCR is described. A rapid, reliable procedure was developed for the extraction of DNA, which could be applied to infected kidney homogenates and head kidney lymphocyte preparations. The target for DNA amplification was a 376-bp region of the gene encoding the 57-kDa major surface antigen (MSA). The PCR was specific for R. salmoninarum and allowed the detection of 10 to 100 cells of the pathogen. Use of the PCR for the examination of experimentally infected rainbow trout showed it to be as reliable as plate culture methods for the detection of R. salmoninarum in infected kidneys. PMID:8899978

  1. Different prevalences of Renibacterium salmoninarum detected by ELISA in Alaskan chinook salmon Oncorhynchus tshawytscha spawned from freshwater and seawater.

    PubMed

    Meyers, T R; Thrower, F; Short, S; Lipson, K; Joyce, J; Farrington, C; Doherty, S

    1999-01-29

    Soluble antigen of Renibacterium salmoninarum (Rs) was detected by a polyclonal enzyme-linked immunosorbent assay (ELISA) at significantly higher prevalences in adult chinook salmon Oncorhynchus tshawytscha that matured in freshwater than in the same cohort of fish spawned after maturation in seawater. The cumulative results were consistent during 4 yr of comparison at the Little Port Walter Hatchery on Baranof Island, Alaska, USA. Possible causes for this difference are discussed. Maturation of chinook salmon broodstock in seawater has become a practical strategy at this hatchery to reduce the prevalence of Rs-positive parent fish and the numbers of culled eggs. PMID:10092972

  2. Renibacterium salmoninarum in spring-summer chinook salmon smolts at dams on the Columbia and Snake Rivers

    USGS Publications Warehouse

    Elliott, D.G.; Pascho, R.J.; Jackson, L.M.; Matthews, G.M.; Harmon, J.R.

    1997-01-01

    We evaluated Renibacterium salmoninarum infection in smolts of hatchery and wild spring-summer chinook salmon Oncorhynchus tshawytscha sampled during most of the out-migration at Little Goose (1988) and Lower Granite dams (1988-1991) on the Snake River and at Priest Rapids and McNary dams on the Columbia River (1988-1990). We sampled 860-2,178 fish per dam each year. Homogenates of kidney-spleen tissue from all fish were tested for the presence of R. salmoninarum antigens by the enzyme-linked immunosorbent assay (ELISA), and homogenates from 10% of the fish were examined by the fluorescent antibody technique (FAT). Although only 1-11% of fish sampled at a given dam during any 1 year exhibited lesions characteristic of bacterial kidney disease, 86-100% of the fish tested positive for R. salmoninarum antigen by ELISA, whereas 4-17% of the fish tested positive by the FAT. During most years, a majority (68-87%) of fish testing positive by the ELISA had low R. salmoninarum antigen levels, but in 1989, 53% of positive fish from Lower Granite Dam and 52% from McNary Dam showed medium-to-high antigen levels. For most years, the highest mean antigen levels were measured in fish sampled after 75% of the total out-migrants had passed a given dam. When the largest numbers of fish were being collected for bypass or downriver transportation, mean antigen levels were relatively low.

  3. Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

    USGS Publications Warehouse

    Elliott, D.G.; Applegate, L.J.; Murray, A.L.; Purcell, M.K.; McKibben, C.L.

    2013-01-01

    No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

  4. Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture.

    PubMed

    Elliott, D G; Applegate, L J; Murray, A L; Purcell, M K; McKibben, C L

    2013-09-01

    No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test. PMID:23346868

  5. Loop-mediated isothermal amplification (LAMP) for rapid detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease.

    PubMed

    Saleh, Mona; Soliman, Hatem; El-Matbouli, Mansour

    2008-08-27

    A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Renibacterium salmoninarum in 1 h without thermal cycling. A fragment of R. salmoninarum p57 gene was amplified at 63 degrees C in the presence of Bst polymerase and a specially designed primer mixture. The specificity of the BKD-LAMP assay was demonstrated by the absence of any cross reaction with other bacterial strains, followed by restriction digestion of the amplified products. Detections of BKD-LAMP amplicons by visual inspection, agrose gel electrophoresis, and real-time monitoring using a turbidimeter were equivalently sensitive. The BKD-LAMP assay has the sensitivity of the nested PCR method, and 10 times the sensitivity of one-round PCR assay. The lower detection limit of BKD-LAMP and nested PCR is 1 pg genomic R. salmoninarum DNA, compared to 10 pg genomic R. salmoninarum DNA for one-round PCR assay. In comparison to other available diagnostic methods, the BKD-LAMP assay is rapid, simple, sensitive, specific, and cost effective with a high potential for field application. PMID:18924379

  6. Elevated temperature treatment as a novel method for decreasing p57 on the cell surface of Renibacterium salmoninarum.

    PubMed

    Piganelli, J D; Wiens, G D; Kaattari, S L

    1999-04-15

    Renibacterium salmoninarum is a Gram-positive diplo-bacillus and the causative agent of bacterial kidney disease, a prevalent disease of salmonid fish. Virulent isolates of R. salmoninarum have a hydrophobic cell surface and express the 57-58 kDa protein (p57). Here we have investigated parameters which effect cell hydrophobicity and p57 degradation. Incubation of R. salmoninarum cells at 37 degrees C for > 4 h decreased cell surface hydrophobicity as measured by the salt aggregation assay, and decreased the amount of cell associated p57. Incubation of cells at lower temperatures (22, 17, 4 or -20 degrees C) for up to 16 h did not reduce hydrophobicity or the amount of cell associated p57. Both the loss of cell surface hydrophobicity and the degradation of p57 were inhibited by pre-incubation with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Cell surface hydrophobicity was specifically reconstituted by incubation with extracellular protein (ECP) concentrated from culture supernatant and was correlated with the reassociation of p57 onto the bacterial cell surface as determined by western blot and total protein stain analyses. The ability of p57 to reassociate suggests that the bacterial cell surface is not irreversibly modified by the 37 degrees C treatment and that p57 contributes to the hydrophobic nature of R. salmoninarum. In summary, we describe parameters effecting the removal of the p57 virulence factor and suggest the utility of this modification for generating a whole cell vaccine against bacterial kidney disease. PMID:10349550

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

    PubMed

    Sandell, Todd A; Jacobson, Kym C

    2011-01-21

    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. PMID:21381519

  8. Host responses to Renibacterium salmoninarum and specific components of the pathogen reveal the mechanisms of immune suppression and activation.

    PubMed

    Grayson, T Hilton; Cooper, Lynne F; Wrathmell, Annette B; Roper, Janet; Evenden, Andrew J; Gilpin, Martyn L

    2002-06-01

    During infection, Renibacterium salmoninarum survives within the pronephric macrophages of salmonid fish. Therefore, to study the initial phases of the interaction we infected macrophages with live bacteria and analysed the responses of host and pathogen. It was found that the expression of msa encoding the p57 antigen of R. salmoninarum, was constitutive, while the expression of hly and rsh, encoding haemolysins, and lysB and grp was reduced after infection. Macrophages showed a rapid inflammatory response in which the expression of interleukin-1beta (IL-1beta), major histocompatibility complex class II (MHC II), inducible cyclo-oxygenase (Cox-2), and inducible nitric oxide synthase (iNOS) was enhanced, but tumour necrosis factor-alpha (TNF-alpha) expression was greatly reduced initially and then increased. After 5 days, except for TNF-alpha and MHC II, expression returned to levels approaching those of uninfected macrophages. We propose that R. salmoninarum survives initial contact with macrophages by avoiding and/or interfering with TNF-alpha-dependent killing pathways. The effects of specific R. salmoninarum components were studied in vivo by injecting fish with DNA vaccine constructs expressing msa, hly, rsh, lysB, or grp. We found that msa reduced the expression of IL-1beta, Cox-2, and MHC II but stimulated TNF-alpha while hly, rsh and grp stimulated MHC II but down-regulated TNF-alpha. Constructs expressing hly or lysB stimulated iNOS expression and additionally, lysB stimulated TNF-alpha. The results show how p57 suppresses the host immune system and suggest that the immune mechanisms for the containment of R. salmoninarum infections rely on MHC II- and TNF-alpha-dependent pathways. Moreover, prolonged stimulation of TNF-alpha may contribute to the chronic inflammatory pathology of bacterial kidney disease. PMID:12047757

  9. Host responses to Renibacterium salmoninarum and specific components of the pathogen reveal the mechanisms of immune suppression and activation

    PubMed Central

    Grayson, T Hilton; Cooper, Lynne F; Wrathmell, Annette B; Roper, Janet; Evenden, Andrew J; Gilpin, Martyn L

    2002-01-01

    During infection, Renibacterium salmoninarum survives within the pronephric macrophages of salmonid fish. Therefore, to study the initial phases of the interaction we infected macrophages with live bacteria and analysed the responses of host and pathogen. It was found that the expression of msa encoding the p57 antigen of R. salmoninarum, was constitutive, while the expression of hly and rsh, encoding haemolysins, and lysB and grp was reduced after infection. Macrophages showed a rapid inflammatory response in which the expression of interleukin-1β (IL-1β), major histocompatibility complex class II (MHC II), inducible cyclo-oxygenase (Cox-2), and inducible nitric oxide synthase (iNOS) was enhanced, but tumour necrosis factor-α (TNF-α) expression was greatly reduced initially and then increased. After 5 days, except for TNF-α and MHC II, expression returned to levels approaching those of uninfected macrophages. We propose that R. salmoninarum survives initial contact with macrophages by avoiding and/or interfering with TNF-α-dependent killing pathways. The effects of specific R. salmoninarum components were studied in vivo by injecting fish with DNA vaccine constructs expressing msa, hly, rsh, lysB, or grp. We found that msa reduced the expression of IL-1β, Cox-2, and MHC II but stimulated TNF-α while hly, rsh and grp stimulated MHC II but down-regulated TNF-α. Constructs expressing hly or lysB stimulated iNOS expression and additionally, lysB stimulated TNF-α. The results show how p57 suppresses the host immune system and suggest that the immune mechanisms for the containment of R. salmoninarum infections rely on MHC II- and TNF-α-dependent pathways. Moreover, prolonged stimulation of TNF-α may contribute to the chronic inflammatory pathology of bacterial kidney disease. PMID:12047757

  10. Immunosuppression in progeny of chinook salmon infected with Renibacterium salmoninarum: re-analysis of a brood stock segregation experiment.

    PubMed

    Hamel, Owen S

    2005-06-14

    Female spawner infection level and temperature variation through rearing are sufficient to explain in-hatchery mortality rates and infection levels and smolt to adult return ratios (SARs) of progeny of Renibacterium salmoninarum infected spring chinook salmon. Data from published reports and manuscripts regarding a 1988 brood stock segregation experiment that held progeny of highly infected female spring chinook salmon spawners separate from progeny of other spawners during 16 mo of hatchery rearing are analyzed to test the hypothesis that immunosuppression could account for differences in survival and infection levels between the 2 segregates. Immunosuppression, caused by the presence of the p57 antigen of R. salmoninarum in sufficient concentration within the salmon egg before spawning, can account for differences in infection levels, mortality rates, and SARs for each hatchery raceway in that study. This immunosuppression may be characterized by immunotolerance, or might only affect cell mediated immunity, which appears the most effective defense mechanism against R. salmoninarum infection, as antibody production can result in tissue damaging antibody-antigen complexes. Low-temperature mediated immunosuppression can account for the nearly identical trajectories of infection and mortality between the 2 segregates during the first 8 mo of hatchery rearing. There is no evidence of widespread vertical infection from spawner to progeny, nor is there evidence that brood stock segregation reduces overall mortality. Rather, the suppression of cell-mediated immune mechanisms may condemn progeny of highly infected female spawners to an almost certain eventual premature demise. PMID:16042041

  11. Effects of Renibacterium salmoninarum on olfactory organs of Chinook salmon (Oncorhynchus tshawytscha) marked with coded wire tags

    USGS Publications Warehouse

    Elliott, Diane G.; Conway, Carla M.

    2014-01-01

    Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum can cause significant morbidity and mortality in Chinook salmon (Oncorhynchus tshawytscha), particularly in Chinook salmon of the stream (spring) life history type, which migrate to sea as yearlings rather than subyearlings. R. salmoninarum can be transmitted vertically from the female parent to the progeny in association with the egg, as well as horizontally from fish to fish. This study was conducted as part of a research project to investigate whether the prevalence and intensity of R. salmoninarum infections in adult spring Chinook salmon could affect the survival and pathogen prevalence and intensity in their progeny (Pascho et al., 1991, 1993; Elliott et al., 1995). Fish from two brood years (1988 and 1989) were reared at Dworshak National Fish Hatchery (Idaho, USA) for about 1-1/2 years, released as yearling smolts, and allowed to migrate to the Pacific Ocean for maturation. The majority of progeny fish were marked with coded wire tags (CWTs) about 4 months before they were released from the hatchery so that adult returns could be monitored. The CWTs were implanted in the snouts of the fish by an experienced team of fish markers using automated wire-tagging machines. The intended placement site was the cartilage, skeletal muscle or loose connective tissue of the snout.

  12. Mapping of neutralizing epitopes on Renibacterium salmoninarum p57 by use of transposon mutagenesis and synthetic peptides.

    PubMed

    Wiens, Gregory D; Owen, Jennifer

    2005-06-01

    Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish. PMID:15932983

  13. Genome sequence of the fish pathogen Renibacterium salmoninarum suggests reductive evolution away from an environmental Arthrobacter ancestor.

    PubMed

    Wiens, Gregory D; Rockey, Daniel D; Wu, Zaining; Chang, Jean; Levy, Ruth; Crane, Samuel; Chen, Donald S; Capri, Gina R; Burnett, Jeffrey R; Sudheesh, Ponnerassery S; Schipma, Matthew J; Burd, Henry; Bhattacharyya, Anamitra; Rhodes, Linda D; Kaul, Rajinder; Strom, Mark S

    2008-11-01

    Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding beta-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids. PMID:18723615

  14. Genome Sequence of the Fish Pathogen Renibacterium salmoninarum Suggests Reductive Evolution away from an Environmental Arthrobacter Ancestor▿ †

    PubMed Central

    Wiens, Gregory D.; Rockey, Daniel D.; Wu, Zaining; Chang, Jean; Levy, Ruth; Crane, Samuel; Chen, Donald S.; Capri, Gina R.; Burnett, Jeffrey R.; Sudheesh, Ponnerassery S.; Schipma, Matthew J.; Burd, Henry; Bhattacharyya, Anamitra; Rhodes, Linda D.; Kaul, Rajinder; Strom, Mark S.

    2008-01-01

    Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding β-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids. PMID:18723615

  15. Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

    PubMed Central

    Wiens, Gregory D.; Owen, Jennifer

    2005-01-01

    Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish. PMID:15932983

  16. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

    USGS Publications Warehouse

    Pascho, R.J.; Chase, D.; McKibben, C.L.

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 3 109 cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 3 104 cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

  17. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmonid ovarian fluid.

    PubMed

    Pascho, R J; Chase, D; McKibben, C L

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 x 10(9) cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 x 10(4) cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods. PMID:9526862

  18. Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

    PubMed Central

    Magnússon, H B; Fridjónsson, O H; Andrésson, O S; Benediktsdóttir, E; Gudmundsdóttir, S; Andrésdóttir, V

    1994-01-01

    An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine kidney tissue. Images PMID:7529017

  19. Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

    PubMed

    Magnússon, H B; Fridjónsson, O H; Andrésson, O S; Benediktsdóttir, E; Gudmundsdóttir, S; Andrésdóttir, V

    1994-12-01

    An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine kidney tissue. PMID:7529017

  20. Enzyme-linked immunosorbent assay for a soluble antigen of Renibacterium salmoninarum, the causative agent for salmonid bacterial kidney disease

    USGS Publications Warehouse

    Pascho, R.J.; Mulcahy, D.

    1987-01-01

    A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of a soluble fraction of Renibacterium salmoninarum was developed from components extracted from the supernatant of an R. salmoninarum broth culture. The Costar® Serocluster™ EIA microplate gave the highest absorbance and signal-to-noise ratios among seven types tested. Including Tween 80 in the wash buffer resulted in higher absorbances than Tween 20 when antigen was present. Background absorbance did not increase when Tween 80 was added to the wash buffer, but did when Tween 80 replaced Tween 20 in antigen and conjugate diluents. Adsorption of coating antibody peaked within 4 h at 37 °C and 16 h at 4 °C. Antigen attachment to antibody-coated microplate wells depended more on incubation temperature than duration; we adopted a 3-h incubation at 25 °C. Conjugate incubation for longer than 1 h at 37 °C or 3 h at 25 °C resulted in unacceptable background levels. No cross-reactions resulted from heat-extracted antigens of 10 other species of bacteria. The optimized ELISA is a 6-h test that enables detection of levels of soluble antigen as low as 2–20 ng.

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

    PubMed

    Rhodes, L D; Grayson, T H; Alexander, S M; Strom, M S

    2000-02-22

    Renibacterium salmoninarum, a slowly growing, Gram-positive bacterium, is responsible for bacterial kidney disease in salmonid fishes world-wide. To date, no mobile genetic elements have been reported for this pathogen. Here, we describe the first insertion sequence (IS) identified from R. salmoninarum. This element, IS994, has a significant predicted amino acid sequence homology (64.8 and 71.9%) to the two open reading frames encoding the transposase of IS6110 of Mycobacterium tuberculosis. Protein parsimony and protein distance matrix analyses show that IS994 is a member of group IS51 of the IS3 family. From a conservative estimate, there are at least 17 chromosomal insertions of IS994 or closely related elements. Sequence analysis of seven of these loci reveals single nucleotide polymorphisms throughout the element (including the terminal inverted repeats), a 15bp insertion in three of the seven loci, and an absence of flanking direct repeats or conserved insertion site. Restriction fragment length polymorphism analysis of XbaI-digested chromosomal DNA shows variations among European and North American isolates, indicating that IS994 may be a useful molecular marker for epizootiological studies. PMID:10689192

  2. Swimming endurance of bull trout, lake trout, arctic char, and rainbow trout following challenge with Renibacterium salmoninarum

    USGS Publications Warehouse

    Jones, D.T.; Moffitt, C.M.

    2004-01-01

    We tested the swimming endurance of juvenile bull trout Salvelinus confluentus, lake trout S. namaycush, Arctic char S. alpinus, and rainbow trout Oncorhynchus mykiss at 9??C and 15??C to determine whether sublethal infection from a moderate challenge of Renibacterium salmoninarum administered months before testing affected the length of time fish could maintain a swimming speed of 5-6 body lengths per second in an experimental flume. Rainbow trout and Arctic char swam longer in trials than did bull trout or lake trout, regardless of challenge treatment. When we tested fish 14-23 weeks postchallenge, we found no measurable effect of R. salmoninarum on the swimming endurance of the study species except for bull trout, which showed a mixed response. We conducted additional trials with bull trout 5-8 weeks postchallenge to determine whether increasing the challenge dose would affect swimming endurance and hematocrit. In those tests, bull trout with clinical signs of disease and those exposed to the highest challenge doses had significantly reduced swimming endurance compared with unchallenged control fish. Fish hematocrit levels measured at the end of all swimming endurance tests varied among species and between test temperatures, and patterns were not always consistent between challenged and control fish.

  3. Membrane filtration-fluorescent antibody staining procedure for detecting and quantifying Renibacterium salmoninarum in coelomic fluid of Chinook salmon Oncorhynchus tshawytscha)

    USGS Publications Warehouse

    Elliott, D.G.; Barila, T.Y.

    1988-01-01

    e developed a rapid method for detecting and quantifying the pathogen Renibacterium salmoninarum in coelomic fluid of spring chinook salmon (Oncorhynchus tshawytscha) by concentrating the bacteria on 0.2-μm polycarbonate filters and staining them with specific fluorescein-labeled antibody. Centrifugation of samples and resuspension of the sedimented material in phosphate-buffered saline containing Triton X-100 increased the ease of filtration. Background fluorescence was reduced by counterstaining filters with Eriochrome black T. Postfiltration staining, rinsing, and counterstaining were done in the syringe-mounted filter holders, reducing handling of the filters and possible loss of bacteria. The number of bacteria detected by the filtration – fluorescent antibody technique in a broth culture of R. salmoninarum ranged from 6.7 × 107to7.6 × 107/mL and was slightly higher than that determined by plate count (9.6 × 106/mL). Increasing the sample dilution or decreasing the number of microscope fields examined generally increased the variability of filter counts of R. salmoninarum. Using the filtration – fluorescent antibody technique, we detected the bacterium in the coelomic fluid of 85% of spawning female spring chinook salmon sampled from a hatchery population. Membrane Filtration – Fluorescent Antibody Staining Procedure for Detecting and Quantifying Renibacterium salmoninarum in Coelomic Fluid of Chinook Salmon (oncorhynchus tshawytscha) (PDF Download Available). 

  4. An investigation into the prevalence of Renibacterium salmoninarum in farmed rainbow trout, Oncorhynchus mykiss (Walbaum), and wild fish populations in selected river catchments in England and Wales between 1998 and 2000.

    PubMed

    Chambers, E; Gardiner, R; Peeler, E J

    2008-02-01

    A cross-sectional survey of Renibacterium salmoninarum infection in farmed rainbow trout (RBT) and wild fish populations was carried out in 10 farms and six river catchments, respectively, in England and Wales. The majority of the wild fish were sampled in 1998 and the farmed fish in 2000. Grayling, Thymallus thymallus, and brown trout, Salmo trutta, were the main wild species sampled. Two fish, one grayling and one salmon, Salmo salar, were R. salmoninarum culture-positive, compared with 40 confirmed polymerase chain reaction-positive wild fish. The highest prevalence of R. salmoninarum infection was found in grayling in rivers with RBT farms with a history of R. salmoninarum infection. One hundred and fifty fish were sampled from each RBT farm, but none of the fish was found to be R. salmoninarum-positive. Evidence was found, for the first time, for the presence of R. salmoninarum in an eel, Anguilla anguilla. PMID:18234016

  5. A sensitive loop-mediated isothermal amplification (LAMP) method for detection of Renibacterium salmoninarum, causative agent of bacterial kidney disease in salmonids.

    PubMed

    Gahlawat, S K; Ellis, A E; Collet, B

    2009-06-01

    Loop-mediated isothermal amplification (LAMP) is a novel technique for nucleic acid amplification with high specificity, sensitivity and rapidity and does not require expensive equipment or reagents. In the present study, we developed and evaluated a LAMP method for the rapid detection of Renibacterium salmoninarum causing the bacterial kidney disease in salmonids. This method was more sensitive than quantitative real-time polymerase chain reaction (qPCR). Using DNA template extracted from cultured R. salmoninarum, the LAMP method gave an amplification signal from template diluted to 10(-8) while the limit of detection of qPCR was10(-7). The LAMP method was also highly specific and did not amplify DNA purified from five other Gram-positive and -negative bacterial fish pathogens. The method also worked well using extracts of macrophages infected with R. salmoninarum and kidney material from rainbow trout, which were positive for R. salmoninarum by qPCR and crude R. salmoninarum culture. There was some evidence for inhibitors of the LAMP reaction in the kidney samples, which was overcome by diluting the sample. PMID:19538642

  6. Mortality and kidney histopathology of Chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains

    USGS Publications Warehouse

    O'Farrell, Caroline L.; Elliott, Diane G.; Landolt, Marsha L.

    2001-01-01

    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.

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

    USGS Publications Warehouse

    O'Farrell, C. L.; Elliott, D.G.; Landolt, M.L.

    2000-01-01

    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 ?? 103 or 1 ?? 106 bacteria fish-1, or by a 24 h immersion in 1 ?? 105 or 1 ?? 107 bacteria ml-1. For 22 wk fish were held in 12??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.

  8. Prevalence and analysis of Renibacterium salmoninarum infection among juvenile Chinook salmon Oncorhynchus tshawytscha in North Puget Sound.

    PubMed

    Rhodes, Linda D; Durkin, Colleen; Nance, Shelly L; Rice, Casimir A

    2006-08-30

    Renibacterium salmoninarum causes bacterial kidney disease (BKD), a chronic and sometimes fatal disease of salmon and trout that could lower fitness in populations with high prevalences of infection. Prevalence of R. salmoninarum infection among juvenile Chinook salmon Oncorhynchus tshawytscha inhabiting neritic marine habitats in North Puget Sound, Washington, USA, was assessed in 2002 and 2003. Fish were collected by monthly surface trawl at 32 sites within 4 bays, and kidney infections were detected by a quantitative fluorescent antibody technique (qFAT). The sensitivity of the qFAT was within an order of magnitude of the quantitative real-time PCR (qPCR) sensitivity. Prevalence of infection was classified by fish origin (marked/hatchery vs. unmarked/likely natural spawn), month of capture, capture location and stock origin. The highest percentages of infected fish (63.5 to 63.8%) and the greatest infection severity were observed for fish collected in Bellingham Bay. The lowest percentages were found in Skagit Bay (11.4 to 13.5%); however, there was no difference in prevalence between marked and unmarked fish among the capture locations. The optimal logistic regression model of infection probabilities identified the capture location of Bellingham Bay as the strongest effect, and analysis of coded wire tagged (CWT) fish revealed that prevalence of infection was associated with the capture location and not with the originating stock. These results suggest that infections can occur during the early marine life stages of Chinook salmon that may be due to common reservoirs of infection or horizontal transmission among fish stocks. PMID:17058599

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

    PubMed

    O'Farrell, C L; Elliott, D G; Landolt, M L

    2000-12-21

    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. PMID:11206735

  10. Levels of Renibacterium salmoninarum antigens in resident and anadromous salmonids in the River Ellidaár system in Iceland.

    PubMed

    Kristmundsson, Á; Árnason, F; Gudmundsdóttir, S; Antonsson, T

    2016-06-01

    In relation to stock enhancement programmes, wild salmon broodfish have been routinely screened for the presence of Renibacterium salmoninarum antigens (Rs-Ag) for decades. A sudden increase in the prevalence of Rs-Ag experienced caused extensive problems to this industry as eggs from positive fish are discarded. The prevalence and level of Rs-Ag were examined in resident and anadromous salmonids in the River Ellidaár system and the progress of Rs-Ag in a cohort of salmon followed. Both prevalence and Rs-Ag levels were high in resident salmonids and emigrating salmon smolts in the river system. When the smolts re-entered their home river as adults the following summer, they were almost free of Rs-Ag, but the longer they stayed in the river, the more Rs-Ag they acquired; the majority being positive at spawning. This study demonstrates a high level of Rs-Ag in salmonids in the River Ellidaár system which significantly reduces in the salmon during its seawater phase. Accordingly, it seems ideal to sample salmon broodfish as soon as possible after ascending the river and subsequently transfer to Rs-free environment for storage until stripping, which could result in lower Rs-prevalence and minimize the problems that stock enhancement programmes have faced due to Rs-positive wild broodfish. PMID:26275672

  11. Vulnerability to predation and physiological stress responses in juvenile chinook salmon (Oncorhynchus tshawytscha) experimentally infected with Renibacterium salmoninarum

    USGS Publications Warehouse

    Mesa, M.G.; Poe, T.P.; Maule, A.G.; Schreck, C.B.

    1998-01-01

    We experimentally infected juvenile chinook salmon (Oncorhynchus tshawytscha) with Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney disease (BKD), to examine the vulnerability to predation of fish with differing levels of Rs infection and assess physiological change during progression of the disease. Immersion challenges conducted during 1992 and 1994 produced fish with either a low to moderate (1992) or high (1994) infection level of Rs during the 14-week postchallenge rearing period. When equal numbers of treatment and unchallenged control fish were subjected to predation by either northern squaw fish (Ptychocheilus oregonensis) or smallmouth bass (Micropterus dolomieui), Rs-challenged fish were eaten in significantly greater numbers than controls by nearly two to one. In 1994, we also sampled fish every 2 weeks after the challenge to determine some stressful effects of Rs infection. During disease progression in fish, plasma cortisol and lactate increased significantly whereas glucose decreased significantly. Our results indicate the role that BKD may play in predator-prey interactions, thus ascribing some ecological significance to this disease beyond that of direct pathogen-related mortality. In addition, the physiological changes observed in our fish during the chronic progression of BKD indicate that this disease is stressful, particularly during the later stages.

  12. Incidence of Renibacterium salmoninarum infections in juvenile hatchery spring chinook salmon in the Columbia and Snake Rivers

    USGS Publications Warehouse

    Maule, A.G.; Rondorf, D.W.; Beeman, J.W.; Haner, P.V.

    1996-01-01

    From 1988 through 1992, we assessed the prevalence (frequency of occurrence) and severity (degree of infection) of Renibacterium salmoninarum (RS) among fish in marked groups of Columbia River basin and Snake River basin hatchery spring chinook salmon Oncorhynchus tshawytscha before release and during their seaward migration. During the study, prevalence of RS infection decreased (from >90% to <65%) in six of the eight hatchery groups. We attributed this decrease to changes in hatchery practices that reduced vertical and horizontal transmission. Fish from Snake River hatcheries had a higher prevalence of infection when sampled at dams (mean >90%) than in the hatchery (mean <70%), but there were no differences in similar comparisons of Columbia River fish. Although prevalence and severity of RS infection were not correlated in the groups studied, it appears that fish from the Snake River were more severely infected than those from the Columbia River. Some groups of Snake River fish had higher severity of infection at dams than in the hatchery, but infection in fish from Columbia River hatcheries did not change. These differences between Snake River and Columbia River fish might have resulted from differences in river conditions and the distances from hatcheries to dams.

  13. The gills are an important site of iNOS expression in rainbow trout Oncorhynchus mykiss after challenge with the gram-positive pathogen Renibacterium salmoninarum.

    PubMed

    Campos-Perez, J J; Ward, M; Grabowski, P S; Ellis, A E; Secombes, C J

    2000-01-01

    Following injection challenge of rainbow trout with the Gram-positive pathogen Renibacterium salmoninarum, serum nitrate levels increased indicative of NO production. The timing and amount of nitrate produced varied with the virulence of the bacterial strain used, with the highest levels seen in fish challenged with the most virulent (autoaggregating) strain. Immunization with a killed R. salmoninarum preparation in Freund's incomplete adjuvant significantly increased nitrate levels after challenge. Inducible nitric oxide synthase (iNOS) transcript expression was detectable in rainbow trout tissues after injection challenge with R. salmoninarum, and its induction in the gills was both quick (between 3 and 6 hr) and relatively prolonged (lasting several days). iNOS expression in the kidney was also seen at a later stage (24 hr) but appeared to switch off relatively rapidly. Bath challenge with R. salmoninarum also induced iNOS expression in gill, and a variable expression in the gut and kidney also occurred. These results highlight the importance of the gills, not only as a point of entry of pathogens but also as a tissue capable of mounting an immune response. PMID:10651954

  14. The gills are an important site of iNOS expression in rainbow trout Oncorhynchus mykiss after challenge with the Gram‐positive pathogen Renibacterium salmoninarum

    PubMed Central

    Campos‐perez, J J; Ward, M; Grabowski, P S; Ellis, A E; Secombes, C J

    2000-01-01

    Following injection challenge of rainbow trout with the Gram‐positive pathogen Renibacterium salmoninarum, serum nitrate levels increased indicative of NO production. The timing and amount of nitrate produced varied with the virulence of the bacterial strain used, with the highest levels seen in fish challenged with the most virulent (autoaggregating) strain. Immunization with a killed R. salmoninarum preparation in Freund’s incomplete adjuvant significantly increased nitrate levels after challenge. Inducible nitric oxide synthase (iNOS) transcript expression was detectable in rainbow trout tissues after injection challenge with R. salmoninarum, and its induction in the gills was both quick (between 3 and 6 hr) and relatively prolonged (lasting several days). iNOS expression in the kidney was also seen at a later stage (24 hr) but appeared to switch off relatively rapidly. Bath challenge with R. salmoninarum also induced iNOS expression in gill, and a variable expression in the gut and kidney also occurred. These results highlight the importance of the gills, not only as a point of entry of pathogens but also as a tissue capable of mounting an immune response. PMID:10651954

  15. Comparison of two fluorescent antibody techniques (FATS) for detection and quantification of Renibacterium salmoninarum in coelomic fluid of spawning chinook salmon Oncorhynchus tshawytscha

    USGS Publications Warehouse

    Elliott, D.G.; McKibben, C.L.

    1997-01-01

    Two versions of the fluorescent antibody technique (FAT) were compared for detection and quantification of Renibacterium salmoninarum in coelomic fluid samples from naturally infected spawning chinook salmon Oncorhynchus tshawytscha. For the membrane filtration-FAT (MF-FAT), trypsin-treated samples were passed through 0.2 ??m polycarbonate filters to concentrate bacteria for direct enumeration by immunofluorescence microscopy. For the smear-FAT (S-FAT), samples were centrifuged at 8800 x g for 10 min and the pelleted material was smeared on slides for immunofluorescence staining Detected prevalences of Renibacterium salmoninarum were 1.8 to 3.4 times higher by the MF-FAT than by the S-FAT: differences were significant at p ??? 0.0002. The S-FAT consistently detected R. salmoninarum only in samples with calculated bacterial concentrations ??? 2.4 x 103 cells ml-1 by MF-FAT testing. Increasing the area examined on a filter or slide from 50 to 100 microscope fields at 1000x magnification resulted in the detection of a maximum of 4% additional positive samples by the MF-FAT and 7% additional positive samples by the S-FAT. In individual samples for which bacterial counts were obtained by both the MF-FAT and the S-FAT, the counts averaged from 47 times (??30 SD) to 175 times (??165 SD) higher by the MF-FAT. Centrifugation of samples at 10000 x g for 10 min resulted in a 4-fold increase in mean bacterial counts by the S-FAT compared with a 10-min centrifugation at 2000 x g, but the highest calculated bacterial concentration obtained by S-FAT testing was more than 6-fold lower than that obtained for the same sample by MF-FAT testing. Because of its greater sensitivity, the MF-FAT is preferable to the S-FAT for use in critical situations requiring the detection of low numbers of R. salmoninarum.

  16. Influence of infection with Renibacterium salmoninarum on susceptibility of juvenile spring chinook salmon to gas bubble trauma

    USGS Publications Warehouse

    Weiland, L.K.; Mesa, M.G.; Maule, A.G.

    1999-01-01

    During experiments in our laboratory to assess the progression and severity of gas bubble trauma (GBT) in juvenile spring chinook salmon Oncorhynchus tshawytscha, we had the opportunity to assess the influence of Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney disease, on the susceptibility of salmon to GBT. We exposed fish with an established infection of Rs to 120% total dissolved gas (TDG) for 96 h and monitored severity of GBT signs in the fins and gills, Rs infection level in kidneys by using an enzyme-linked immunosorbent assay (ELISA), and mortality. Mortality occurred rapidly after exposure to 120% TDG, with a LT20 (time necessary to kill 20% of the population) of about 37 h, which is at a minimum about 16% earlier than other bioassays we have conducted using fish that had no apparent signs of disease. Fish that died early (from 31 to 36 h and from 49 to 52 h) had significantly higher infection levels (mean ?? SE ELISA absorbance = 1.532 ?? 0.108) than fish that survived for 96h (mean ?? SE ELISA absorbance = 0.828 ?? 0.137). Fish that died early also had a significantly greater number of gill filaments occluded with bubbles than those that survived 96 h. Conversely, fish that survived for 96 h had a significantly higher median fin severity ranking than those that died early. Our results indicate that fish with moderate to high levels of Rs infection are more vulnerable to the effects of dissolved gas supersaturation (DGS) and die sooner than fish with lower levels of Rs infection. However, there is a substantial amount of individual variation in susceptibility to the apparent cumulative effects of DGS and Rs infection. Collectively, our findings have important implications to programs designed to monitor the prevalence and severity of GBT in juvenile salmonids in areas like the Columbia River basin and perhaps elsewhere.

  17. Interaction of infection with Renibacterium salmoninarum and physical stress in juvenile chinook salmon: Physiological responses, disease progression, and mortality

    USGS Publications Warehouse

    Mesa, M.G.; Maule, A.G.; Schreck, C.B.

    2000-01-01

    We experimentally infected juvenile spring chinook salmon Oncorhynchus tshawytscha with Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney disease (BKD), in order to compare the physiological responses of Rs-infected and Rs-noninfected fish to a series of multiple, acute stressors and to determine whether exposure to these stressors worsens the infection and leads to increased mortality. After subjecting groups of fish to a waterborne challenge of Rs, we sampled them biweekly to monitor infection levels, mortality, and some stress-related physiological changes. As infections worsened, fish developed decreased hematocrits and blood glucose levels and increased levels of cortisol and lactate, indicating that BKD is stressful, particularly during the later stages. Eight weeks after the challenge, when fish had moderate to high infection levels, we subjected them, along with unchallenged control fish, to three 60-s bouts of hypoxia, struggling, and mild agitation that were separated by 48-72 h. Our results indicate that the imposition of these stressors on Rs-infected fish did not lead to higher infection levels or increased mortality when compared with diseased fish that did not receive the stressors. Furthermore, the kinetics of plasma cortisol, glucose, and lactate over a 24-h period following each application of the stressor were similar between fish with moderate to high Rs infections and those that had low or no detectable infection. Some differences in the stress responses of these two groups did exist, however. Most notably, fish with moderate to high Rs infections had higher titers of cortisol and lactate prior to each application of the stressor and also were unable to consistently elicit a significant hyperglycemia in response to the stressors. Collectively, our results should be important in understanding the impact that BKD has on the survival of juvenile salmonids, but we caution that our results represent the combined effects of one

  18. A sensitive nested reverse transcriptase PCR assay to detect viable cells of the fish pathogen Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.).

    PubMed

    Cook, M; Lynch, W H

    1999-07-01

    A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission. PMID:10388701

  19. In vitro growth of the bacterial kidney disease organism Renibacterium salmoninarum on a nonserum, noncharcoal-based "homospecies-metabolite" medium.

    PubMed

    Teska, J D

    1994-07-01

    Laboratory and field trials were conducted to evaluate in vitro growth of Renibacterium salmoninarum in media without serum or charcoal. Growth of this bacterium, the cause of bacterial kidney disease (BKD) in salmonids, is accelerated by addition of a growth enhancing "metabolite" of unknown composition to KDM2 medium, the medium commonly used for isolation of R. salmoninarum. KDM2 medium supplemented with greater than 1% (v/v) metabolite enhanced growth even without addition of either serum or charcoal. Medium containing 5% metabolite (denoted Five-M) allowed optimal growth in laboratory studies and was further evaluated as a primary plating medium for recovery of the bacterium isolated from chinook salmon (Oncorhynchus tshawytscha) exhibiting clinical BKD. Recovery rates of R. salmoninarum using Five-M medium were 4% and 36% higher, respectively, than comparable rates using a serum-based medium for the two salmon populations evaluated. Five-M medium is an effective, inexpensive alternative to serum-based or charcoal-based media. PMID:7933282

  20. A Sensitive Nested Reverse Transcriptase PCR Assay To Detect Viable Cells of the Fish Pathogen Renibacterium salmoninarum in Atlantic Salmon (Salmo salar L.)

    PubMed Central

    Cook, Marcia; Lynch, William H.

    1999-01-01

    A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission. PMID:10388701

  1. Sensitive detection of Renibacterium salmoninarum in whole fry, blood, and other tissues of pacific salmon by reverse transcription-polymerase chain reaction.

    PubMed

    Rhodes, L D; Nilsson, W B; Strom, M S

    1998-12-01

    A sensitive, reproducible assay for detecting Renibacterium salmoninarum in a variety of tissues, including blood, has been developed. This assay, based on reverse transcription-polymerase chain reaction (RT-PCR) of 16S ribosomal RNA, exhibited sensitivity to

  2. Temperature-mediated differences in bacterial kidney disease expression and survival in Renibacterium salmoninarum-challenged bull trout and other salmonids

    USGS Publications Warehouse

    Jones, D.T.; Moffitt, C.M.; Peters, K.K.

    2007-01-01

    Resource managers considering restoration and reconnection of watersheds to protect and enhance threatened populations of bull trout Salvelinus confluentus have little information about the consequences of bacterial kidney disease (BKD) caused by Renibacterium salmoninarum. To better understand the response of bull trout to R. salmoninarum challenge, we conducted several laboratory experiments at two water temperatures. The extent, severity, and lethality of BKD in bull trout were compared with those of similarly challenged lake trout S. namaycush, Arctic char S. alpinus, Chinook salmon Oncorhynchus tshawytscha, and rainbow trout O. mykiss. The lethal dose of bacterial cells necessary to induce 50% mortality (LD50) was 10-fold lower at the 15??C challenge than at the 9??C challenge. Of the species tested, bull trout were relatively resistant to BKD, Arctic char were the most susceptible among Salvelinus species, and Chinook salmon were the most susceptible among Oncorhynchus species tested. Mean time to death was more rapid for all fish tested at 15??C than for fish challenged at 9??C. These results suggest that infection of bull trout with BKD likely poses a low risk to successful restoration of threatened populations. ?? Copyright by the American Fisheries Society 2007.

  3. Detection of Renibacterium salmoninarum in tissue samples by sequence capture and fluorescent PCR based on the 16S rRNA gene.

    PubMed

    Königsson, Malin Heldtander; Ballagi, Andras; Jansson, Eva; Johansson, Karl-Erik

    2005-02-25

    The 16S rRNA genes from eight isolates of Renibacterium salmoninarum with different origins and dates of isolation were sequenced to evaluate the possibility to construct a diagnostic PCR system with target sites within this gene. The sequences were found to be identical but for one single position in one of the isolates, and two regions with an adequate number of nucleotide differences as compared to closely related species were identified. Species-specific fluorescent PCR primers complementary to these regions were constructed as well as oligonucleotides for DNA preparation by sequence capture. A mimic molecule was constructed to be used as an internal control. The PCR was specific and allowed the detection of DNA equivalent to 1-10 R. salmoninarum genomes per reaction. The DNA preparation with sequence capture and analysis by PCR with a mimic was found to be a reliable method for analysis of kidneys from fish with BKD. The amount of PCR inhibiting substances present in the tissue was reduced, and the relevant DNA was concentrated in the capture step. Furthermore, the use of the mimic molecule in the system assured that false negative results could be identified. PMID:15708821

  4. Infections by Renibacterium salmoninarum and Nanophyetus salmincola Chapin are associated with reduced growth of juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), in the Northeast Pacific Ocean.

    PubMed

    Sandell, T A; Teel, D J; Fisher, J; Beckman, B; Jacobson, K C

    2015-04-01

    We examined 1454 juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), captured in nearshore waters off the coasts of Washington and Oregon (USA) from 1999 to 2004 for infection by Renibacterium salmoninarum, Nanophyetus salmincola Chapin and skin metacercariae. The prevalence and intensities for each of these infections were established for both yearling and subyearling Chinook salmon. Two metrics of salmon growth, weight residuals and plasma levels of insulin-like growth factor-1, were determined for salmon infected with these pathogens/parasites, both individually and in combination, with uninfected fish used for comparison. Yearling Chinook salmon infected with R. salmoninarum had significantly reduced weight residuals. Chinook salmon infected with skin metacercariae alone did not have significantly reduced growth metrics. Dual infections were not associated with significantly more severe effects on the growth metrics than single infections; the number of triple infections was very low and precluded statistical comparison. Overall, these data suggest that infections by these organisms can be associated with reduced juvenile Chinook salmon growth. Because growth in the first year at sea has been linked to survival for some stocks of Chinook salmon, the infections may therefore play a role in regulating these populations in the Northeast Pacific Ocean. PMID:24720546

  5. Differential expression of the virulence-associated protein p57 and characterization of its duplicated gene rosa in virulent and attenuated strains of Renibacterium salmoninarum

    USGS Publications Warehouse

    O'Farrell, C. L.; Strom, M.S.

    1999-01-01

    Virulence mechanisms utilized by the salmonid fish pathogen Renibacterium salmoninarum are poorly understood. One potential virulence factor is p57 (also designated MSA for major soluble antigen), an abundant 57 kDa soluble protein that is predominately localized on the bacterial cell surface with significant levels released into the extracellular milieu. Previous studies of an attenuated strain, MT 239, indicated that it differs from virulent strains in the amount of surface-associated p57. In this report, we show overall expression of p57 in R. salmoninarum MT 239 is considerably reduced as compared to a virulent strain, ATCC 33209. The amount of cell-associated p57 is decreased while the level of p57 in the culture supernatant is nearly equivalent between the strains. To determine if lowered amount of cell-associated p57 was due to a sequence defect in p57, a genetic comparison was performed. Two copies of the gene encoding p57 (msa1 and msa2) were found in 33209 and MT 239, as well as in several other virulent isolates. Both copies from 33209 and MT 239 were cloned and sequenced and found to be identical to each other, and identical between the 2 strains. A comparison of msa1 and msa2 within each strain showed that their sequences diverge 40 base pairs 5, to the open reading frame, while sequences 3' to the open reading frame are essentially identical for at least 225 base pairs. Northern blot analysis showed no difference in steady state levels of rosa mRNA between the 2 strains. These data suggest that while cell-surface localization of p57 may be important for R. salmoninarum virulence, the differences in localization, and total p57 expression between 33209 anti MT 239 are not due to differences in rosa sequence or differences in steady state transcript levels.

  6. Differential expression of the virulence-associated protein p57 and characterization of its duplicated gene msa in virulent and attenuated strains of Renibacterium salmoninarum.

    PubMed

    O'Farrell, C L; Strom, M S

    1999-11-01

    Virulence mechanisms utilized by the salmonid fish pathogen Renibacterium salmoninarum are poorly understood. One potential virulence factor is p57 (also designated MSA for major soluble antigen), an abundant 57 kDa soluble protein that is predominately localized on the bacterial cell surface with significant levels released into the extracellular milieu. Previous studies of an attenuated strain, MT 239, indicated that it differs from virulent strains in the amount of surface-associated p57. In this report, we show overall expression of p57 in R. salmoninarum MT 239 is considerably reduced as compared to a virulent strain, ATCC 33209. The amount of cell-associated p57 is decreased while the level of p57 in the culture supernatant is nearly equivalent between the strains. To determine if the lowered amount of cell-associated p57 was due to a sequence defect in p57, a genetic comparison was performed. Two copies of the gene encoding p57 (msa1 and msa2) were found in 33209 and MT 239, as well as in several other virulent isolates. Both copies from 33209 and MT 239 were cloned and sequenced and found to be identical to each other, and identical between the 2 strains. A comparison of msa1 and msa2 within each strain showed that their sequences diverge 40 base pairs 5' to the open reading frame, while sequences 3' to the open reading frame are essentially identical for at least 225 base pairs. Northern blot analysis showed no difference in steady state levels of msa mRNA between the 2 strains. These data suggest that while cell-surface localization of p57 may be important for R. salmoninarum virulence, the differences in localization and total p57 expression between 33209 and MT 239 are not due to differences in msa sequence or differences in steady state transcript levels. PMID:10598282

  7. Development of a nested polymerase chain reaction for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that provides a highly sensitive method for detection of the bacterium in salmonid kidney

    USGS Publications Warehouse

    Chase, D.M.; Pascho, R.J.

    1998-01-01

    Nucleic acid-based assays have shown promise for diagnosing Renibacterium salmoninarum in tissues and body fluids of salmonids. DeVelopment of a nested polymerase chain reaction (PCR) method to detect a 320 bp DNA segment of the gene encoding the p57 protein of R. salmoninarum is described. Whereas a conventional PCR for a 383 bp segment of the p57 gene reliably detected 1000 R. salmoninarum cells per reaction in kidney tissue, the nested PCR detected as few as 10 R. salmoninarum per reaction in kidney tissue. Two DNA extraction methods for the nested PCR were compared and the correlation between replicate samples was generally higher in samples extracted by the QIAamp system compared with those extracted by the phenol/chloroform method. The specificity of the nested PCR was confirmed by testing DNA extracts of common bacterial fish pathogens and a panel of bacterial species reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for R. salmoninarum. Kidney samples from 74 naturally infected chinook Salmon were examined by the nested PCR, the ELISA, and the FAT, and the detected prevalences of R. salmoninarum were 61, 47, and 43%, respectively.

  8. Development of a nested polymerase chain reaction for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that provides a highly sensitive method for detection of the bacterium in salmonid kidney.

    PubMed

    Chase, D M; Pascho, R J

    1998-11-30

    Nucleic acid-based assays have shown promise for diagnosing Renibacterium salmoninarum in tissues and body fluids of salmonids. Development of a nested polymerase chain reaction (PCR) method to detect a 320 bp DNA segment of the gene encoding the p57 protein of R. salmoninarum is described. Whereas a conventional PCR for a 383 bp segment of the p57 gene reliably detected 1000 R. salmoninarum cells per reaction in kidney tissue, the nested PCR detected as few as 10 R. salmoninarum per reaction in kidney tissue. Two DNA extraction methods for the nested PCR were compared and the correlation between replicate samples was generally higher in samples extracted by the QIAamp system compared with those extracted by the phenol/chloroform method. The specificity of the nested PCR was confirmed by testing DNA extracts of common bacterial fish pathogens and a panel of bacterial species reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for R. salmoninarum. Kidney samples from 74 naturally infected chinook salmon were examined by the nested PCR, the ELISA, and the FAT, and the detected prevalences of R. salmoninarum were 61, 47, and 43%, respectively. PMID:9925428

  9. PCR and probe-PCR assays to monitor broodstock Atlantic salmon (Salmo salar L.) ovarian fluid and kidney tissue for presence of DNA of the fish pathogen Renibacterium salmoninarum.

    PubMed Central

    Miriam, A; Griffiths, S G; Lovely, J E; Lynch, W H

    1997-01-01

    A simple, rapid PCR assay for the identification of Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.) tissues detected DNA extracted from between 4 and 40 bacterial cells. PCR was at least as sensitive as culture when it was used to identify subclinically infected fish experimentally challenged with R. salmoninarum. However, PCR identified much higher numbers of kidney tissue and ovarian fluid samples from commercially reared broodstock fish to be positive for R. salmoninarum than did culture. This difference may be due to the antibiotic chemotherapy of broodstock fish used by the industry in 1994 to control the vertical transmission of R. salmoninarum. A much closer relationship between PCR and culture results was observed for ovarian fluid samples collected from broodstock fish in 1993. Also, PCR scored a much higher percentage of kidney tissue samples than ovarian fluid samples from 1994 broodstock fish positive for R. salmoninarum, which may reflect the uneven distribution of the pathogen in different fish tissues. Inclusion of a nested probe to identify the PCR-positive 1994 ovarian fluid samples increased the sensitivity of detection to between one and four cells and the number of samples that scored positive by almost threefold. These data indicate that many infected ovarian fluid samples contained very low numbers of R. salmoninarum cells and, because almost all these samples were culture negative, that PCR may have detected dead or otherwise unculturable bacterial cells. PMID:9163437

  10. PCR and probe-PCR assays to monitor broodstock Atlantic salmon (Salmo salar L.) ovarian fluid and kidney tissue for presence of DNA of the fish pathogen Renibacterium salmoninarum.

    PubMed

    Miriam, A; Griffiths, S G; Lovely, J E; Lynch, W H

    1997-06-01

    A simple, rapid PCR assay for the identification of Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.) tissues detected DNA extracted from between 4 and 40 bacterial cells. PCR was at least as sensitive as culture when it was used to identify subclinically infected fish experimentally challenged with R. salmoninarum. However, PCR identified much higher numbers of kidney tissue and ovarian fluid samples from commercially reared broodstock fish to be positive for R. salmoninarum than did culture. This difference may be due to the antibiotic chemotherapy of broodstock fish used by the industry in 1994 to control the vertical transmission of R. salmoninarum. A much closer relationship between PCR and culture results was observed for ovarian fluid samples collected from broodstock fish in 1993. Also, PCR scored a much higher percentage of kidney tissue samples than ovarian fluid samples from 1994 broodstock fish positive for R. salmoninarum, which may reflect the uneven distribution of the pathogen in different fish tissues. Inclusion of a nested probe to identify the PCR-positive 1994 ovarian fluid samples increased the sensitivity of detection to between one and four cells and the number of samples that scored positive by almost threefold. These data indicate that many infected ovarian fluid samples contained very low numbers of R. salmoninarum cells and, because almost all these samples were culture negative, that PCR may have detected dead or otherwise unculturable bacterial cells. PMID:9163437

  11. Production of the 57 kDa major surface antigen by a non-agglutinating strain of the fish pathogen Renibacterium salmoninarum.

    PubMed

    Senson, P R; Stevenson, R M

    1999-10-11

    The major surface antigen of Renibacterium salmoninarum, p57, is associated with cell autoagglutination and implicated as a virulence factor in fish infections. An autoagglutinating strain, JD24, caused 92% mortality when 2 x 10(7) cells were injected intraperitoneally into rainbow trout Oncorhynchus mykiss, while a non-agglutinating strain, MT 239, produced only 7% mortality after 100 d. The p57 antigen was present in the supernates of broth cultures of both strains when examined by western immunoblotting, and the gene for p57 was detected in both strains by PCR. Electron microscopy of cryopreserved thin sections showed an amorphous layer associated with the cell surface of JD24 which was not seen with MT 239. While p57 from JD24 could reassociate with cells of both strains, p57 from MT 239 failed to restore haemagglutination activity to either strain. Biotinylation of bacterial surfaces demonstrated the presence of a carbohydrate component of p57 from JD24 which was absent from the p57 produced by MT 239. The higher virulence of JD24 may depend not only on the production of p57, but also its direct association with the bacterial cell surface. PMID:10590925

  12. Characterization of susceptibility and carrier status of burbot, Lota lota (L.), to IHNV, IPNV, Flavobacterium psychrophilum, Aeromonas salmonicida and Renibacterium salmoninarum.

    PubMed

    Polinski, M P; Fehringer, T R; Johnson, K A; Snekvik, K R; Lapatra, S E; Lafrentz, B R; Ireland, S C; Cain, K D

    2010-07-01

    In this study, susceptibility and potential carrier status of burbot, Lota lota, were assessed for five important fish pathogens. Burbot demonstrated susceptibility and elevated mortality following challenge with infectious haematopoietic necrosis virus (IHNV) by immersion and to Aeromonas salmonicida by intraperitoneal (i.p.) injection. IHNV persisted in fish for at least 28 days, whereas A. salmonicida was not re-isolated beyond 17 days post-challenge. In contrast, burbot appeared refractory to Flavobacterium psychrophilum following intramuscular (i.m.) injection and to infectious pancreatic necrosis virus (IPNV) by immersion. However, i.p injection of IPNV resulted in re-isolation of virus from fish for the duration of the 28 day challenge. Renibacterium salmoninarum appeared to induce an asymptomatic carrier state in burbot following i.p. injection, but overt manifestation of disease was not apparent. Viable bacteria persisted in fish for at least 41 days, and bacterial DNA isolated by diagnostic polymerase chain reaction was detected from burbot kidney tissue 90 days after initial exposure. This study is the first to investigate susceptibility of burbot to selected fish pathogens, and this information will aid in efforts to culture and manage this species. PMID:20367740

  13. Detection of humoral antibodies to Renibacterium salmoninarum in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar challenged by immersion and in naturally infected populations.

    PubMed

    Jansson, E; Ljungberg, O

    1998-06-19

    Humoral antibodies to heat-stable antigens of Renibacterium salmoninarum (Rs) were detected by enzyme-linked immunosorbent assay (ELISA) in rainbow trout Oncorhynchus mykiss and in Atlantic salmon Salmo salar challenged by immersion. A slow antibody response was found: 3% (1/30) was positive 4 wk after immersion and 72% (26/36) was positive after 8 wk. All 30 fish sampled after 4 wk were found to be infected, as determined by bacterial culture and/or the presence of soluble antigens in the kidney. At 6, 8 and 12 wk after immersion the proportion of positives indicated by ELISA was 58%. The Rs infection was detected by cultivation in 36% of sampled fish collected on the same occasion. Elevated antibody titres to Rs were detected in samples from both Atlantic salmon (59% in 1 farm) and from rainbow trout (20% in 1 of 5 sampled farms) in naturally exposed populations all of which classified positive for bacterial kidney disease (BKD). Elevated antibody titres were detected among sampled fish from populations of rainbow trout and salmon with clinical BKD. Samples collected from farm populations of rainbow trout, salmon and brown trout Salmo trutta, exposed to Rs but without clinical BKD, were negative in the ELISA, although Rs bacteria or soluble antigens were detected at the same sampling. The antibody ELISA method cannot be recommended for general fish health monitoring purposes, but may be a valuable tool for monitoring the disease progression during controlled experiments. PMID:9684315

  14. Immunohistochemical identification of Renibacterium salmoninarum by monoclonal antibodies in paraffin-embedded tissues of Atlantic salmon (Salmo salar L.), using paired immunoenzyme and paired immunofluorescence techniques.

    PubMed

    Evensen, O; Dale, O B; Nilsen, A

    1994-01-01

    Renibacterium salmoninarum was identified in situ by immunoenzymatic and immunofluorescence techniques in paraffin-embedded tissue specimens collected during a natural outbreak of bacterial kidney disease (BKD) and from an experimental infection in Atlantic salmon (Salmo salar L.). Monoclonal antibodies (MAbs) 4D3 and 2G5 were used in this study, both specific for the 57-58-kD outer membrane protein (p57) of the bacterium. Both MAbs revealed positive staining in ethanol-fixed tissue specimens, but only the epitope identified by MAb 4D3 was formalin resistant. Pretreatment with trypsin did not reestablish the antigenicity for the epitope identified by Mab 2G5. Paired immunoenzymatic staining for identification of the bacterium in sequential incubation steps on ethanol-fixed tissue specimens using an avidin-biotin-peroxidase system was obtained after serial dilution of the Mab (2G5) or the chromagen, amino ethyl carbazole, in the first sequence. Paired immunofluorescence staining with well-balanced color mixing was easily obtained on ethanol-fixed tissue specimens using sequential incubations. Single exposures gave blue (aminomethyl coumarin acetic acid) and green (fluorescein isothiocyanate) fluorescence for MAbs 2G5 and biotinylated 4D3, respectively. Color mixing was revealed as a turquoise staining. Studies on method sensitivity was performed by incorporating a known amount of a protein preparation of p57 into an inert matrix, creating an artificial test substrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8011782

  15. Molecular cloning and sequence analysis of the gene coding for the 57-kDa major soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum.

    PubMed

    Chien, M S; Gilbert, T L; Huang, C; Landolt, M L; O'Hara, P J; Winton, J R

    1992-09-15

    The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum, was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated M(r) value of 57,190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27-61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein is synthesized as a 557-amino acid precursor and processed to produce a mature protein of M(r) 54,505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene. PMID:1383085

  16. Renibacterium salmoninarum isolates from different sources possess two highly conserved copies of the rRNA operon .

    PubMed

    Grayson, T H; Alexander, S M; Cooper, L F; Gilpin, M L

    2000-07-01

    The nucleotide sequences of the rRNA genes and the 5' flanking region were determined for R. salmoninarum ATCC 33209T from overlapping products generated by PCR amplification from the genomic DNA. Comparison of the sequences with rRNA genes from a variety of bacteria demonstrated the close relatedness between R. salmoninarum and the high G+C group of the actinobacteria, in particular, Arthrobacter species. A regulatory element within the 5' leader of the rRNA operon was identical to an element, CL2, described for mycobacteria. PCR, DNA sequence analysis, and DNA hybridisation were performed to examine variation between isolates from diverse sources which represented the four 16S-23S rRNA intergenic spacer sequevars previously described for R. salmoninarum. Two 23S-5S rRNA intergenic spacer sequevars of identical length were found. DNA hybridisation using probes complementary to 23S rDNA and 16S rDNA identified two rRNA operons which were identical or nearly identical amongst 40 isolates sourced from a variety of countries. PMID:11016696

  17. A single Ala139-to-Glu substitution in the Renibacterium salmoninarum virulence-associated protein p57 results in antigenic variation and is associated with enhanced p57 binding to chinook salmon leukocytes.

    PubMed

    Wiens, Gregory D; Pascho, Ron; Winton, James R

    2002-08-01

    The gram-positive bacterium Renibacterium salmoninarum produces relatively large amounts of a 57-kDa protein (p57) implicated in the pathogenesis of salmonid bacterial kidney disease. Antigenic variation in p57 was identified by using monoclonal antibody 4C11, which exhibited severely decreased binding to R. salmoninarum strain 684 p57 and bound robustly to the p57 proteins of seven other R. salmoninarum strains. This difference in binding was not due to alterations in p57 synthesis, secretion, or bacterial cell association. The molecular basis of the 4C11 epitope loss was determined by amplifying and sequencing the two identical genes encoding p57, msa1 and msa2. The 5' and coding sequences of the 684 msa1 and msa2 genes were identical to those of the ATCC 33209 msa1 and msa2 genes except for a single C-to-A nucleotide mutation. This mutation was identified in both the msa1 and msa2 genes of strain 684 and resulted in an Ala(139)-to-Glu substitution in the amino-terminal region of p57. We examined whether this mutation in p57 altered salmonid leukocyte and rabbit erythrocyte binding activities. R. salmoninarum strain 684 extracellular protein exhibited a twofold increase in agglutinating activity for chinook salmon leukocytes and rabbit erythrocytes compared to the activity of the ATCC 33209 extracellular protein. A specific and quantitative p57 binding assay confirmed the increased binding activity of 684 p57. Monoclonal antibody 4C11 blocked the agglutinating activity of the ATCC 33209 extracellular protein but not the agglutinating activity of the 684 extracellular protein. These results indicate that the Ala139-to-Glu substitution altered immune recognition and was associated with enhanced biological activity of R. salmoninarum 684 p57. PMID:12147498

  18. A Single Ala139-to-Glu Substitution in the Renibacterium salmoninarum Virulence-Associated Protein p57 Results in Antigenic Variation and Is Associated with Enhanced p57 Binding to Chinook Salmon Leukocytes

    PubMed Central

    Wiens, Gregory D.; Pascho, Ron; Winton, James R.

    2002-01-01

    The gram-positive bacterium Renibacterium salmoninarum produces relatively large amounts of a 57-kDa protein (p57) implicated in the pathogenesis of salmonid bacterial kidney disease. Antigenic variation in p57 was identified by using monoclonal antibody 4C11, which exhibited severely decreased binding to R. salmoninarum strain 684 p57 and bound robustly to the p57 proteins of seven other R. salmoninarum strains. This difference in binding was not due to alterations in p57 synthesis, secretion, or bacterial cell association. The molecular basis of the 4C11 epitope loss was determined by amplifying and sequencing the two identical genes encoding p57, msa1 and msa2. The 5′ and coding sequences of the 684 msa1 and msa2 genes were identical to those of the ATCC 33209 msa1 and msa2 genes except for a single C-to-A nucleotide mutation. This mutation was identified in both the msa1 and msa2 genes of strain 684 and resulted in an Ala139-to-Glu substitution in the amino-terminal region of p57. We examined whether this mutation in p57 altered salmonid leukocyte and rabbit erythrocyte binding activities. R. salmoninarum strain 684 extracellular protein exhibited a twofold increase in agglutinating activity for chinook salmon leukocytes and rabbit erythrocytes compared to the activity of the ATCC 33209 extracellular protein. A specific and quantitative p57 binding assay confirmed the increased binding activity of 684 p57. Monoclonal antibody 4C11 blocked the agglutinating activity of the ATCC 33209 extracellular protein but not the agglutinating activity of the 684 extracellular protein. These results indicate that the Ala139-to-Glu substitution altered immune recognition and was associated with enhanced biological activity of R. salmoninarum 684 p57. PMID:12147498

  19. A new real time PCR-based assay for diagnosing Renibacterium salmoninarum in rainbow trout (Oncorhynchus mykiss) and comparison with other techniques.

    PubMed

    Halaihel, Nabil; Vendrell, Daniel; Ruiz-Zarzuela, Imanol; de Blas, Ignacio; Alonso, José Luis; Gironés, Olivia; Pérez, Tania; Muzquiz, José Luis

    2009-01-01

    Bacterial Kidney Disease of salmonid is caused by a slow-growing gram-positive bacterium, Renibacterium salmoninarum. This bacterium lives both extra-cellular and intra-cellular in the host. Serological and molecular diagnostic methods to detect the bacterium major surface protein antigen p57 have been developed. In the present work, a newly developed quantitative Reverse Transcriptase-PCR (RT-QPCR), using self-quenched fluorescent primer (Lux), a nested PCR (NPCR), a commercial ELISA and recently commercially available Immune-chromatographic strip test(IC-Strip) were compared for their ability to detect BKD in kidney tissue samples obtained from experimentally infected fish. ELISA test resulted to be rapid, simple and indicative for the bacterial load. The IC-Strip test had similar characteristics for bacterial detection. Both tests are a good option for rapid and relatively inexpensive screening studies, despite the one and two log decrease in bacterial detection limits compared to NPCR and RT-QPCR, respectively. The use of Lux primers in the newly developed RT-QPCR revealed to be a cost-effective alternative to other fluorescence-based PCR techniques. The option of generating a melting temperature curve with the real time PCR instrument confirmed the specificity of the PCR product. The RT-QPCR technique had the advantage of detecting low numbers of viable bacterial mRNA which implied a higher capacity of detecting chronically infected animals. For instance, some fish in the group infected by cohabitation had very low bacterial load and were only detected by this technique. PMID:18938198

  20. A single Alal 39-to-Glu substitution in the Renibacterium salmoninarum virulence-associated protein p57 results in antigenic variation and is associated with enhanced p57 binding to Chinook salmon leukocytes

    USGS Publications Warehouse

    Wiens, Gregory D.; Pascho, Ron; Winton, James R.

    2002-01-01

    The gram-positive bacterium Renibacterium salmoninarum produces relatively large amounts of a 57-kDa protein (p57) implicated in the pathogenesis of salmonid bacterial kidney disease. Antigenic variation in p57 was identified by using monoclonal antibody 4C11, which exhibited severely decreased binding to R. salmoninarum strain 684 p57 and bound robustly to the p57 proteins of seven other R. salmoninarum strains. This difference in binding was not due to alterations in p57 synthesis, secretion, or bacterial cell association. The molecular basis of the 4C11 epitope loss was determined by amplifying and sequencing the two identical genes encoding p57, msa1 and msa2. The 5′ and coding sequences of the 684 msa1 and msa2 genes were identical to those of the ATCC 33209 msa1and msa2 genes except for a single C-to-A nucleotide mutation. This mutation was identified in both the msa1 and msa2 genes of strain 684 and resulted in an Ala139-to-Glu substitution in the amino-terminal region of p57. We examined whether this mutation in p57 altered salmonid leukocyte and rabbit erythrocyte binding activities. R. salmoninarum strain 684 extracellular protein exhibited a twofold increase in agglutinating activity for chinook salmon leukocytes and rabbit erythrocytes compared to the activity of the ATCC 33209 extracellular protein. A specific and quantitative p57 binding assay confirmed the increased binding activity of 684 p57. Monoclonal antibody 4C11 blocked the agglutinating activity of the ATCC 33209 extracellular protein but not the agglutinating activity of the 684 extracellular protein. These results indicate that the Ala139-to-Glu substitution altered immune recognition and was associated with enhanced biological activity of R. salmoninarum 684 p57.

  1. Prevalence and levels of Renibacterium salmoninarum in spring-summer Chinook salmon (Oncorhynchus tshawytscha) smolts at dams on the Columbia and Snake Rivers.

    USGS Publications Warehouse

    Elliott, D.G.; Pascho, R.J.; Jackson, L.M.; Mathews, G.M.; Harmon, J.R.

    1997-01-01

    We evaluated Renibaeterium salmoninarum infection in smolts of hatchery and wild spring-summer Chinook salmon Oncorhynchus tshawytscha sampled during most of the outmigration at Little Goose (1988) and Lower Granite dams (1988–1991) on the Snake River and at Priest Rapids and McNary dams on the Columbia River (1988–1990). We sampled 860–2,178 fish per dam each year. Homogenates of kidney–spleen tissue from all fish were tested for the presence of R. salmoninarum antigens by the enzyme-linked immunosorbent assay (ELISA), and homogenates from 10% of the fish were examined by the fluorescent antibody technique (FAT). Although only 1–11% of fish sampled at a given dam during any l year exhibited lesions characteristic of bacterial kidney disease, 86–100% of the fish tested positive for R. salmoninarum antigen by ELISA, whereas 4–17% of the fish tested positive by the FAT. During most years, a majority (68–87%) of fish testing positive by the ELISA had low R. salmoninarum antigen levels, but in 1989, 53% of positive fish from Lower Granite Dam and 52% from McNary Dam showed medium-to-high antigen levels. For most years, the highest mean antigen levels were measured in fish sampled after 75% of the total out-migrants had passed a given dam. When the largest numbers of fish were being collected for bypass or downriver transportation, mean antigen levels were relatively low.

  2. A field evaluation of an indirect fluorescent antibody-based broodstock screening test used to control the vertical transmission of Renibacterium salmoninarium in Chinook salmon (Oncorhynchus tshawytscha).

    PubMed Central

    Armstrong, R D; Martin, S W; Evelyn, T P; Hicks, B; Dorward, W J; Ferguson, H W

    1989-01-01

    Ovarian fluid samples from erythromycin treated and untreated spawning three year old Chinook salmon were screened independently by two laboratories for the presence of Renibacterium salmoninarum using the indirect fluorescent antibody technique (IFAT). Agreement between the results of the two laboratories could be explained by chance when R. salmoninarum cell numbers as low as one per sample were considered sufficient to represent a positive result. If a positive result was considered to be the detection of larger numbers of R. salmoninarum cells (greater than 51 cells per sample), agreement increased and there was a statistically significant association between the results of the two laboratories. However, the level of agreement did not reach satisfactory levels for a population screening test. Furthermore, approximately 60% of the samples yielded false negative results when IFAT results were compared with positive culture results. These results led to the conclusion that the IFAT screening procedure, as carried out, was unsuitable for the purposes intended. Erythromycin injection of the spawning fish had no statistically significant effect on the results of the IFAT screening test. PMID:2686828

  3. Distinguished Books.

    ERIC Educational Resources Information Center

    Marles, Daisy; Ink, Gary

    1999-01-01

    Presents the following lists of distinguished books: "Best Books of 1998"; "Best Young Adult Books"; "Notable Children's Videos"; "Best Children's Books"; "Notable Recordings for Children"; "Notable Software and Web Sites for Children"; "Quick Picks for Reluctant Young Adult Readers"; "Bestsellers of 1998"; and "Literary Prizes, 1998." (AEF)

  4. Characterization of susceptibility and carrier status of burbot, Lota lota (L.), to IHNV, IPNV, Flavobacterium psychrophilum, Aeromonas salmonicida, and Renibacterium salmoninarum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, susceptibility and potential carrier status of burbot, Lota lota, were assessed for five important fish pathogens. Burbot demonstrated susceptibility and elevated mortality following challenge with infectious haematopoietic necrosis virus (IHNV) by immersion and to Aeromonas salmonici...

  5. Distinguished Cooperating Teacher Program.

    ERIC Educational Resources Information Center

    Chicago State Univ., IL.

    The Distinguished Cooperating Teacher Program at Chicago State University was developed to train cooperating teachers to supervise student teachers. The program departs from traditional practice by changing the roles of the classroom teacher and the university field supervisor. The supervisor's role becomes that of coordinator while the teacher…

  6. Distinguishing Radiculopathies from Mononeuropathies

    PubMed Central

    Robblee, Jennifer; Katzberg, Hans

    2016-01-01

    Identifying “where is the lesion” is particularly important in the approach to the patient with focal dysfunction where a peripheral localization is suspected. This article outlines a methodical approach to the neuromuscular patient in distinguishing focal neuropathies versus radiculopathies, both of which are common presentations to the neurology clinic. This approach begins with evaluation of the sensory examination to determine whether there are irritative or negative sensory signs in a peripheral nerve or dermatomal distribution. This is followed by evaluation of deep tendon reflexes to evaluate if differential hyporeflexia can assist in the two localizations. Finally, identification of weak muscle groups unique to a nerve or myotomal pattern in the proximal and distal extremities can most reliably assist in a precise localization. The article concludes with an application of the described method to the common scenario of distinguishing radial neuropathy versus C7 radiculopathy in the setting of a wrist drop and provides additional examples for self-evaluation and reference. PMID:27468275

  7. Distinguishing heroes from celebrities.

    PubMed

    North, Adrian C; Bland, Victoria; Ellis, Nicky

    2005-02-01

    Although numerous studies have concerned both 'greatness' and 'celebrity worship,' little previous research has overtly investigated the psychological bases for distinguishing 'heroes' from 'celebrities'. Study 1 indicated that, in response to 13 attitudinal measures, participants' responses to heroes could be summarized in terms of emotional attachment, drive for affiliation, and ordinariness, whereas the corresponding factors for celebrities were drive for affiliation, disdain, and ordinariness. Study 2 investigated how certain individuals might come to be perceived as producing 'great' outputs. Using a vignette, manipulations of 'dead versus alive,' and 'good conduct versus bad conduct' mediated perceptions of the emotional significance of a pop song. The results are used to generate some tentative theoretical explanations for the differences between heroism and celebrity. PMID:15826323

  8. Application of isotope coded affinity tag (ICAT) analysis for the identification of differentially expressed proteins following infection of atlantic salmon (Salmo salar) with infectious hematopoietic necrosis virus (IHNV) or Renibacterium salmoninarum (BKD).

    PubMed

    Booy, A T; Haddow, J D; Ohlund, L B; Hardie, D B; Olafson, R W

    2005-01-01

    Aquaculture and commercial fisheries worldwide suffer from significant economic loss due to diseases of net-pen reared fish. In British Columbia, infectious hematopoietic necrosis (IHN) and bacterial kidney disease (BKD) epidemics occur because there are currently no commercially available drugs or fully licensed vaccines to treat these diseases. With a better understanding of the molecular mechanisms underlying these diseases, this circumstance might be significantly improved. In the present study, we have used a proteomics approach in an effort to identify and quantitate differentially expressed proteins in the liver and kidneys of diseased and healthy Atlantic salmon (Salmo salar). Isotope coded affinity tagging (ICAT), 2D gel electrophoresis, and multidimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC MS/MS) were used to identify hundreds of differentially expressed proteins. While the direct significance of changes in expression levels of many proteins remains to be elucidated, others appear to be more clearly related to the infectious process. Examples of the latter are discussed here and include, a natural killer cell enhancement factor (NKEF), procathepsin L, superoxide-producing NADPH oxidase and interferon-induced viral resistance protein Mx (IFI-Mx). PMID:15822907

  9. Vaccination against salmonid bacterial kidney disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial kidney disease (BKD) of salmonid fishes, caused by Renibacterium salmoninarum, has presented challenges for development of effective vaccines, despite several decades of research. The only vaccine against BKD that is commercially licensed is an injectable preparation containing live cells ...

  10. Wigner-Araki-Yanase theorem on distinguishability

    SciTech Connect

    Miyadera, Takayuki; Imai, Hideki

    2006-08-15

    The presence of an additive-conserved quantity imposes a limitation on the measurement process. According to the Wigner-Araki-Yanase theorem, perfect repeatability and distinguishability of the apparatus cannot be attained simultaneously. Instead of repeatability, in this paper, the distinguishability in both systems is examined. We derive a trade-off inequality between the distinguishability of the final states on the system and the one on the apparatus. An inequality shows that perfect distinguishability of both systems cannot be attained simultaneously.

  11. Distinguishing cell type using epigenotype

    NASA Astrophysics Data System (ADS)

    Wytock, Thomas; Motter, Adilson E.

    Recently, researchers have proposed that unique cell types are attractors of their epigenetic dynamics including gene expression and chromatin conformation patterns. Traditionally, cell types have been classified by their function, morphology, cytochemistry, and other macroscopically observable properties. Because these properties are the result of many proteins working together, it should be possible to predict cell types from gene expression or chromatin conformation profiles. In this talk, I present a maximum entropy approach to identify and distinguish cell type attractors on the basis of correlations within these profiles. I will demonstrate the flexibility of this method through its separate application to gene expression and chromatin conformation datasets. I show that our method out-performs other machine-learning techniques and uncorrelated benchmarks. We adapt our method to predict growth rate from gene expression in E. coli and S. cerevisiae and compare our predictions with those from metabolic models. In addition, our method identifies a nearly convex region of state-space associated with each cell type attractor basin. Estimates of the growth rate and attractor basin make it possible to rationally control gene regulatory networks independent of a model. This research was supported by NSF-GRFP, NSF-GK12, GAANN, and Northwestern's NIH-NIGMS Molecular Biophysics Training Grant.

  12. Distinguishability of generic quantum states

    NASA Astrophysics Data System (ADS)

    Puchała, Zbigniew; Pawela, Łukasz; Życzkowski, Karol

    2016-06-01

    Properties of random mixed states of dimension N distributed uniformly with respect to the Hilbert-Schmidt measure are investigated. We show that for large N , due to the concentration of measure, the trace distance between two random states tends to a fixed number D ˜=1 /4 +1 /π , which yields the Helstrom bound on their distinguishability. To arrive at this result, we apply free random calculus and derive the symmetrized Marchenko-Pastur distribution, which is shown to describe numerical data for the model of coupled quantum kicked tops. Asymptotic value for the root fidelity between two random states, √{F }=3/4 , can serve as a universal reference value for further theoretical and experimental studies. Analogous results for quantum relative entropy and Chernoff quantity provide other bounds on the distinguishablity of both states in a multiple measurement setup due to the quantum Sanov theorem. We study also mean entropy of coherence of random pure and mixed states and entanglement of a generic mixed state of a bipartite system.

  13. Distinguishing psychogenic and essential tremor.

    PubMed

    Kenney, Christopher; Diamond, Alan; Mejia, Nicte; Davidson, Anthony; Hunter, Christine; Jankovic, Joseph

    2007-12-15

    In contrast to essential tremor (ET), psychogenic tremor (PT) is often manifested by shaking with variable amplitude and frequency, distractibility, suggestibility, and entrainment. The sensitivity and specificity of these findings in differentiating PT and ET have not been systematically examined. In order to determine clinical features that reliably distinguish PT from ET, we collected patient information regarding tremor onset, spontaneous remissions, family history, and employment history. A "blinded" rater evaluated video segments of subjects using a standardized protocol with special attention to distractibility, suggestibility, or entrainment. A total of 45 subjects with ET or PT were enrolled in this study: 33 met clinical criteria for ET with a mean age of 56.8+/-17.0 years and 12 met clinical criteria for PT with a mean age of 42.5+/-11.0 years. PT subjects were significantly more likely to relay a history of sudden onset (p=0.03), spontaneous remissions (p=0.03), and shorter duration of tremor (p=0.001). Family history of tremor was significantly more common in the ET group (p=0.001). A moderate-to-marked degree of distraction with alternate finger tapping (p=0.01) and mental concentration on serial 7 s (p=0.01) was more common in PT. Furthermore, suggestibility with a tuning fork (p=0.04) and exacerbation with hyperventilation (p=0.06) seemed predictive of PT. Entrainment was not different in the two groups. In conclusion, a history of tremor with sudden onset and spontaneous remissions along with distractibility and suggestibility on examination are good predictors of PT and help differentiate it from ET. PMID:17604055

  14. RESTRICTION FRAGMENT LENGTH POLYMORPHISMS DISTINGUISH ECTOMYCORRHIZAL FUNGI

    EPA Science Inventory

    Basidiomycetous fungi, two saprophytes and three mycorrhizal, were used to assess the specificity of DNA hybridization for distinguishing genera from one another. nterspecific comparisons were done with several isolates of mycorrhizal fungi, Laccaria bicolor and L. laccata, colle...

  15. Stanislas Dehaene: Award for Distinguished Scientific Contributions.

    PubMed

    2015-11-01

    The APA Awards for Distinguished Scientific Contributions are presented to persons who, in the opinion of the Committee on Scientific Awards, have made distinguished theoretical or empirical contributions to basic research in psychology. One of the 2015 award winners is Stanislas Dehaene, who received this award for "outstanding empirical and theoretical contributions to not just one but three fields that are central to the enterprises of psychology and cognitive neuroscience." Dehaene's award citation, biography, and a selected bibliography are presented here. PMID:26618941

  16. Orthogonality and distinguishability: Criterion for local distinguishability of arbitrary orthogonal states

    SciTech Connect

    Chen Pingxing; Li Chengzu

    2003-12-01

    We consider the relation between the orthogonality and the distinguishability of a set of arbitrary states (including multipartite states). It is shown that if a set of arbitrary states can be distinguished by local operations and classical communication (LOCC), each of the states can be written as a linear combination of product vectors such that all product vectors of one of the states are orthogonal to the other states. With this result we then prove a simple necessary condition for LOCC distinguishability of a class of orthogonal states. These conclusions may be useful in discussing the distinguishability of orthogonal quantum states further, understanding the essence of nonlocality and discussing the distillation of entanglement.

  17. Children distinguish between positive pride and hubris.

    PubMed

    Nelson, Nicole L; Russell, James A

    2015-11-01

    Adults distinguish expressions of hubris from those of positive pride. To determine whether children (N = 183; 78-198 months old) make a similar distinction, we asked them to attribute emotion labels and a variety of social characteristics to dynamic expressions intended to convey hubris and positive pride. Like adults, children attributed different emotion labels to the expressions, and this tendency increased with age. Girls were more likely to distinguish between the expressions than boys were. Children also associated more positive social characteristics with the expression of positive pride and more negative characteristics with the expression of hubris. PMID:26347987

  18. Beyond Benford's Law: Distinguishing Noise from Chaos.

    PubMed

    Li, Qinglei; Fu, Zuntao; Yuan, Naiming

    2015-01-01

    Determinism and randomness are two inherent aspects of all physical processes. Time series from chaotic systems share several features identical with those generated from stochastic processes, which makes them almost undistinguishable. In this paper, a new method based on Benford's law is designed in order to distinguish noise from chaos by only information from the first digit of considered series. By applying this method to discrete data, we confirm that chaotic data indeed can be distinguished from noise data, quantitatively and clearly. PMID:26030809

  19. Beyond Benford's Law: Distinguishing Noise from Chaos

    PubMed Central

    Li, Qinglei; Fu, Zuntao; Yuan, Naiming

    2015-01-01

    Determinism and randomness are two inherent aspects of all physical processes. Time series from chaotic systems share several features identical with those generated from stochastic processes, which makes them almost undistinguishable. In this paper, a new method based on Benford's law is designed in order to distinguish noise from chaos by only information from the first digit of considered series. By applying this method to discrete data, we confirm that chaotic data indeed can be distinguished from noise data, quantitatively and clearly. PMID:26030809

  20. Michael Tomasello: Award for Distinguished Scientific Contributions.

    PubMed

    2015-11-01

    The APA Awards for Distinguished Scientific Contributions are presented to persons who, in the opinion of the Committee on Scientific Awards, have made distinguished theoretical or empirical contributions to basic research in psychology. One of the 2015 award winners is Michael Tomasello, who received this award for "outstanding empirical and theoretical contributions to understanding what makes the human mind unique. Michael Tomasello's pioneering research on the origins of social cognition has led to revolutionary insights in both developmental psychology and primate cognition." Tomasello's award citation, biography, and a selected bibliography are presented here. PMID:26618943

  1. 10 CFR 1002.22 - Use of distinguishing flag.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 4 2011-01-01 2011-01-01 false Use of distinguishing flag. 1002.22 Section 1002.22 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) OFFICIAL SEAL AND DISTINGUISHING FLAG Distinguishing Flag § 1002.22 Use of distinguishing flag. (a) DOE distinguishing flags may be used only: (1) In the offices of...

  2. 10 CFR 1002.22 - Use of distinguishing flag.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 4 2014-01-01 2014-01-01 false Use of distinguishing flag. 1002.22 Section 1002.22 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) OFFICIAL SEAL AND DISTINGUISHING FLAG Distinguishing Flag § 1002.22 Use of distinguishing flag. (a) DOE distinguishing flags may be used only: (1) In the offices of...

  3. 10 CFR 1002.22 - Use of distinguishing flag.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 4 2012-01-01 2012-01-01 false Use of distinguishing flag. 1002.22 Section 1002.22 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) OFFICIAL SEAL AND DISTINGUISHING FLAG Distinguishing Flag § 1002.22 Use of distinguishing flag. (a) DOE distinguishing flags may be used only: (1) In the offices of...

  4. 10 CFR 1002.22 - Use of distinguishing flag.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 4 2013-01-01 2013-01-01 false Use of distinguishing flag. 1002.22 Section 1002.22 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) OFFICIAL SEAL AND DISTINGUISHING FLAG Distinguishing Flag § 1002.22 Use of distinguishing flag. (a) DOE distinguishing flags may be used only: (1) In the offices of...

  5. 10 CFR 1002.22 - Use of distinguishing flag.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Use of distinguishing flag. 1002.22 Section 1002.22 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) OFFICIAL SEAL AND DISTINGUISHING FLAG Distinguishing Flag § 1002.22 Use of distinguishing flag. (a) DOE distinguishing flags may be used only: (1) In the offices of...

  6. Characteristics of Distinguished Programs of Agricultural Education

    ERIC Educational Resources Information Center

    Birkenholz, Robert J.; Simonsen, Jon C.

    2011-01-01

    Academic program rankings are highly anticipated by many university administrators, faculty, and alumni. This study analyzed the perceptions of agricultural education departmental contact persons to identify esteemed post-secondary agricultural education programs and the distinguishing characteristics of each program. The ten most distinguished…

  7. Entropy of Mixing of Distinguishable Particles

    ERIC Educational Resources Information Center

    Kozliak, Evguenii I.

    2014-01-01

    The molar entropy of mixing yields values that depend only on the number of mixing components rather than on their chemical nature. To explain this phenomenon using the logic of chemistry, this article considers mixing of distinguishable particles, thus complementing the well-known approach developed for nondistinguishable particles, for example,…

  8. Distinguishing the Spending Preferences of Seniors.

    ERIC Educational Resources Information Center

    Zimmer, Zachary; Chappell, Neena L.

    1996-01-01

    The consumer spending preferences of 1,406 senior Canadians were surveyed. Age distinguished those who had product-specific preferences. Income and health status separated those interested in recreational spending from those more interested in basic needs. Diversity of health and social characteristics in this population extends to their…

  9. The Humanity of English. 1972 Distinguished Lectures.

    ERIC Educational Resources Information Center

    National Council of Teachers of English, Urbana, IL.

    This is a collection of lectures by distinguished members of the English profession who were invited to lecture to schools located far from large urban and cultural centers. Included are papers by: John H. Fisher, "Truth Versus Beauty: An Inquiry into the Function of Language and Literature in an Articulate Society"; Walter Loban, "The Green…

  10. Distinguishing Disability: Parents, Privilege, and Special Education

    ERIC Educational Resources Information Center

    Ong-Dean, Colin

    2009-01-01

    Students in special education programs can have widely divergent experiences. For some, special education amounts to a dumping ground where schools unload their problem students, while for others, it provides access to services and accommodations that drastically improve chances of succeeding in school and beyond. "Distinguishing Disability"…

  11. Local distinguishability of generic unentangled orthonormal bases

    NASA Astrophysics Data System (ADS)

    Lebl, Jiří; Shakeel, Asif; Wallach, Nolan

    2016-01-01

    An orthonormal basis consisting of unentangled (pure tensor) elements in a tensor product of Hilbert spaces is an unentangled orthonormal basis (UOB). In general, for n qubits, we prove that in its natural structure as a real variety, the space of UOB is a bouquet of products of Riemann spheres parametrized by a class of edge colorings of hypercubes. Its irreducible components of maximum dimension are products of 2n-1 two spheres. Using a theorem of Walgate and Hardy, we observe that the UOB whose elements are distinguishable by local operations and classical communication (called locally distinguishable or LOCC distinguishable UOB) are exactly those in the maximum dimensional components. Bennett et al. [Phys. Rev. A 59, 1070 (1999)., 10.1103/PhysRevA.59.1070], in their in-depth study of quantum nonlocality without entanglement, include a specific three-qubit example UOB which is not LOCC distinguishable; we construct certain generalized counterparts of this UOB in n qubits.

  12. Repetitive sequence environment distinguishes housekeeping genes

    PubMed Central

    Eller, C. Daniel; Regelson, Moira; Merriman, Barry; Nelson, Stan; Horvath, Steve; Marahrens, York

    2007-01-01

    Housekeeping genes are expressed across a wide variety of tissues. Since repetitive sequences have been reported to influence the expression of individual genes, we employed a novel approach to determine whether housekeeping genes can be distinguished from tissue-specific genes their repetitive sequence context. We show that Alu elements are more highly concentrated around housekeeping genes while various longer (>400-bp) repetitive sequences ("repeats"), including Long Interspersed Nuclear Element 1 (LINE-1) elements, are excluded from these regions. We further show that isochore membership does not distinguish housekeeping genes from tissue-specific genes and that repetitive sequence environment distinguishes housekeeping genes from tissue-specific genes in every isochore. The distinct repetitive sequence environment, in combination with other previously published sequence properties of housekeeping genes, were used to develop a method of predicting housekeeping genes on the basis of DNA sequence alone. Using expression across tissue types as a measure of success, we demonstrate that repetitive sequence environment is by far the most important sequence feature identified to date for distinguishing housekeeping genes. PMID:17141428

  13. Children Distinguish between Positive Pride and Hubris

    ERIC Educational Resources Information Center

    Nelson, Nicole L.; Russell, James A.

    2015-01-01

    Adults distinguish expressions of hubris from those of positive pride. To determine whether children (N = 183; 78-198 months old) make a similar distinction, we asked them to attribute emotion labels and a variety of social characteristics to dynamic expressions intended to convey hubris and positive pride. Like adults, children attributed…

  14. Distinguishing Provenance Equivalence of Earth Science Data

    NASA Technical Reports Server (NTRS)

    Tilmes, Curt; Yesha, Ye; Halem, M.

    2010-01-01

    Reproducibility of scientific research relies on accurate and precise citation of data and the provenance of that data. Earth science data are often the result of applying complex data transformation and analysis workflows to vast quantities of data. Provenance information of data processing is used for a variety of purposes, including understanding the process and auditing as well as reproducibility. Certain provenance information is essential for producing scientifically equivalent data. Capturing and representing that provenance information and assigning identifiers suitable for precisely distinguishing data granules and datasets is needed for accurate comparisons. This paper discusses scientific equivalence and essential provenance for scientific reproducibility. We use the example of an operational earth science data processing system to illustrate the application of the technique of cascading digital signatures or hash chains to precisely identify sets of granules and as provenance equivalence identifiers to distinguish data made in an an equivalent manner.

  15. The ambiguity of "distinguishability" in statistical mechanics

    NASA Astrophysics Data System (ADS)

    Swendsen, Robert H.

    2015-06-01

    Differences of opinion concerning fundamental issues in statistical mechanics directly related to the thermodynamic entropy have persisted through more than a century of debate. One reason is the lack of consensus on the definitions of key terms, especially the terms "distinguishable," "indistinguishable," and "identical." Several definitions occur in the literature, but are not always made explicit. The multiplicity of definitions has created confusion about the basic conditions under which entropy is to be defined. In this paper, I present an overview of definitions in current use for terms associated with distinguishability and relate them to various definitions that have been suggested for entropy. My hope is that consensus will be achievable if the definitions are clarified and agreed upon.

  16. Distinguishing between applied research and practice

    PubMed Central

    Johnston, James M.

    1996-01-01

    Behavior-analytic research is often viewed along a basic—applied continuum of research goals and methods. The applied portion of this continuum has evolved in ways that combine applied research and service delivery. Although these two facets of applied behavior analysis should be closely related, more clearly distinguishing between them, particularly in how we conceptualize and conduct applied research, may enhance the continuing development of each. This differentiation may improve the recruitment and training of graduate students. PMID:22478238

  17. Familial Identification: Population Structure and Relationship Distinguishability

    PubMed Central

    Rohlfs, Rori V.; Fullerton, Stephanie Malia; Weir, Bruce S.

    2012-01-01

    With the expansion of offender/arrestee DNA profile databases, genetic forensic identification has become commonplace in the United States criminal justice system. Implementation of familial searching has been proposed to extend forensic identification to family members of individuals with profiles in offender/arrestee DNA databases. In familial searching, a partial genetic profile match between a database entrant and a crime scene sample is used to implicate genetic relatives of the database entrant as potential sources of the crime scene sample. In addition to concerns regarding civil liberties, familial searching poses unanswered statistical questions. In this study, we define confidence intervals on estimated likelihood ratios for familial identification. Using these confidence intervals, we consider familial searching in a structured population. We show that relatives and unrelated individuals from population samples with lower gene diversity over the loci considered are less distinguishable. We also consider cases where the most appropriate population sample for individuals considered is unknown. We find that as a less appropriate population sample, and thus allele frequency distribution, is assumed, relatives and unrelated individuals become more difficult to distinguish. In addition, we show that relationship distinguishability increases with the number of markers considered, but decreases for more distant genetic familial relationships. All of these results indicate that caution is warranted in the application of familial searching in structured populations, such as in the United States. PMID:22346758

  18. Quantum correlations and distinguishability of quantum states

    SciTech Connect

    Spehner, Dominique

    2014-07-15

    A survey of various concepts in quantum information is given, with a main emphasis on the distinguishability of quantum states and quantum correlations. Covered topics include generalized and least square measurements, state discrimination, quantum relative entropies, the Bures distance on the set of quantum states, the quantum Fisher information, the quantum Chernoff bound, bipartite entanglement, the quantum discord, and geometrical measures of quantum correlations. The article is intended both for physicists interested not only by collections of results but also by the mathematical methods justifying them, and for mathematicians looking for an up-to-date introductory course on these subjects, which are mainly developed in the physics literature.

  19. Distinguishing ankle and knee articular cartilage.

    PubMed

    Cole, Ada A; Margulis, Arkady; Kuettner, Klaus E

    2003-06-01

    Degenerative changes in the tall and femoral distal cartilages of more than 2,000 tissue donors were graded based on the appearance of articular cartilage and osteophytes. In the ankle and the knee the degenerative changes increased with age; however, the rate of degeneration in the ankle was slower than in the knee. The degenerative changes in the ankle were more severe in men than in women, were predominantly bilateral, and seemed to be correlated with weight. The slower rate of change in the ankle may be caused, in part, by the biochemical and biomechanical tissue properties that distinguish ankle cartilage from that of the knee. PMID:12911243

  20. Can cosmic parallax distinguish between anisotropic cosmologies?

    SciTech Connect

    Fontanini, Michele; West, Eric J.; Trodden, Mark

    2009-12-15

    In an anisotropic universe, observers not positioned at a point of special symmetry should observe cosmic parallax--the relative angular motion of test galaxies over cosmic time. It was recently argued that the nonobservance of this effect in upcoming precision astrometry missions such as GAIA may be used to place strong bounds on the position of off-center observers in a void-model universe described by the Lemaitre-Tolman-Bondi metric. We consider the analogous effect in anisotropic cosmological models described by an axisymmetric homogeneous Bianchi type I metric and discuss whether any observation of cosmic parallax would distinguish between different anisotropic evolutions.

  1. Techniques to Distinguish Apoptosis from Necroptosis.

    PubMed

    Feoktistova, Maria; Wallberg, Fredrik; Tenev, Tencho; Geserick, Peter; Leverkus, Martin; Meier, Pascal

    2016-04-01

    The processes by which cells die are as tightly regulated as those that govern cell growth and proliferation. Recent studies of the molecular pathways that regulate and execute cell death have uncovered a plethora of signaling cascades that lead to distinct modes of cell death, including "apoptosis," "necrosis," "autophagic cell death," and "mitotic catastrophe." Cells can readily switch from one form of death to another; therefore, it is vital to have the ability to monitor the form of death that cells are undergoing. A number of techniques are available that allow the detection of cell death and when combined with either knockdown approaches or inhibitors of specific signaling pathways, such as caspase or RIP kinase pathways, they allow the rapid dissection of divergent cell death pathways. However, techniques that reveal the end point of cell death cannot reconstruct the sequence of events that have led to death; therefore, they need to be complemented with methods that can distinguish all forms of cell death. Apoptotic cells frequently undergo secondary necrosis under in vitro culture conditions; therefore, novel methods relying on high-throughput time-lapse fluorescence video microscopy are necessary to provide temporal resolution to cell death events. Further, visualizing the assembly of multiprotein signaling hubs that can execute apoptosis or necroptosis helps to explore the underlying processes. Here we introduce a suite of techniques that reliably distinguish necrosis from apoptosis and secondary necrosis, and that enable investigation of signaling platforms capable of instructing apoptosis or necroptosis. PMID:27037077

  2. Distinguishing Feedback Mechanisms in Clock Models

    NASA Astrophysics Data System (ADS)

    Golden, Alexander; Lubensky, David

    Biological oscillators are very diverse but can be classified based on dynamical motifs such as type of feedback. The S. Elongatus circadian oscillator is a novel circadian oscillator that can operate at constant protein number by modifying covalent states. It can be reproduced in vitro with only 3 different purified proteins: KaiA, KaiB, and KaiC. We use computational and analytic techniques to compare models of the S. Elongatus post-translational oscillator that rely on positive feedback with models that rely on negative feedback. We show that introducing a protein that binds competitively with KaiA to the KaiB-KaiC complex can distinguish between positive and negative feedback as the primary driver of the rhythm, which has so far been difficult to address experimentally. NSF Grant DMR-1056456.

  3. Distinguishing Asthma Phenotypes Using Machine Learning Approaches.

    PubMed

    Howard, Rebecca; Rattray, Magnus; Prosperi, Mattia; Custovic, Adnan

    2015-07-01

    Asthma is not a single disease, but an umbrella term for a number of distinct diseases, each of which are caused by a distinct underlying pathophysiological mechanism. These discrete disease entities are often labelled as 'asthma endotypes'. The discovery of different asthma subtypes has moved from subjective approaches in which putative phenotypes are assigned by experts to data-driven ones which incorporate machine learning. This review focuses on the methodological developments of one such machine learning technique-latent class analysis-and how it has contributed to distinguishing asthma and wheezing subtypes in childhood. It also gives a clinical perspective, presenting the findings of studies from the past 5 years that used this approach. The identification of true asthma endotypes may be a crucial step towards understanding their distinct pathophysiological mechanisms, which could ultimately lead to more precise prevention strategies, identification of novel therapeutic targets and the development of effective personalized therapies. PMID:26143394

  4. Do open clusters have distinguishable chemical signatures?

    NASA Astrophysics Data System (ADS)

    Blanco-Cuaresma, S.; Soubiran, C.; Heiter, U.

    2014-07-01

    Past studies have already shown that stars in open clusters are chemically homogeneous (e.g. De Silva et al. 2006, 2007 and 2009). These results support the idea that stars born from the same giant molecular cloud should have the same chemical composition. In this context, the chemical tagging technique was proposed by Freeman et al. (2002). The principle is to recover disrupted stellar clusters by looking only to the stellar chemical composition. In order to evaluate the feasibility of this approach, it is necessary to test if we can distinguish between stars born from different molecular clouds. For this purpose, we studied the chemical composition of stars in 32 old and intermediate-age open clusters, and we applied machine learning algorithms to recover the original cluster by only considering the chemical signatures.

  5. Inequality indicators and distinguishability in economics

    NASA Astrophysics Data System (ADS)

    Rosenblatt, J.; Martinás, K.

    2008-03-01

    Money has a material counterpart, such as banknotes or coins, and an ideal expression, monetary units. In the latter case, it is boson-like: individual incomes have no a priori limit, and their units are not distinguishable from each other in economic processes. Individuals, on the other hand, usually occupy one job at a time which makes them akin to fermions. We apply to individual incomes down-to-earth statistical calculations, similar to those for quantum particles, and obtain expressions for the cumulative distribution function, probability density and Lorenz function resulting from the simultaneous use of both statistics. They provide extremely good fits to corresponding data on French income distributions. On this basis, we propose a new entropic inequality indicator.

  6. Distinguishing Solar Cycle Effects in Planetary Atmospheres

    NASA Astrophysics Data System (ADS)

    Aplin, K. L.; Harrison, R. G.

    2008-12-01

    As solar radiation decreases with distance from the Sun, other sources of energy, such as ionization from galactic cosmic rays (GCR), assume a greater relative importance than at the terrestrial planets. Charged particle effects could therefore be more relevant to the formation of clouds and haze at the outer planets. The long-term solar modulation of Neptune's albedo is thought to be caused by either ion-induced nucleation of cloud-forming particles, or ultraviolet (UV) radiation effects on the colour of the clouds. On the basis of the 11 year solar cycle, the statistical evidence was slightly in favour of the UV mechanism, however distinguishing unambiguously between the two mechanisms will require more than the solar cycle variation alone. A 1.68 year quasi-periodicity, uniquely present at some times from heliospheric modulation of GCR, has previously been used to discriminate between solar UV and GCR effects in terrestrial data. The cosmic ray proton monitor data from both the Voyager spacecraft show this 1.68 year modulation during the 1980s when the spacecraft were close to the outer planets, indicating the possibility for applying a similar technique as far out as Neptune.

  7. How do recovery definitions distinguish recovering individuals?

    PubMed Central

    Witbrodt, Jane; Kaskutas, Lee Ann; Grella, Christine E.

    2015-01-01

    Background Six percent of American adults say they are “in recovery” from an alcohol or drug problem yet only a scant emergent literature has begun to ask how they define “recovery” or explored whether there is heterogeneity among their definitions. Methods Secondary analysis of the What Is Recovery? online survey employed Latent Class Analysis (LCA) to identify typologies of study participants based on their actual endorsement of 39 recovery elements and to compare the composition of these typologies in terms of distinguishing personal characteristics. Results A 5-class solution provided the best fit and conceptual representation for the recovery definitions. Classes were labeled 12-Step Traditionalist (n=4912); 12-Step Enthusiast (n=2014); Secular (n=980); Self-Reliant (n=1040); and Atypical (n=382) based on patterns of endorsement of the recovery elements. Abstinence, spiritual, and social interaction elements differentiated the classes most (as did age and recovery duration but to a lesser extent). Although levels and patterns of endorsement to the elements varied by class, a rank-ordering of the top 10 elements indicated that four elements were endorsed by all five classes: being honest with myself, handling negative feelings without using, being able to enjoy life, and process of growth and development. Conclusions The results of the LCA demonstrate the diversity of meanings, and varying degrees of identification with, specific elements of recovery. As others have found, multiple constituents are invested in how recovery is defined and this has ramifications for professional, personal, and cultural processes related to how strategies to promote recovery are implemented. PMID:25630961

  8. LDRD 149045 final report distinguishing documents.

    SciTech Connect

    Mitchell, Scott A.

    2010-09-01

    This LDRD 149045 final report describes work that Sandians Scott A. Mitchell, Randall Laviolette, Shawn Martin, Warren Davis, Cindy Philips and Danny Dunlavy performed in 2010. Prof. Afra Zomorodian provided insight. This was a small late-start LDRD. Several other ongoing efforts were leveraged, including the Networks Grand Challenge LDRD, and the Computational Topology CSRF project, and the some of the leveraged work is described here. We proposed a sentence mining technique that exploited both the distribution and the order of parts-of-speech (POS) in sentences in English language documents. The ultimate goal was to be able to discover 'call-to-action' framing documents hidden within a corpus of mostly expository documents, even if the documents were all on the same topic and used the same vocabulary. Using POS was novel. We also took a novel approach to analyzing POS. We used the hypothesis that English follows a dynamical system and the POS are trajectories from one state to another. We analyzed the sequences of POS using support vector machines and the cycles of POS using computational homology. We discovered that the POS were a very weak signal and did not support our hypothesis well. Our original goal appeared to be unobtainable with our original approach. We turned our attention to study an aspect of a more traditional approach to distinguishing documents. Latent Dirichlet Allocation (LDA) turns documents into bags-of-words then into mixture-model points. A distance function is used to cluster groups of points to discover relatedness between documents. We performed a geometric and algebraic analysis of the most popular distance functions and made some significant and surprising discoveries, described in a separate technical report.

  9. Charles L. Brewer Award for Distinguished Teaching of Psychology

    ERIC Educational Resources Information Center

    American Psychologist, 2009

    2009-01-01

    The American Psychological Foundation (APF) Charles L. Brewer Distinguished Teaching of Psychology Award recognizes an outstanding career contribution to the teaching of psychology. The 2009 recipient of the Distinguished Teaching Award is William Buskist. Dorothy W. Cantor, president of the APF, will present the APF Teaching Award at the 117th…

  10. 28 CFR 301.318 - Civilian compensation laws distinguished.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Civilian compensation laws distinguished... Civilian compensation laws distinguished. The Inmate Accident Compensation system is not obligated to... under civilian workmen's compensation laws in that hospitalization is usually completed prior to...

  11. 29 CFR 779.328 - Retail and wholesale distinguished.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 3 2011-07-01 2011-07-01 false Retail and wholesale distinguished. 779.328 Section 779.328... AS APPLIED TO RETAILERS OF GOODS OR SERVICES Exemptions for Certain Retail or Service Establishments ârecognizedâ As Retail âin the Particular Industryâ § 779.328 Retail and wholesale distinguished. (a)...

  12. Distinguishability of Biological Material Using Ultraviolet Multi-Spectral Fluorescence

    SciTech Connect

    Gray, P.C.; Heinen, R.J.; Rigdon, L.D.; Rosenthal, S.E.; Shokair, I.R.; Siragusa, G.R.; Tisone, G.C.; Wagner, J.S.

    1998-10-14

    Recent interest in the detection and analysis of biological samples by spectroscopic methods has led to questions concerning the degree of distinguishability and biological variability of the ultraviolet (W) fluorescent spectra from such complex samples. We show that the degree of distinguishability of such spectra is readily determined numerically.

  13. Liberating the Publications of a Distinguished Scholar: A Pilot Project

    ERIC Educational Resources Information Center

    Kelly, Julie

    2014-01-01

    Many distinguished scholars published the primary corpus of their work before the advent of online journals, which makes it more challenging to access. Upon being approached by a distinguished Emeritus Professor seeking advice about getting his work posted online, librarians at the University of Minnesota worked to gain copyright permissions to…

  14. Excellence in Teacher Education. 1973 Distinguished Achievement Awards Program.

    ERIC Educational Resources Information Center

    American Association of Colleges for Teacher Education, Washington, DC.

    The State University of New York College of Cortland received the 1973 Distinguished Achievement Award for its Project Change, a performance-based early childhood teacher education program. Also cited for distinguished achievement were a) Teachers College of Ball State University, Indiana for an experimental program designed to include…

  15. Charles L. Brewer Award for Distinguished Teaching of Psychology.

    PubMed

    2008-01-01

    The American Psychological Foundation (APF) Charles L. Brewer Distinguished Teaching of Psychology Award recognizes an outstanding career contribution to the teaching of psychology. The 2008 recipient of the Distinguished Teaching Award is Scott Plous. A citation, biography, and selected bibliography for Scott Plous are provided in this article. (PsycINFO Database Record (c) 2008 APA, all rights reserved). PMID:18665671

  16. John Glenn: Presented with NASA Distinguished Service Medal

    NASA Technical Reports Server (NTRS)

    1963-01-01

    John Glenn tours with his family, meets JFK and is presented with the NASA distinguished Service Medal. From: The John Glenn Story: Summary of astronaut John Glenn's flying career, from naval aviation training to space flight. The Mercury project is featured as John Glenn flies the Friendship 7 spacecraft. President John F. Kennedy presents the NASA Distinguished service Medal to Astronaut John Glenn.

  17. 32 CFR 22.205 - Distinguishing assistance from procurement.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 1 2010-07-01 2010-07-01 false Distinguishing assistance from procurement. 22... Instrument § 22.205 Distinguishing assistance from procurement. Before using a grant or cooperative agreement... procurement contract, is the appropriate instrument, based on the following: (a) Purpose. (1) The...

  18. Distinguishability of countable quantum states and von Neumann lattice

    NASA Astrophysics Data System (ADS)

    Kawakubo, Ryûitirô; Koike, Tatsuhiko

    2016-07-01

    The condition for distinguishability of a countably infinite number of pure states by a single measurement is given. Distinguishability is to be understood as the possibility of an unambiguous measurement. For a finite number of states, it is known that the necessary and sufficient condition of distinguishability is that the states are linearly independent. For an infinite number of states, several natural classes of distinguishability can be defined. We give a necessary and sufficient condition for a system of pure states to be distinguishable. It turns out that each level of distinguishability naturally corresponds to one of the generalizations of linear independence to families of infinite vectors. As an important example, we apply the general theory to von Neumann’s lattice, a subsystem of coherent states which corresponds to a lattice in the classical phase space. We prove that the condition for distinguishability is that the area of the fundamental region of the lattice is greater than the Planck constant, and also find subtle behavior on the threshold. These facts reveal the measurement theoretical meaning of the Planck constant and give a justification for the interpretation that it is the smallest unit of area in the phase space. The cases of uncountably many states and of mixed states are also discussed.

  19. Direct fluorescent antibody technique for the detection of bacterial kidney disease in paraffin-embedded tissues

    USGS Publications Warehouse

    Ochiai, T.; Yasutake, W.T.; Gould, R.W.

    1985-01-01

    The direct fluorescent antibody technique (FAT) was successfully used to detect the causative agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, in Bouin's solution flexed and paraffinembedded egg and tissue sections. This method is superior to gram stain and may be particularly useful in detecting the BKD organism in fish with low-grade infection.

  20. Strain gage network distinguishes between thermal and mechanical deformations

    NASA Technical Reports Server (NTRS)

    Cepollina, F. J.

    1966-01-01

    Strain gage network measures the thermal coefficient of linear expansion of composite metal structures. The network consists of a test gage and two dummy gages arranged to distinguish thermally induced deformation from mechanical strain.

  1. Charles L. Brewer Award for Distinguished Teaching of Psychology

    ERIC Educational Resources Information Center

    American Psychologist, 2007

    2007-01-01

    This article announces the 2007 recipient of the Charles L. Brewer Award for Distinguished Teaching of Psychology: Baron Perlman. A brief biography, highlighting areas of special focus in Perlman's work, is provided.

  2. Edna B. Foa: Award for Distinguished Scientific Contributions.

    PubMed

    2015-11-01

    The APA Awards for Distinguished Scientific Contributions are presented to persons who, in the opinion of the Committee on Scientific Awards, have made distinguished theoretical or empirical contributions to basic research in psychology. One of the 2015 award winners is Edna B. Foa, who received this award for "her outstanding and innovative research on the nature, measurement, and treatment of anxiety." Foa's award citation, biography, and a selected bibliography are presented here. PMID:26618942

  3. Carol A. Barnes: Award for Distinguished Scientific Contributions.

    PubMed

    2014-11-01

    The APA Awards for Distinguished Scientific Contributions are presented to persons who, in the opinion of the Committee on Scientific Awards, have made distinguished theoretical or empirical contributions to basic research in psychology. One of the 2014 award winners is Carol A. Barnes, who received this award for her "groundbreaking work on the neurobiological mechanisms underlying memory changes in normal aging." Barnes' award citation, biography, and a selected bibliography are presented here. PMID:25486138

  4. Richard N. Aslin: Award for Distinguished Scientific Contributions.

    PubMed

    2014-11-01

    The APA Awards for Distinguished Scientific Contributions are presented to persons who, in the opinion of the Committee on Scientific Awards, have made distinguished theoretical or empirical contributions to basic research in psychology. One of the 2014 award winners is Richard N. Aslin, who received this award for "elegance of thought in providing new ways to think about the relationships among learning, development, and biology." Aslin's award citation, biography, and a selected bibliography are presented here. PMID:25486136

  5. John A. Bargh: Award for Distinguished Scientific Contributions.

    PubMed

    2014-11-01

    The APA Awards for Distinguished Scientific Contributions are presented to persons who, in the opinion of the Committee on Scientific Awards, have made distinguished theoretical or empirical contributions to basic research in psychology. One of the 2014 award winners is John A. Bargh, who received this award for his "groundbreaking work on the automaticity of social cognition, emotion, motivation, and behavior." Bargh's award citation, biography, and a selected bibliography are presented here. PMID:25486137

  6. G. Terence Wilson: Award for Distinguished Scientific Applications of Psychology.

    PubMed

    2014-11-01

    The APA Award for Distinguished Scientific Applications of Psychology is presented to a person who, in the opinion of the Committee on Scientific Awards, has made distinguished theoretical or empirical advances leading to the understanding or amelioration of important practical problems. The 2014 recipient is G. Terence Wilson, who received the award for "his outstanding contributions to the nature and theory of behavior therapy." Wilson's award citation, biography, and a selected bibliography are presented here. PMID:25486140

  7. Distinguishing grammatical constructions with fMRI pattern analysis.

    PubMed

    Allen, Kachina; Pereira, Francisco; Botvinick, Matthew; Goldberg, Adele E

    2012-12-01

    All linguistic and psycholinguistic theories aim to provide psychologically valid analyses of particular grammatical patterns and the relationships that hold among them. Until recently, no tools were available to distinguish neural correlates of particular grammatical constructions that shared the same content words, propositional meaning, and degree of surface complexity, such as the dative (e.g., Sally gave the book to Joe) and the ditransitive (e.g., Sally gave Joe a book). We report the first fMRI data that distinguish such closely related, abstract grammatical patterns. Multi-voxel pattern analysis (MVPA) proved capable of discriminating at above-chance levels between activity patterns arising during reading of dative and ditransitive sentences. Region-of-interest analyses reveal that the union of certain language-relevant areas, anterior and posterior BA22, BA44/45 and BA47, yield classification accuracy above chance and above that of control conditions in the left hemisphere but not in the right. Looking more closely at the LH ROIs, we find that the combination of areas aBA22 and BA47 is sufficient to distinguish the two constructions better than the controls and better than chance. The fact that both of these areas-particularly BA47-have been implicated in semantics, lends support to claims that the two constructions are distinguishable semantically. More generally, the ability to distinguish closely related grammatical constructions using MVPA offers the promise of addressing traditional theoretical questions on a neuroscientifically grounded basis. PMID:23010489

  8. Two-Year-Olds Distinguish Pretending and Joking.

    PubMed

    Hoicka, Elena; Martin, Catriona

    2016-05-01

    Although children understand intentions to joke and pretend by 2 or 3 years, it is unclear whether they distinguish these intentional acts. Using a normativity paradigm, this study found (N = 72) 2-year-olds protest against jokes more than pretending, suggesting, for the first time, they distinguish these acts. Furthermore, toddlers protested more generally after pretend than literal or joke contexts but only if intentional cues were used. Additionally, children objected more to joking than pretending after pretend and literal contexts but not after joke contexts. Thus, toddlers distinguish the intentional nature of pretending and joking. Furthermore, a pretend intentional context establishes specific rules to be followed, whereas a joke intentional context allows an open space to perform various types of acts. PMID:27170036

  9. Entanglement as a resource to distinguish orthogonal product states

    PubMed Central

    Zhang, Zhi-Chao; Gao, Fei; Cao, Tian-Qing; Qin, Su-Juan; Wen, Qiao-Yan

    2016-01-01

    It is known that there are many sets of orthogonal product states which cannot be distinguished perfectly by local operations and classical communication (LOCC). However, these discussions have left the following open question: What entanglement resources are necessary and/or sufficient for this task to be possible with LOCC? In m ⊗ n, certain classes of unextendible product bases (UPB) which can be distinguished perfectly using entanglement as a resource, had been presented in 2008. In this paper, we present protocols which use entanglement more efficiently than teleportation to distinguish some classes of orthogonal product states in m ⊗ n, which are not UPB. For the open question, our results offer rather general insight into why entanglement is useful for such tasks, and present a better understanding of the relationship between entanglement and nonlocality. PMID:27458034

  10. Distinguishing succulent plants from crop and woody plants

    NASA Technical Reports Server (NTRS)

    Gausman, H. W.; Escobar, D. E.; Everitt, J. H.; Richardson, A. J.; Rodriguez, R. R.

    1978-01-01

    We compared laboratory spectrophotometrically measured leaf reflectances of six succulents (peperomia, possum-grape, prickly pear, spiderwort, Texas tuberose, wolfberry) with those of four nonsucculents (cenizo, honey mesquite, cotton, sugarcane) for plant species discrimination. Succulents (average leaf water content of 92.2 percent) could be distinguished from nonsucculents (average leaf water content of 71.2 percent) within the near-infrared water absorption waveband (1.35 to 2.5 microns). This was substantiated by field spectrophotometric reflectances of plant canopies. Sensor bands encompassing either the 1.6- or 2.2-wavelengths may be useful to distinguish succulent from nonsucculent plant species.

  11. The distinguishable cluster approach from a screened Coulomb formalism.

    PubMed

    Kats, Daniel

    2016-01-28

    The distinguishable cluster doubles equations have been derived starting from an effective screened Coulomb formalism and a particle-hole symmetric formulation of the Fock matrix. A perturbative triples correction to the distinguishable cluster with singles and doubles (DCSD) has been introduced employing the screened integrals. It is shown that the resulting DCSD(T) method is more accurate than DCSD for reaction energies and is less sensitive to the static correlation than coupled cluster with singles and doubles with a perturbative triples correction. PMID:26827197

  12. Recent Detrimental and Distinguished Books about Hispanic People and Cultures.

    ERIC Educational Resources Information Center

    Schon, Isabel

    1981-01-01

    The article discusses nine detrimental and six distinguished books about Hispanic people and cultures, published since 1979 for young readers. It is suggested that many recent books that depict Hispanic people and cultures repeat the same stereotypes, misconceptions and insensibilities that were prevalent in books published in the 1960s and early…

  13. Elliptic cylinder geometry for distinguishability analysis in impedance tomography.

    PubMed

    Saka, Birsen; Yilmaz, Atila

    2004-01-01

    Electrical impedance tomography (EIT) is a technique that computes the cross-sectional impedance distribution within the body by using current and voltage measurements made on the body surface. It has been reported that the image reconstruction is distorted considerably when the boundary shape is considered to be more elliptical than circular as a more realistic shape for the measurement boundary. This paper describes an alternative framework for determining the distinguishability region with a finite measurement precision for different conductivity distributions in a body modeled by elliptic cylinder geometry. The distinguishable regions are compared in terms of modeling error for predefined inhomogeneities with elliptical and circular approaches for a noncircular measurement boundary at the body surface. Since most objects investigated by EIT are noncircular in shape, the analytical solution for the forward problem for the elliptical cross section approach is shown to be useful in order to reach a better assessment of the distinguishability region defined in a noncircular boundary. This paper is concentrated on centered elliptic inhomogeneity in the elliptical boundary and an analytic solution for this type of forward problem. The distinguishability performance of elliptical cross section with cosine injected current patterns is examined for different parameters of elliptical geometry. PMID:14723501

  14. Bifunctional mesoporous silicas with clearly distinguished localization of grafted groups

    NASA Astrophysics Data System (ADS)

    Roik, N. V.; Belyakova, L. A.

    2013-12-01

    Bifunctional mesoporous silicas with clearly distinguished localization of grafted groups on the surface of particles and inside their pores were obtained by means of sol-gel synthesis with postsynthetic vapor-phase treatment in vacuum. It was found that the synthesized materials have the hexagonally ordered porous structure typical of MCM-41 type silica.

  15. Distinguishing Grammatical Constructions with fMRI Pattern Analysis

    ERIC Educational Resources Information Center

    Allen, Kachina; Pereira, Francisco; Botvinick, Matthew; Goldberg, Adele E.

    2012-01-01

    All linguistic and psycholinguistic theories aim to provide psychologically valid analyses of particular grammatical patterns and the relationships that hold among them. Until recently, no tools were available to distinguish neural correlates of particular grammatical constructions that shared the same content words, propositional meaning, and…

  16. Distinguishing between Poor/Dysfunctional Parenting and Child Emotional Maltreatment

    ERIC Educational Resources Information Center

    Wolfe, David A.; McIsaac, Caroline

    2011-01-01

    Objective: This paper was intended to distinguish between poor parenting and child emotional maltreatment (CEM), to inform child welfare and public health policymakers of the need for differentiated responses. Methods: Scientific literature was integrated with current practice and assumptions relating to poor/dysfunctional parenting and child…

  17. Award for Distinguished Scientific Contributions: Alice H. Eagly

    ERIC Educational Resources Information Center

    American Psychologist, 2009

    2009-01-01

    Alice H. Eagly, winner of the Award for Distinguished Scientific Contributions, is cited for her work in the field of social psychology, the psychology of gender, and the use of meta-analytic techniques. She envisions a psychology that extends from individual cognitions to societal structures. In addition to the citation, a biography and selected…

  18. A Conceptual and Psychometric Framework for Distinguishing Categories and Dimensions

    ERIC Educational Resources Information Center

    De Boeck, Paul; Wilson, Mark; Acton, G. Scott

    2005-01-01

    An important, sometimes controversial feature of all psychological phenomena is whether they are categorical or dimensional. A conceptual and psychometric framework is described for distinguishing whether the latent structure behind manifest categories (e.g., psychiatric diagnoses, attitude groups, or stages of development) is category-like or…

  19. A Tribute to My Ag Teacher: 2011 AAAE Distinguished Lecture

    ERIC Educational Resources Information Center

    Barrick, R. Kirby

    2012-01-01

    The author is a product of school-based agricultural education. In a way, this distinguished lecture could also be called a tribute to his high school ag teacher, John Stimpert. Mr. Stimpert was a true professional and an excellent teacher. He changed and he changed the program with the changing school and community. The more the author became…

  20. Distinguishing Schemes and Tasks in Children's Development of Multiplicative Reasoning

    ERIC Educational Resources Information Center

    Tzur, Ron; Johnson, Heather L.; McClintock, Evan; Kenney, Rachael H.; Xin, Yan P.; Si, Luo; Woordward, Jerry; Hord, Casey; Jin, Xianyan

    2013-01-01

    We present a synthesis of findings from constructivist teaching experiments regarding six schemes children construct for reasoning multiplicatively and tasks to promote them. We provide a task-generating platform game, depictions of each scheme, and supporting tasks. Tasks must be distinguished from children's thinking, and learning situations…

  1. Michael J. Meaney: Award for Distinguished Scientific Contributions

    ERIC Educational Resources Information Center

    American Psychologist, 2012

    2012-01-01

    Presents Michael J. Meaney as one of the winners of the American Psychological Association's Award for Distinguished Scientific Contributions (2012). Michael J. Meaney has taken the phenomenon of "handling" of newborn rats and opened a new area of investigation that has given new meaning to epigenetics via his work demonstrating transgenerational…

  2. Award for Distinguished Scientific Contributions: Susan E. Carey

    ERIC Educational Resources Information Center

    American Psychologist, 2009

    2009-01-01

    Susan E. Carey, winner of the 2009 Award for Distinguished Scientific Contributions, is cited for groundbreaking studies of the nature of concepts and conceptual change. Her research deepens understanding of the development of concepts, and of the belief systems in which they are embedded, over human childhood, over the history of science, and…

  3. The Identification of Conductor-Distinguished Functions of Conducting

    ERIC Educational Resources Information Center

    Gumm, Alan J.; Battersby, Sharyn L.; Simon, Kathryn L.; Shankles, Andrew E.

    2011-01-01

    The purpose of the present study was to identify whether conductors distinguish functions of conducting similarly to functions implied in previous research. A sample of 84 conductors with a full range of experience levels (M = 9.8) and of a full range of large ensemble types and ensemble age levels rated how much they pay attention to 82…

  4. Distinguishing between Realistic and Fantastical Figures in Iran

    ERIC Educational Resources Information Center

    Davoodi, Telli; Corriveau, Kathleen H.; Harris, Paul L.

    2016-01-01

    Children in the United States come to distinguish historical from fictional story figures between the ages of 3 and 5 years, guided by the plausibility of the story events surrounding the figure (Corriveau, Kim, Schwalen, & Harris, 2009; Woolley & Cox, 2007). However, U.S. children vary in their reactions to stories that include…

  5. Distinguishing between Dirac and Majorana neutrinos withtwo-particle interferometry

    SciTech Connect

    Gutierrez, Thomas D.

    2006-03-02

    Two-particle interferometry, a second-order interferenceeffect, is explored as another possible tool to distinguish betweenmassive Dirac and Majorana neutrinos. A simple theoretical framework isdiscussed in the context of several gedanken experiments. The method canin principle provide both the mass scale and the quantum nature of theneutrino for a certain class of incoherent left-handed sourcecurrents.

  6. Daniel L. Schacter: Award for Distinguished Scientific Contributions

    ERIC Educational Resources Information Center

    American Psychologist, 2012

    2012-01-01

    Presents Daniel L. Schacter as one of the winners of the American Psychological Association's Award for Distinguished Scientific Contributions (2012). Daniel L. Schacter's major theoretical and empirical contributions include groundbreaking research on the psychological and neural foundations of implicit and explicit memory, memory distortions and…

  7. Edward F. Diener: Award for Distinguished Scientific Contributions

    ERIC Educational Resources Information Center

    American Psychologist, 2012

    2012-01-01

    Presents Edward F. Diener as one of the winners of the American Psychological Association's Award for Distinguished Scientific Contributions (2012). Edward F. Diener has been a leader in every aspect of well-being research. He provided an influential conception of well-being as consisting of cognitive and emotional elements. A citation, biography,…

  8. Award for Distinguished Scientific Contributions: Steven F. Maier

    ERIC Educational Resources Information Center

    American Psychologist, 2009

    2009-01-01

    Steven F. Maier, winner of the Award for Distinguished Scientific Contributions, is cited for his work in the fields of learned helplessness; cytokines, depressed mood, and cognitive interference; and the brain structures that produce and counteract learned helplessness. In addition to the citation, a biography and selected bibliography of Maier's…

  9. 32 CFR 22.205 - Distinguishing assistance from procurement.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... research and development, the appropriate use of grants and cooperative agreements therefore is almost exclusively limited to the performance of selected basic, applied, and advanced research projects. Development... 32 National Defense 1 2013-07-01 2013-07-01 false Distinguishing assistance from procurement....

  10. 32 CFR 22.205 - Distinguishing assistance from procurement.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... research and development, the appropriate use of grants and cooperative agreements therefore is almost exclusively limited to the performance of selected basic, applied, and advanced research projects. Development... 32 National Defense 1 2014-07-01 2014-07-01 false Distinguishing assistance from procurement....