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bacterial kidney disease

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bacterial kidney disease

Summary

  • Last modified
  • 14 July 2018
  • Datasheet Type(s)
  • Animal Disease
  • Preferred Scientific Name
  • bacterial kidney disease
  • Overview
  • Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a prevalent disease that impacts the sustainable production of salmonid fish for consumption and species conservation efforts. The disease...

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PictureTitleCaptionCopyright
Kidney section from infected coho salmon reared in a commercial net-pen farm in Puget Sound, Washington State. Note granulomatous foci scattered throughout the kidney.
TitlePathology
CaptionKidney section from infected coho salmon reared in a commercial net-pen farm in Puget Sound, Washington State. Note granulomatous foci scattered throughout the kidney.
CopyrightJ. Heidel/R. Elston
Kidney section from infected coho salmon reared in a commercial net-pen farm in Puget Sound, Washington State. Note granulomatous foci scattered throughout the kidney.
PathologyKidney section from infected coho salmon reared in a commercial net-pen farm in Puget Sound, Washington State. Note granulomatous foci scattered throughout the kidney.J. Heidel/R. Elston
Exposed midsection of an infected coho salmon. Note granulomatous lesions in liver and spleen.
TitlePathology
CaptionExposed midsection of an infected coho salmon. Note granulomatous lesions in liver and spleen.
CopyrightJ. Heidel/R. Elston
Exposed midsection of an infected coho salmon. Note granulomatous lesions in liver and spleen.
PathologyExposed midsection of an infected coho salmon. Note granulomatous lesions in liver and spleen. J. Heidel/R. Elston
(a) Section of the kidney of an infected coho salmon reared in a commercial net-pen salmon farm in Puget Sound, Washington State. Note the granulomatous foci scattered throughout the kidney (arrow). Lesions are typically found in naturally infected and fish experimentally infected with a low dose of R. salmoninarum. (b) Exposed midsection of an infected coho salmon. Arrows denote granulomatous lesions in the liver (left) and within the spleen (right). Photos are courtesy of Dr J. Heidel, Department of Veterinary Medicine, Oregon State University, Oregon, and Dr R. Elston, Battelle Pacific Northwest Laboratories, Washington State.
TitleInfected coho salmon
Caption(a) Section of the kidney of an infected coho salmon reared in a commercial net-pen salmon farm in Puget Sound, Washington State. Note the granulomatous foci scattered throughout the kidney (arrow). Lesions are typically found in naturally infected and fish experimentally infected with a low dose of R. salmoninarum. (b) Exposed midsection of an infected coho salmon. Arrows denote granulomatous lesions in the liver (left) and within the spleen (right). Photos are courtesy of Dr J. Heidel, Department of Veterinary Medicine, Oregon State University, Oregon, and Dr R. Elston, Battelle Pacific Northwest Laboratories, Washington State.
CopyrightG. D. Wiens & S. L. Kaattari
(a) Section of the kidney of an infected coho salmon reared in a commercial net-pen salmon farm in Puget Sound, Washington State. Note the granulomatous foci scattered throughout the kidney (arrow). Lesions are typically found in naturally infected and fish experimentally infected with a low dose of R. salmoninarum. (b) Exposed midsection of an infected coho salmon. Arrows denote granulomatous lesions in the liver (left) and within the spleen (right). Photos are courtesy of Dr J. Heidel, Department of Veterinary Medicine, Oregon State University, Oregon, and Dr R. Elston, Battelle Pacific Northwest Laboratories, Washington State.
Infected coho salmon(a) Section of the kidney of an infected coho salmon reared in a commercial net-pen salmon farm in Puget Sound, Washington State. Note the granulomatous foci scattered throughout the kidney (arrow). Lesions are typically found in naturally infected and fish experimentally infected with a low dose of R. salmoninarum. (b) Exposed midsection of an infected coho salmon. Arrows denote granulomatous lesions in the liver (left) and within the spleen (right). Photos are courtesy of Dr J. Heidel, Department of Veterinary Medicine, Oregon State University, Oregon, and Dr R. Elston, Battelle Pacific Northwest Laboratories, Washington State.G. D. Wiens & S. L. Kaattari
Schematic diagram of the theory and practice of the monoclonal antibody-based ELISA described in Rockey et al. (1991a). An individual well of an ELISA plate is depicted after each step in the assay. (1) The wells are coated with a primary monoclonal antibody (MAb 4D3) and the unbound sites on the plate are then blocked with a non-specific protein, bovine serum albumin (not shown). (2) Serial dilutions of a standard p57 preparation or dilutions of clinical samples are added. The primary antibody 4D3 binds to an amino-proximal epitope on p57, specifically retaining p57 but not other proteins in the sample. (3) A secondary biotinylated MAb (3H1) is used to recognize a specific epitope in the middle of p57. B, biotin. (4) MAb 3H1 is biotinylated, which is bound subsequently by an avidin-enzyme conjugate. (5) A chromogenic substrate is added and the colorimetric reaction is measured on an ELISA reader.
TitleTheory and practice of the monoclonal antibody-based ELISA
CaptionSchematic diagram of the theory and practice of the monoclonal antibody-based ELISA described in Rockey et al. (1991a). An individual well of an ELISA plate is depicted after each step in the assay. (1) The wells are coated with a primary monoclonal antibody (MAb 4D3) and the unbound sites on the plate are then blocked with a non-specific protein, bovine serum albumin (not shown). (2) Serial dilutions of a standard p57 preparation or dilutions of clinical samples are added. The primary antibody 4D3 binds to an amino-proximal epitope on p57, specifically retaining p57 but not other proteins in the sample. (3) A secondary biotinylated MAb (3H1) is used to recognize a specific epitope in the middle of p57. B, biotin. (4) MAb 3H1 is biotinylated, which is bound subsequently by an avidin-enzyme conjugate. (5) A chromogenic substrate is added and the colorimetric reaction is measured on an ELISA reader.
CopyrightG. D. Wiens & S. L. Kaattari
Schematic diagram of the theory and practice of the monoclonal antibody-based ELISA described in Rockey et al. (1991a). An individual well of an ELISA plate is depicted after each step in the assay. (1) The wells are coated with a primary monoclonal antibody (MAb 4D3) and the unbound sites on the plate are then blocked with a non-specific protein, bovine serum albumin (not shown). (2) Serial dilutions of a standard p57 preparation or dilutions of clinical samples are added. The primary antibody 4D3 binds to an amino-proximal epitope on p57, specifically retaining p57 but not other proteins in the sample. (3) A secondary biotinylated MAb (3H1) is used to recognize a specific epitope in the middle of p57. B, biotin. (4) MAb 3H1 is biotinylated, which is bound subsequently by an avidin-enzyme conjugate. (5) A chromogenic substrate is added and the colorimetric reaction is measured on an ELISA reader.
Theory and practice of the monoclonal antibody-based ELISASchematic diagram of the theory and practice of the monoclonal antibody-based ELISA described in Rockey et al. (1991a). An individual well of an ELISA plate is depicted after each step in the assay. (1) The wells are coated with a primary monoclonal antibody (MAb 4D3) and the unbound sites on the plate are then blocked with a non-specific protein, bovine serum albumin (not shown). (2) Serial dilutions of a standard p57 preparation or dilutions of clinical samples are added. The primary antibody 4D3 binds to an amino-proximal epitope on p57, specifically retaining p57 but not other proteins in the sample. (3) A secondary biotinylated MAb (3H1) is used to recognize a specific epitope in the middle of p57. B, biotin. (4) MAb 3H1 is biotinylated, which is bound subsequently by an avidin-enzyme conjugate. (5) A chromogenic substrate is added and the colorimetric reaction is measured on an ELISA reader.G. D. Wiens & S. L. Kaattari
Photo of an ELISA plate after substrate addition. The dark colour of the substrate indicates the presence of R. salmoninarum antigen. On the bottom of the plate (rows F, G, H) is a dilution of a p57 standard (six wells per dilution), while kidney homogenates of indivdual fish samples are on the remainder of the plate rows A-E (samples are in duplicate). Comparison of the colour intensity of the clinical samples with the standard allows for precise quantification of antigen levels in fish tissues. The image was supplied courtesy of Dr John Reddington, Diaxotics, Wilton, Connecticut.
TitleTheory and practice of the monoclonal antibody-based ELISA
CaptionPhoto of an ELISA plate after substrate addition. The dark colour of the substrate indicates the presence of R. salmoninarum antigen. On the bottom of the plate (rows F, G, H) is a dilution of a p57 standard (six wells per dilution), while kidney homogenates of indivdual fish samples are on the remainder of the plate rows A-E (samples are in duplicate). Comparison of the colour intensity of the clinical samples with the standard allows for precise quantification of antigen levels in fish tissues. The image was supplied courtesy of Dr John Reddington, Diaxotics, Wilton, Connecticut.
CopyrightG. D. Wiens & S. L. Kaattari
Photo of an ELISA plate after substrate addition. The dark colour of the substrate indicates the presence of R. salmoninarum antigen. On the bottom of the plate (rows F, G, H) is a dilution of a p57 standard (six wells per dilution), while kidney homogenates of indivdual fish samples are on the remainder of the plate rows A-E (samples are in duplicate). Comparison of the colour intensity of the clinical samples with the standard allows for precise quantification of antigen levels in fish tissues. The image was supplied courtesy of Dr John Reddington, Diaxotics, Wilton, Connecticut.
Theory and practice of the monoclonal antibody-based ELISAPhoto of an ELISA plate after substrate addition. The dark colour of the substrate indicates the presence of R. salmoninarum antigen. On the bottom of the plate (rows F, G, H) is a dilution of a p57 standard (six wells per dilution), while kidney homogenates of indivdual fish samples are on the remainder of the plate rows A-E (samples are in duplicate). Comparison of the colour intensity of the clinical samples with the standard allows for precise quantification of antigen levels in fish tissues. The image was supplied courtesy of Dr John Reddington, Diaxotics, Wilton, Connecticut.G. D. Wiens & S. L. Kaattari
Model of structural regions of p57 while associated with the bacterial cell surface. The amino-terminal region is surface-exposed and available to bind leucocytes. Reprinted with permission from Wiens and Kaattari (1991).
TitleModel of structural regions of p57
CaptionModel of structural regions of p57 while associated with the bacterial cell surface. The amino-terminal region is surface-exposed and available to bind leucocytes. Reprinted with permission from Wiens and Kaattari (1991).
CopyrightG. D. Wiens & S. L. Kaattari
Model of structural regions of p57 while associated with the bacterial cell surface. The amino-terminal region is surface-exposed and available to bind leucocytes. Reprinted with permission from Wiens and Kaattari (1991).
Model of structural regions of p57Model of structural regions of p57 while associated with the bacterial cell surface. The amino-terminal region is surface-exposed and available to bind leucocytes. Reprinted with permission from Wiens and Kaattari (1991).G. D. Wiens & S. L. Kaattari
Model of one possible mechanism of action of p57. When associated with the bacterial cell surface, p57 may enhance binding, either specifically or non-specifically, to phagocytic cells, which would facilitate uptake or invasion by R. salmoninarum. Evidence suggests that R. salmoninarum is able to escape from the phagolysosome, which would allow access to intracellular nutrients and escape from damage by lysosomal enzymes and oxidative species.
TitleModel of possible mechanism of action of p57
CaptionModel of one possible mechanism of action of p57. When associated with the bacterial cell surface, p57 may enhance binding, either specifically or non-specifically, to phagocytic cells, which would facilitate uptake or invasion by R. salmoninarum. Evidence suggests that R. salmoninarum is able to escape from the phagolysosome, which would allow access to intracellular nutrients and escape from damage by lysosomal enzymes and oxidative species.
CopyrightG. D. Wiens & S. L. Kaattari
Model of one possible mechanism of action of p57. When associated with the bacterial cell surface, p57 may enhance binding, either specifically or non-specifically, to phagocytic cells, which would facilitate uptake or invasion by R. salmoninarum. Evidence suggests that R. salmoninarum is able to escape from the phagolysosome, which would allow access to intracellular nutrients and escape from damage by lysosomal enzymes and oxidative species.
Model of possible mechanism of action of p57Model of one possible mechanism of action of p57. When associated with the bacterial cell surface, p57 may enhance binding, either specifically or non-specifically, to phagocytic cells, which would facilitate uptake or invasion by R. salmoninarum. Evidence suggests that R. salmoninarum is able to escape from the phagolysosome, which would allow access to intracellular nutrients and escape from damage by lysosomal enzymes and oxidative species.G. D. Wiens & S. L. Kaattari

Identity

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Preferred Scientific Name

  • bacterial kidney disease

International Common Names

  • English: corynebacterial kidney disease; Dee disease; Renibacterium salmoninarum infection; salmonid kidney disease; white boil disease

English acronym

  • BKD

Overview

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Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a prevalent disease that impacts the sustainable production of salmonid fish for consumption and species conservation efforts. The disease is chronic in nature and mortality most often occurs in 6-12 month old juvenile salmonids and prespawning adults (Evelyn, 1993). R. salmoninarum is a small (0.3-0.1 µm by 1.0-1.5 µm), nonmotile, nonspore-forming, nonacid-fast, Gram-positive diplobacillus (Fryer and Sanders, 1981; Euzeby, 2005). There is only one species described in the genus to date. Little is known about R. salmoninarum due, in part, to the technical difficulties of working with this slow growing and fastidious microorganism. R. salmoninarum was recently selected for complete genome sequencing, which will provide a first step toward a more comprehensive understanding of the biology of this microbe (http://micro.nwfsc.noaa.gov/rs-genome/).

The kidney disease bacterium can be transmitted both horizontally from infected fish sharing a water supply (Bell et al., 1984; Mitchum and Sherman, 1981) and vertically via association with eggs from infected parents (Evelyn et al., 1986b; Pascho et al., 1991b). The spread of BKD has followed the expansion of salmonid culture (Rohovec and Fryer, 1988; Evenden et al., 1993) and most recorded outbreaks of BKD have occurred in fish culture facilities. Losses as high as 80% in stocks of Pacific salmon and 40% in stocks of Atlantic salmon (Salmo salar) have been reported (Evenden et al., 1993). Salmonids vary in their susceptibility to BKD: Pacific salmon species are the most susceptible, while Atlantic salmon and rainbow trout are considered more resistant (Evelyn, 1993; Fryer and Lannan, 1993; Sakai et al., 1991; Starliper et al., 1997). The disease has also been documented in naturally spawning populations that have never been supplemented with hatchery fish (Evelyn et al., 1973; Souter et al., 1987). The chronic nature of the disease has hindered accurate estimates of fish losses, particularly in feral fish populations (Banner et al., 1986; Maclean and Yoder, 1970; Mitchum et al., 1979; Pippy, 1969). BKD is also a concern for several endangered salmonid species protection programs (Flagg et al., 1995).

As with other infectious diseases of salmonids that are difficult or impossible to treat, avoidance is recommended for the control of BKD in cultured salmonid stocks (Ahne et al., 1989; Evelyn, 1993). Because R. salmoninarum can be enzootic in wild salmonid populations (Bruneau et al., 1999; Evelyn et al., 1973; Jonsdottir et al., 1998), measures to control losses from BKD may be defeated by constant exposure of hatchery fish to waterborne bacteria shed into the water supply by wild fish residing upstream from the hatchery (Hastein and Lindstad, 1991; Mitchum and Sherman, 1981). Brood stock segregation or culling is now used to select egg lots to retain as a source of juvenile fish for hatchery rearing (Meyers et al., 2003; Gudmundsdottir et al., 2000; Pascho et al., 1991b; Warren, 1991). The selection process is aimed at rearing egg lots from mating pairs with undetectable or very low levels of R. salmoninarum. Losses from BKD among the progeny of parents with very low levels of R. salmoninarum have been less than those among the progeny of parents with very high infection levels. The aquaculturist must be aware, however, that brood stock segregation may not completely eliminate BKD from an affected population and the impact of segregation on other traits is unknown. Because the broodstock used for commercial fish farming should be free of the kidney disease bacterium, it may be necessary to repopulate a contaminated facility with brood fish from a BKD-free population. The regular fallowing, particularly of marine sites, may also help break the disease cycle (Bruno, 2004).

TAXONOMY

Historical background

Bacterial kidney disease, also referred to as white boil disease, salmonid kidney disease, corynebacterial kidney disease or Dee disease, was first reported in 1933 in Atlantic salmon (Mackie et al., 1933). The first report in the U.S. was by Belding and Merrill (1935) who observed a Gram-positive bacterium associated with necrotic kidney lesions of salmonids, but they were unable to culture the disease agent. Cultivation of the organism on cysteine-blood-enriched medium made possible the completion of Koch’s postulates and identification of the aetiological agent as a slow-growing Gram-positive bacterium (Rucker et al., 1954; Ordal and Earp, 1956). For additional information on historical aspects of bacterial kidney disease, readers are referred to reviews by Fryer and Sanders (1981), Klontz (1983), Bullock and Herman (1988).

Current classification

Initially, Ordal and Earp (1956) classified the organism as a Corynebacterium sp. Smith (1964) concurred with this classification based on specific characteristics of the bacterium, such as aerobic growth, lack of endospores, production of metachromatic granules, reproduction by binary fission and pathogenic capability. Young and Chapman (1978), however, were unable to find metachromatic granules or the postfission snapping process associated with corynebacteria. Further, the absence of mycolic acids (Goodfellow et al., 1976; Fryer and Sanders, 1981), the presence of lysine in the peptidoglycan instead of m-diaminopimelic acid (Sanders and Fryer, 1980) and the difference in lipid composition (Collins, 1982; Embley et al., 1983) do not support the placement of the bacterium in the genus Corynebacterium or in the genus Listeria, as proposed by Bullock et al. (1974). Sanders and Fryer (1980) concluded that the organism belonged to a unique genus and named it Renibacterium salmoninarum (kidney bacterium of salmon). Molecular cataloguing and sequencing of the 16S ribosomal ribonucleic acid (RNA) from R. salmoninarum (Stackebrandt et al., 1988, Gutenberger et al., 1991) and recalculation of the guanine plus cytosine (G + C) content (Banner et al., 1991) support the placing of the organism as a member of the high G + C content, Gram-positive, eubacterial subdivision of the actinomycetes. The closest known relatives of R. salmoninarum include Arthrobacter and Micrococcus. As yet, additional species have not been included in this genus, nor have subspecies been identified. The availability in the near future of the whole genome sequence may shed further light on the evolutionary relationship of R. salmoninarum to other high G + C Gram-positive bacteria.

[Based upon material originally published in Woo PTK, Bruno DW, eds, 1999. Fish diseases and disorders, Vol. 3 Viral, bacterial and fungal infections. Wallingford, UK: CABI Publishing.]

Hosts/Species Affected

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Salmonids of the genera Oncorhynchus, Salmo and Salvelinus appear to be the primary hosts of R. salmoninarum (reviewed in Fryer and Sanders, 1981). Natural infections have also been documented in Hucho hucho (Danube salmon; Pfeil-Putzien et al., 1985), Thymallus thymallus (grayling; Kettler et al., 1986), Plecoglossus altivelis (ayu; Nagai and Iida, 2002), and Coregonus lavaretus (whitefish; Rimaila-Parnanen, 2002). Experimental infection and mortality have been induced in a non-salmonid species - Anoplopoma fimbria (sablefish; Bell et al., 1990) and Clupea harengus pallasii (Pacific herring; Evelyn, 1993) - but not in Cyprinus carpio (carp; Sakai et al., 1989b) or Lampetra tridentata (lamprey; Bell and Traxler, 1986). Sakai and Kobayashi (1992) found R. salmoninarum antigen in Patinopecten yessoensis (scallops), Platycephalus indicus (flathead) and Cottus japonicus (Japanese sculpin) sampled near a coho salmon salt-water net pen facility. However, the importance of these species as reservoirs of infection has yet to be established as viable R. salmoninarum could not be cultured. Efforts to identify R. salmoninarum in mussels (Mytilus edulis), and non-salmonid fin-fish which reside in and around sea-water net pens in the Pacific Northwest, have not been successful (Paclibare et al., 1988; Kent et al., 1998). In addition, three species of fresh water bivalves challenged with R. salmoninarum failed to show infection or transmit R. salmoninarum to rainbow trout by cohabitation (Starliper and Morrison, 2000). These studies collectively indicate that R. salmoninarum is highly adapted for infection and persistence in salmonids.

Distribution

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Bacterial kidney disease is commonly observed in cultured salmonid stocks from North America, continental Europe, Japan (Fryer and Sanders, 1981), South America (Sanders and Barros, 1986), Scotland (Bruno, 1986c) and Scandinavia (Ljungberg et al., 1990; Gudmundsdóttir et al., 1993). Significant populations of free-ranging fish also harbour R. salmoninarum (Pippy, 1969; Evelyn et al., 1973; Ellis et al., 1978; Mitchum et al., 1979; Paterson et al., 1979, 1981b; Banner et al., 1986; Souter et al., 1987; Elliott and Pascho, 1991; Sanders et al., 1992, Meyers et al., 1993). BKD has not been reported in Ireland, Australia, New Zealand or the former Soviet Union (Bruno, 2004).

Distribution Table

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The distribution in this summary table is based on all the information available. When several references are cited, they may give conflicting information on the status. Further details may be available for individual references in the Distribution Table Details section which can be selected by going to Generate Report.

Pathology

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Fish with severe R. salmoninarum infections may show no obvious external signs, or they may exhibit one or more of the following: lethargy; skin darkening; abdominal distension due to ascites; pale gills associated with anaemia; exophthalmos; haemorrhages around the vent; and cystic cavities in the skeletal muscle. Internal examination usually reveals the presence of focal to multifocal grayish-white nodular lesions in the kidney (see figure: kidney section from infected coho salmon), and sometimes in the spleen and liver (see figure: exposed midsection of an infected coho salmon). In addition, there may be turbid fluid in the abdominal cavity, haemorrhages on the abdominal wall and in the viscera, and a diffuse white membranous layer (pseudomembrane) on one or more of the internal organs (Fryer and Sanders, 1981; Evelyn, 1993; Evenden et al., 1993).

Infection changes a number of haematological and serum parameters in both experimentally and naturally infected fish. Circulating erythrocytes decrease 59-66% during infection (Bruno, 1986a, b). In addition, erythrocyte diameter decreases from 16.6 µm to 14.5 µm and erythrocyte sedimentation rate increases. Increased progression of disease correlates with decreased haematocrit, cholesterol, sodium and electrophoretically faster migrating serum proteins, as well as an increase in serum bilirubin, blood urea nitrogen and potassium concentrations (Hunn, 1964; Suzumoto et al., 1977; Aldrin et al., 1978; Bruno, 1986a; Turaga et al., 1987a). No changes in small and large lymphocyte numbers occur, but there is a transitory increase in neutrophils, monocytes and thrombocytes after bacterial injection (Bruno and Munro, 1986a). Increased levels of p57 protein correlate with the severity of infection and also a decrease in haematocrit (Turaga et al., 1987b). High levels of p57 are found in experimentally and naturally infected fish (Turaga et al., 1987b; Rockey et al., 1991b), and humoral immunity to p57 has been hypothesized to result in immune complex formation and subsequent hypersensitivity reactions in the glomeruli of the kidney (Turaga, 1989; Kaattari and Piganelli, 1997). Electron-dense subendothelial deposits that resemble immune complexes in experimentally and naturally infected fish support this possibility (Young and Chapman, 1978; Sami et al., 1992). However, isolation of immune complexes has not yet been formally demonstrated.

Humoral and cell-mediated immunity

The analysis of fish immunity to R. salmoninarum is an active area of research and may provide a unique model for understanding mechanisms of lower vertebrate cell-mediated immunity against a natural bacterial pathogen. Circumstantial evidence suggests that infected salmonids can mount a protective immune response. Munro and Bruno (1988) described a natural epizootic of R. salmoninarum in Atlantic salmon, which occurred during smoltification and resulted in 18% cumulative mortality. Subsequently, fish were found to be Gram- and IFAT-negative up to 69 weeks post-sea-water transfer; however, 100% of the fish had agglutinin response and demonstrated a resolution of granulomatous lesions. Antibody responses are induced by i.p. injection of killed R. salmoninarum (Evelyn, 1971) or by immersion challenge (Jansson and Ljungberg, 1998; Jansson et al., 2003). However, antibody responses are generally slow and dependent on the fish rearing temperature (Alcorn et al., 2002). Fish produce a robust antibody response against p57 as well as to peptidoglycan, but not to the galactose-rich polysaccharide (Sorum et al., 1998a). R. salmoninarum is resistant to normal and immune serum killing (Bandin et al., 1995), and at present, there is no evidence that humoral immunity is protective.

Recently, Renibacterium has emerged as a model system for understanding mechanisms of fish cellular immunity against a facultative intracellular pathogen (Ellis, 1999; Campos-Perez et al., 2000; Grayson et al., 2002; Jansson et al., 2003). An R. salmoninarum responsive cell population was identified in the rainbow trout spleens using the 1C2 MAb (Jansson et al., 2003). This MAb recognizes the TCR ß-chain and also a subpopulation of Ig+ cells. 1C2+ cells initially increased after challenge then decreased by 10 weeks post challenge but could be restimulated again after a secondary challenge with heat-killed bacteria. Inducible nitric oxide synthase mRNA is robustly upregulated after Renibacterium injection or bath challenge (Campos-Perez et al., 2000). Determination of the nature and function of these responding cells will advance our understanding of cell mediated immunity in fish. In a comprehensive study, bacterial and macrophage gene expression were examined simultaneously following in vitro infection (Grayson et al., 2002). Macrophages demonstrated a rapid inflammatory response including up-regulation of interleukin-1ß (IL-1ß), major histocompatibility complex class II (MHC II), inducible cyclo-oxygenase (Cox-2), inducible nitric oxide synthase (iNOS), CXC chemokine receptor 4 (CXC-R4), CC chemokine receptor 7 (CC-R7), and Mx 1-3 genes. Tumor necrosis factor-alpha (TNF-alpha) expression was suppressed at 2 h but recovered by 24 h post infection. The Renibacterium p57 gene was constitutively expressed during infection. From these and other studies, the investigators propose a model in which R. salmoninarum activates an inflammatory response but survives initial contact with macrophages by avoiding and/or interfering with TNF-alpha-dependent killing pathways. The development of host-pathogen specific microarrays will be a next step to fully understand the complex changes in gene expression occurring during infection.


Virulence factors



A number of enzymatic activities of R. salmoninarum have been identified which may contribute to virulence. These include haemolytic (Bruno and Munro, 1982), proteolytic (Smith, 1964; Bruno and Munro, 1986c; Rockey et al., 1991b), exotoxin (Shieh, 1988b), catalase (Bruno and Munro, 1982), DNAse (Bruno and Munro, 1982) and iron reductase activities (Grayson et al., 1995b). Several putative virulence genes have been cloned including the major soluble antigen (msa; Chien et al., 1992), a hemolysin (rsh; Evenden et al., 1990; Grayson et al., 1995a), a zinc-metalloprotease (hly; Grayson et al., 1995d) and a glucose kinase (Maulen et al., 1996; Concha and Leon, 2000). In addition, a 50-100 nm capsule, a common virulence factor of other bacteria, has been observed using electron microscopy (Dubreuil et al., 1990b). However, conclusive proof of the contribution of these enzymes, cloned genes, or the capsule to isolate virulence has not been reported.

Several investigators have observed pathological changes in infected fish consistent with in vivo secretion of a toxin (Bruno, 1986a; Bell et al., 1990). Bruno (1986a) observed an accumulation of erythrocytes in the spleen of experimentally infected rainbow trout and suggested that a toxin may be damaging erythrocytes, resulting in their sequestration within the spleen. Additionally, the lack of histologically detectable bacterial cells in the brain of experimentally infected sable fish which developed meningitis suggests the presence of a toxin (Bell et al., 1990). Shieh (1988b) identified a putative exotoxin isolated from R. salmoninarum culture supernatant, which at a dose of 160 µg was lethal for Atlantic salmon fingerlings (9-12 g). Unfortunately, a control extract was not prepared to rule out the possibility that the exotoxin was a component of the semidefined medium (Shieh, 1988a). In contrast to the work of Shieh, other investigators have been unable to demonstrate toxicity with extracellular product (ECP) either by i.p. injection of rainbow trout fingerlings or by addition of ECP to fish cell lines (Bandin et al., 1991). We have not found R. salmoninarum ECP to exhibit cytotoxic effects when added to in vitrocultures of salmonid leukocytes using trypan blue staining (Turaga et al., 1987a; Wiens and Kaattari, 1991). These experiments, however, did not rule out the possibility of toxicity to a small set of leukocytes. The purification of extracellular components to homogeneity, as demonstrated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), is needed to identify exotoxin(s) produced by R. salmoninarum.

In bacterial species that are difficult to genetically manipulate such as Renibacterium (Rhodes et al., 2002), one strategy for the identification of putative virulence factors is to isolate spontaneous mutants with reduced virulence and to compare these to virulent isolates using biochemical or DNA analysis (Smith, 1989). Several spontaneous R. salmoninarum mutants have been identified which have reduced virulence by i.p. challenge (Bruno, 1988; Griffiths et al., 1998; Daly et al., 2001). In rainbow trout, low-virulence isolates MT238, MT239 and MT240 caused 8-18% total mortality compared to virulent isolates that produced 73- 81% mortality (Bruno, 1988). Strain MT239 also has reduced virulence in chinook salmon, a species highly susceptible to BKD (O’Farrell et al., 2000). In rainbow trout, gross lesions were observed in fish injected with virulent or low-virulence isolates. The low virulence isolates were catalase positive and haemolytic against horse and sheep blood. Interestingly, the isolates with low virulence no longer possessed the typical R. salmoninarum trait of autoagglutination in culture, had reduced cell-surface hydrophobicity (Bruno, 1988), and lack an amorphous layer associated with the cell surface (Senson and Stevenson, 1999). Renibacterium salmoninarum reisolated from fish infected with these reduced-virulence isolates did not revert to the original phenotype and were stable during an 18 month period. The absence of a saline-extractable 57 kDa protein was correlated with the reduction of virulence, suggesting that a cell-associated 57 kDa protein may be a virulence factor (Bruno, 1990). In support of this possibility, R. salmoninarum isolates containing three copies of the msa gene encoding p57 are more virulent than isolates containing two copies (Rhodes et al., 2004a).


The 57/58-kilodalton protein (p57)



The most thoroughly characterized protein produced by R. salmoninarum is the 57/58 kDa protein (p57). This protein is an immunodominant antigen which is both secreted and present on the bacterial cell surface (Getchell et al., 1985; Turaga et al., 1987b; Wiens and Kaattari, 1989). The protein can either be extracted by washing cells in an acidic pH (Daly and Stevenson, 1990) or be concentrated from culture supernatant (Getchell et al., 1985). When extracts are electrophoresed under reducing and denaturing conditions, a predominant 57 kDa band is apparent, as well as a minor 58 kDa band (Getchell et al., 1985; Wiens and Kaattari, 1989; Daly and Stevenson, 1990). The gene coding p57 has been cloned and sequenced (msa, major soluble antigen), and it encodes a protein of 557 amino acids with a calculated Mr of 57,190 (Chien et al., 1992). Surprisingly, two copies of msa are present in most strains (O’Farrell and Strom, 1999; Wiens et al., 2002) and recently, strains have been identified containing at least 3 copies of msa (Rhodes et al., 2004a and Wiens, unpublished). Amino acids 1-26 encode a putative leader peptide sequence, which, after processing, results in a mature 54,505 Da protein. The amino-terminal sequence derived from microsequencing p57 agrees with the predicted sequence of the protein starting at residue 27 (Radacovici and Dubreuil, 1991; Wiens and Kaattari, 1991; Chien et al., 1992). It is unclear whether the 58 kDa protein represents the unprocessed protein containing the leader sequence or is the result of another type of modification of the mature protein. The 57 and 58 kDa proteins are not complexed by disulfide bonding, as the addition of beta-mercaptoethanol does not change the electrophoretic migration (Daly and Stevenson, 1990). P57 contains two sets sequence repeats: there are two direct, A repeats which are each 81 amino acids in length, and there are five B repeats which are approximately 25 amino acids in length (Chien et al., 1992; Wiens et al., 1999). Interestingly, the A repeats contain a transcription factor immunoglobulin (TIG)-like domain which is found in the extracellular domain of members of the plexin protein family of adhesion-repulsion molecules (Wiens et al., 2002). The B repeats do not share significant sequence homology to other proteins.

It is unclear if p57 is post-translationally modified or if the protein assembles as part of a larger structure. Schiff staining for carbohydrate has been negative (Dubreuil et al., 1990a) however, a carbohydrate biotinylation assay stained p57 from strain JD24 but not MT239 (Senson and Stevenson, 1999). Since p57 does not reassemble onto strain MT239, a potential role for carbohydrate in the reassociation of p57 to the bacterial cell surface has been proposed (Senson and Stevenson, 1999). However, the nature of this association has not been biochemically defined. Based on the gene sequence analysis, the theoretical isoelectric point (pI) of p57 is 4.6. The amino acid composition of the protein is rich in glycine, valine, tryptophan, alanine and serine (Chien et al., 1992). Thirty-eight per cent of the amino acids are hydrophobic, possibly contributing to the hydrophobic nature of R. salmoninarum cells. Fimbriae are often hydrophobic (Jones and Isaacson, 1984) and it has been suggested that the short peritrichous fimbriae observed using electron microscopy may be composed of p57 (Dubreuil et al., 1990b). A fimbriae structure is not required for biological activity as we have found that p57 monomer is sufficient for agglutinating activity (Wiens et al., 1999).

A number of investigators have shown that p57 is unstable and susceptible to degradation while attached to the bacterial cell surface, secreted into culture or secreted during infection (Dubrieul et al., 1990a; Griffiths and Lynch, 1991; Rockey et al., 1991b). Freezing or heating decreases the stability of the 57 kDa protein (Griffiths and Lynch, 1991). Also, more lower-molecular-weight bands can be found in aged culture supernatants. Proteolysis can be inhibited by the addition of the serine protease inhibitor, phenylethylsulfonyl fluoride (Griffiths and Lynch, 1991; Rockey et al., 1991b). In an attempt to identify the responsible protease, Griffiths and Lynch (1991) resolved the ECP using two-dimensional electrophoresis. Breakdown of the 57 kDa protein occurred after the first dimension resolution and was apparent in the 57 and 33-37 kDa bands, suggesting that p57 might be autolytic. In contrast, Rockey et al. (1991b) identified a high-molecular-weight protease independent of p57. This protease has low activity against p57 at 17°C and is highly active at 37°C. The protease was inhibited by phenylethylsulfonyl fluoride, methanol, ethanol and 10 min incubation at temperatures greater than 65°C. No degradation has been observed of purified p57 preparations, suggesting that p57 is not autolytic or that the autolytic activity was destroyed during purification (Rockey et al., 1991b; Wiens et al., 1999). Addition of the R. salmoninarum protease to purified p57 at 17°C yielded a spectrum of its breakdown products similar in molecular mass and antigenicity to those seen in the extracellular protein (Rockey et al., 1991b). However, p57 was completely degraded into numerous smaller molecular weight bands when enzymatic reactions were performed at 37°C. This suggests that either the protease has high activity at elevated temperatures, or p57 is more susceptible to degradation due to denaturation and increased proteolytic site exposure. The physiological function of the protease is unknown, but one possibility is that it modulates the amount of functionally active p57 on the bacterial cell surface. Interestingly, the processing of p57 is reduced in iron-restricted cultures (Grayson et al., 1995c). Since iron-limited conditions occur in fish serum, this may facilitate expression or stability of p57 early during the infectious process prior to intracellular invasion.

Further research on the proteolytic products has helped define the structural and functional domains of p57 (Wiens and Kaattari, 1991). Digestion of p57 at 17°C with the endogenous serine protease typically produces predominant breakdown products with approximate molecular weights of 45, 36, 34, 25 and 20 kDa. Monoclonal antibodies were categorized into three groups, based on differential recognition of these fragments as determined using Western blotting. Group I MAbs recognize a region proximal to the amino terminus of the protein and two of these antibodies block the leukoagglutination activity. This suggests that the leukocyte-binding domain is located near the amino terminal portion of the protein. Two lines of evidence suggest that the carboxy terminus of p57 is associated with the bacterial cell surface. First, group III antibodies are unable to bind to p57 on the cell surface as determined by immunofluorescence assays indicating that the epitope(s) are sterically unavailable. Second, the proteolytic fragments p36 and p25, recognized by group III MAbs, are retained on washed R. salmoninarum cells, as determined by SDS-PAGE (Wiens and Kaattari, 1991). The retention of these fragments by the bacterial cell surface suggests that they contain an attachment domain which functions even after partial proteolysis of the whole protein. This model has been confirmed by overexpression of p57, and p57 fragments, in E. coli (Wiens et al., 1999). The precise locations of the neutralizing epitopes have been recently mapped by transposon mutagenesis (Wiens and Owen, 2005). The order of the epitopes from the amino terminus is 4H8, 4C11 and 4D3.

The in vivo function of p57 is uncertain, but the concentration in fish tissues increases during disease progression (Turaga et al., 1987b). The protein has been associated with a number of biological activities in vitro which may be responsible for some of the observed pathology. Among these activities are mammalian erythrocyte agglutination (Daly and Stevenson, 1987; Daly and Stevenson, 1990), salmonid spermatocyte and leukocyte agglutination (Daly and Stevenson, 1989; Wiens and Kaattari, 1991), non-specific suppression of salmonid antibody responses (Rockey et al., 1991b; Fredriksen et al., 1997), suppression of phagocytic cell bactericidal activity (Siegel and Congleton, 1997) and suppression of respiratory burst activity (Densmore et al., 1998). In an interesting study, Brown et al. (1996) injected p57 into coho salmon eggs which were then fertilized, raised and challenged. Fish which had been injected with 100 ng p57 demonstrated a significantly higher cumulative percent mortality than those from the saline-injected eggs. The p57-injected fish also produced lower levels of antibodies against p57, although not against whole cells, and exhibited a lower phagocytic response. These data suggest that early egg exposure may result in long-term immunosuppression and a decreased ability to resist subsequent challenge with R. salmoninarum.

Diagnosis

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Crucial to the success of any BKD control program is the application of reliable diagnostic methods that can detect low levels of R. salmoninarum in a variety of sample types. For that reason, fish health specialists and researchers have long been interested in developing methods for rapid and reliable detection of R. salmoninarum infections (Gudmundsdottir et al., 1993; Pascho et al., 1998; Pascho et al., 1987; Sakai et al., 1989a; White et al., 1995). As each new test has been developed, however, there has been a tendency to reject older techniques. Nevertheless, no single ideal diagnostic test has yet been developed for the evaluation of multiple sample types for the presence of BKD (reviewed in Pascho et al., 2002).

Historically, two general strategies for diagnosis of infection have been pursued: (1) direct detection of R. salmoninarum or antigens using culture methods, fluorescent antibody staining, ELISA or PCR; and, (2) detection of fish antibodies to R. salmoninarum. Of the two, methods for the direct detection of R. salmoninarum and its products are the most widely used for identifying infected fish. Early efforts relied on the observation of Gram-positive diplobacilli and the presence of clinical signs (Earp et al., 1953; Bell, 1961; Pippy, 1969). The use of Gram stain is limited because of its low sensitivity (1 × 107-9 bacterial cells g-1 tissue) (Bullock et al., 1980; Pascho et al., 1987; Sakai et al., 1987), and the presence of melanin granules in kidney tissue can obscure low numbers of organisms. A periodic acid-Schiff stain can locate R. salmoninarum in tissue sections, however it is not a specific stain (Bruno and Munro, 1982).



Culture of R. salmoninarum from fish tissues, followed by serological identification or PCR, is considered the definitive test (Fryer and Sanders, 1981; OIE 2003). Several different media have been used to culture the bacterium. Originally, Ordal and Earp (1956) used a complex blood medium supplemented with cysteine (kidney disease medium 1 (KDM-1)). Subsequently, Evelyn (1977) devised a kidney disease medium containing 1% (w/v) peptone, 0.05% (w/v) yeast extract, 0.1% (w/v) cysteine and 20% serum (KDM-2), which allowed the primary isolation of bacteria from fish tissues. The serum component can be replaced by charcoal (Daly and Stevenson, 1985; Daly, 1989) and attempts have been made to define specific components required for growth (Embley et al., 1982; Shieh, 1988a). Primary isolation is also enhanced by a heavy inoculum of a ‘nurse culture’ in the centre of the Petri dish (Evelyn et al., 1989) or the addition of 1.5 to 5% spent media to culture plates (Evelyn et al., 1990; Teska, 1994). Enhanced growth under these conditions is presumably due to the action of a diffusible factor which is able either to inactivate a toxic component in the medium or to stimulate growth of the bacterium. The nature of this component has not yet been identified.

Regardless of the medium used, primary isolation of R. salmoninarum is difficult and time consuming. Isolation of colonies from a highly infected fish takes from approximately 2 weeks (Evelyn, 1977) to as long as 19 weeks for subclinical cases (Benediktsdóttir et al., 1991). One problem is contamination with heterotrophic bacteria. The bacteria grow much faster than R. salmoninarum and tend to overgrow Petri dishes. Addition of four antimicrobials (0.005% w/v cycloheximide, 0.00125% w/v D-cycloserine, 0.0025% polymyxin B sulfate and 0.00025% oxolinic acid) to KDM-2 allows the selective isolation of R. salmoninarum (Austin et al., 1983). When using culture techniques, care should be taken in interpreting the results from kidney, as this tissueis inhibitory toin vitro R. salmoninarum growth (Evelyn et al., 1981; Daly and Stevenson, 1988). Additionally, R. salmoninarum is extremely sensitive to some media components, for example, the bacteria grow poorly in some batches of peptone (Evelyn and Prosperi-Porta, 1989). In general, the fastidious culture requirements, slow growth of R. salmoninarum and common overgrowth of Petri dishes with contaminating organisms limit the usefulness of culture as a rapid method to screen large numbers of fish.


Immunodiagnosis



Since culture of R. salmoninarum is difficult and Gram stain is insensitive, a number of immunodiagnostic assays have been developed for the detection of R. salmoninarum. Bullock and Stuckey (1975) first described the direct fluorescent antibody technique (FAT) to directly visualize bacterial cells. The FAT is more sensitive than the Gram stain and can detect subclinical infections (Bullock and Stuckey, 1975; Laidler, 1980). Several methods to quantify R. salmoninarum employing fluorescent antibodies have been used, including a subjective scoring of fluorescence intensity (1+ to 4+) of tissue smears (Bullock et al., 1980) and a membrane FAT procedure. In the latter procedure, bacteria are immobilized on filter-paper grids and titres expressed as cells per unit of tissue or ovarian fluid (Elliott and Barila, 1987). This assay detects less than 102 bacterial cells ml-1 of coelomic fluid (Elliott and Barila, 1987) and has a similar sensitivity when it is used to enumerate R. salmoninarum cells in tissues (Lee, 1989). While the FAT is a very sensitive test for R. salmoninarum, it suffers from being tedious to perform, labour-intensive when large numbers of samples are examined, and is less accurate with low levels of R. salmoninarum (Armstrong et al., 1989).

An alternate method of diagnosis is the detection of soluble antigens. Soluble antigens were first detected using the immunodiffusion technique with samples from the kidney, liver, spleen and blood of infected fish (Chen et al., 1974). Kidney and liver tissue contained higher concentrations of these antigens and were the most useful for diagnosis. A 100% correlation (n = 30) was observed between positive precipitin reactions, the presence of clinical signs, and a positive Gram stain (Kimura et al., 1978). A major limitation, however, is the inability of the immunodiffusion technique to detect low levels of R. salmoninarum (Fryer and Sanders, 1981; Cipriano et al., 1985; Sakai et al., 1989a). Counterimmunoelectrophoresis is more sensitive than immunodiffusion or bacterial culture (Cipriano et al., 1985), but can be variable (Pascho et al., 1987). Detection of subclinically infected fish was accomplished by Kimura and Yoshimizu (1981) using the staphylococcal co-agglutination technique. This qualitative test requires 1.5 h to complete and is as sensitive as the FAT (Sakai et al., 1987).

The most widely used assays for rapid detection of R. salmoninarum antigen in large surveys are the dot-blot and the enzyme-linked immunosorbent assay (ELISA). The dot-blot technique qualitatively detects antigen after samples are coated on to nitrocellulose (Sakai et al., 1987). Enzyme-linked immunosorbent assays are more advantageous, as they allow precise quantitative detection of antigen (Dixon, 1987; Pascho and Mulcahy, 1987; Turaga et al., 1987b; Hsu and Bowser, 1991; Rockey et al., 1991b; Gudmundsdóttir et al., 1993; Olea et al., 1993). The ELISA developed by Dixon rapidly detects antigen in 0.5 h but is unable to detect low levels associated with subclinically infected fish. Other assays (Pascho and Mulcahy, 1987; Turaga et al., 1987b) use longer incubation periods and detect antigens in amounts as low as 2-20 ng and 10 ng, respectively. Rockey et al. (1991a) developed an antigen-capture, MAb-based ELISA which is specific for two epitopes present on p57. The technology of ELISA is considered to possess equivalent or greater sensitivity than either culture or the FAT (Pascho et al., 1987; Rockey et al., 1991a).

Indirect detection of R. salmoninarum infection by the identification of specific salmonid antibodies has not met with widespread success. Salmonids produce agglutinating antibodies to R. salmoninarum (Evelyn, 1971), however, antibody levels do not seem to correlate with the level of infection. Banowetz (1974) assayed 207 yearling coho salmon from a population during an epizootic of BKD and found that fish agglutinin titres equal or higher than 1:128 generally had no detectable bacteria. Conversely, fish which were R. salmoninarum-positive had low agglutinin titres. Similar findings were reported by Bruno (1987), who found that serum agglutinins were not detected in heavily infected Atlantic salmon (Salmo salar) smolts, but that the titre of agglutinins increased prior to the end of an epizootic. The variable levels of serum agglutinins in apparently healthy fish (Evelyn et al., 1981; Paterson et al., 1981a; Bruno, 1987) and the low levels of detectable agglutinins in infected fish suggest that serum agglutination assays are of limited value for fish health monitoring (Banowetz, 1974; Bruno, 1987; Jansson and Ljungberg, 1998). However, since recovering fish produce a humoral response to R. salmoninarum, detection of seropositive fish using agglutination, ELISA (Bartholomew et al., 1991; Jansson et al., 2003) or electroimmunotransfer blot assays (Olivier et al., 1992) may be useful methods for monitoring disease progression under experimental conditions.

Antigenic cross-reactivity

The development of highly sensitive ELISA techniques has identified many more antigen positive fish than had previously been identified by FAT or culture techniques (Meyers et al., 1993; Gudmundsdóttir et al., 1993). The antigen positive samples that cannot be confirmed using another technique should be interpreted with caution. It should not be assumed that these fish will at some point develop clinical BKD, as the fish might harbour cross-reactive substances which interfere with the ELISA and/or antigen may be present but the bacteria may be non-viable.

The sensitivity and specificity of the ELISA relies heavily on the quality of antisera used. Numerous reports of cross-reactive organisms have been documented in the literature for both polyclonal antibodies and MAbs. Cross-reactive Gram-positive, Gram-negative and Gram-undetermined organisms have been identified using FAT (Bullock et al., 1980; Evelyn et al., 1981; Austin et al., 1985; Yoshimizu et al., 1987; Brown et al., 1995; Teska et al., 1995) and ELISA (Brown et al., 1995; Bandin et al., 1996). In general, the cross-reactive component(s) have not been identified but may include common carbohydrate molecules, such as galactose (Fiedler and Draxl, 1986) or heat-shock proteins (Wood et al., 1995). Dixon (1987) found that preadsorption of polyclonal antisera with Rothia dentocariosa and Bacillus sphaericus increased specificity of the ELISA. In addition to cross-reactivity with microorganisms, cross-reactivity of polyclonal antisera has been observed in the ELISA with feather meal components present in certain commercial diets (Pascho et al., 1991a) and with fish serum components (Turaga et al., 1987a). Thus, affinity purification of polyclonal antisera is probably necessary to ensure adequate specificity. The use of MAbs may increase the specificity of the immunoassay, as these antibodies recognize precise, selected epitopes. Judicious choice of MAbs is necessary, however, as Arakawa et al. (1987) identified a MAb that recognized a cross-reactive determinant on R. salmoninarum and three other Gram-positive organisms.

Since cross-reactive antigens exist, it is essential that independent assays are utilized to confirm ELISA results. Western blotting is one technique for the confirmation of R. salmoninarum p57 in ELISA-positive samples, since p57 can be identified on the basis of both molecular mass and immunoreactivity (Wiens et al., 1990). A disadvantage of this technique is that it is still three- to fourfold less sensitive than the monoclonal-based ELISA (Wiens, 1992). Alternatively, PCR is a sensitive method for detection of the msa gene encoding p57. However, even if the p57 protein or gene is confirmed, the presence of viable organisms is still in question. Therefore, it is recommended that if putative R. salmoninarum-free stocks are identified as ELISA-positive, Western blot or PCR, in conjunction with culture techniques, be used to confirm the presence of p57 and viable R. salmoninarum. Using this type of combined approach for detecting R. salmoninarum, Griffiths et al. (1996) have found that incubation of ovarian fluid cellular debris in selective kidney disease medium (SKDM) broth, followed by Western blotting, increased the total numbers of positive samples by 32% over SKDM agar culture or indirect FAT (IFAT).


Molecular probes



The availability of gene sequences from R. salmoninarum has facilitated the development of nucleic-acid-based diagnostic assays. These assays are generally highly specific and can be rapidly performed (Pascho et al., 2002). Several nucleic acid hybridization approaches have been reported (Etchegary et al., 1991; Mattsson et al., 1993; Leon et al., 1994a,b and Hariharan et al., 1995); however, there is little information on the relative sensitivities of these assays compared to traditional diagnostic methods (Pascho et al., 2002). Nucleic acid technologies that amplify a target sequence using the polymerase chain reaction (PCR) are generally more sensitive than DNA probes. Both PCR and reverse-transcription PCR assays have been developed to measure either the 16s RNA gene (Magnusson et al., 1994; Rhodes et al., 1998) or the gene encoding p57 (Brown et al., 1994; Brown et al., 1995; McIntosh et al., 1996; Miriam et al., 1997; Chase and Pascho, 1998; Cook and Lynch, 1999). Most PCR assays that detect R. salmoninarum have been shown to be very sensitive for detecting the pathogen in a variety of tissue types. Sensitivity of nested PCR amplification of p57 is equal to or greater than detection by ELISA or FAT (Chase and Pascho, 1998). PCR sensitivity is also equal to or greater than that of bacteriological culture using kidney tissue (McIntosh et al., 1996; Miriam et al., 1997; Cook and Lynch, 1999) or coelomic fluid (Miriam et al., 1997). PCR may be especially useful for detecting R. salmoninarum in ovarian fluid (Miriam et al., 1997; Pascho et al., 1998) as both polyclonal and monoclonal-based ELISA do not consistently detect low bacterial cell concentrations (Wiens, 1992; Griffiths et al., 1996; Pascho et al., 1998;). A limitation of genomic DNA amplification, similar to ELISA and FAT, is that the viability of the pathogen is unknown. Reverse-transcription PCR however, requires the presence of bacteria RNA which is usually degraded rapidly once a bacterium is nonviable. Cook and Lynch (1999) demonstrated that RT-PCR detection of the msa genes required viable R. salmoninarum and was reduced by rifampin or erythromycin treatment paralleling the loss of bacterial cell viability. This assay should be useful for distinguishing the continued presence of viable bacteria particularly in samples from broodstock treated with antibiotics prior to spawning. Currently, PCR assays are considered impractical for large-scale brood stock screening programs due to equipment capacity limitations, the manipulations required to extract samples and concerns about cross-contamination (Pascho et al., 2002). However, PCR is being rapidly accepted as a confirmatory test for the ELISA or FAT. The ability to detect and distinguish multiple pathogens at once (Nilsson and Strom, 2002) and the use of fluorescent PCR (Konigsson et al., 2005) will increased the utility of these molecular-based assays.

List of Symptoms/Signs

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SignLife StagesType
Finfish / Build up of bloody fluids - Body Cavity and Muscle Aquatic:Adult,Aquatic:Fry Sign
Finfish / Build up of bloody fluids - Body Cavity and Muscle Aquatic:Adult,Aquatic:Fry Sign
Finfish / Generalised lethargy - Behavioural Signs Aquatic:Adult,Aquatic:Broodstock Sign
Finfish / Haemorrhagic lesions - Skin and Fins Aquatic:Adult,Aquatic:Broodstock Sign
Finfish / Haemorrhagic lesions - Skin and Fins Aquatic:Adult,Aquatic:Broodstock Sign
Finfish / Haemorrhaging - Body Cavity and Muscle Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Sign
Finfish / Haemorrhaging - Body Cavity and Muscle Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Sign
Finfish / Kidney - white-grey patches (haemorrhage / necrosis / tissue damage) - Organs Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Diagnosis
Finfish / Kidney - white-grey patches (haemorrhage / necrosis / tissue damage) - Organs Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Diagnosis
Finfish / Kidney swelling / oedema - Organs Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Diagnosis
Finfish / Kidney swelling / oedema - Organs Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Diagnosis
Finfish / Liver - white / grey patches (haemorrhage / necrosis / tissue damage) - Organs Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Sign
Finfish / Liver - white / grey patches (haemorrhage / necrosis / tissue damage) - Organs Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Sign
Finfish / Mortalities -Miscellaneous Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Diagnosis
Finfish / Mortalities -Miscellaneous Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Diagnosis
Finfish / Necrotic musculature - Body cavity and muscle Aquatic:Adult,Aquatic:Fry Sign
Finfish / Necrotic musculature - Body cavity and muscle Aquatic:Adult,Aquatic:Fry Sign
Finfish / Paleness - Gills Aquatic:Adult,Aquatic:Fry Sign
Finfish / Paleness - Gills Aquatic:Adult,Aquatic:Fry Sign
Finfish / Pop-eye - Eyes Aquatic:Adult,Aquatic:Fry Sign
Finfish / Pop-eye - Eyes Aquatic:Adult,Aquatic:Fry Sign
Finfish / Spleen white-grey patches (haemorrhage / necrosis / tissue damage) - Organs Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Diagnosis
Finfish / Spleen white-grey patches (haemorrhage / necrosis / tissue damage) - Organs Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Diagnosis
Finfish / White-grey patches (necrosis / tissue damage) - Organs Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Diagnosis
Finfish / White-grey patches (necrosis / tissue damage) - Organs Aquatic:Adult,Aquatic:Broodstock,Aquatic:Fry Diagnosis

Disease Course

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Process of infection

BKD can exhibit either an acute or chronic pathology, which in many aspects, is similar to mycobacterial pathology observed in mammals. Infections are often characterized by chronic granulomatous, inflammatory reactions with tissue necrosis caused by intracellular infection. In Pacific salmon, the granulomas are diffuse with poorly defined borders, whereas the granulomas in Atlantic salmon are more encapsulated with caseation in the centres (Kent, 1992). The histopathological changes that occur after intraperitoneal injection (i.p.) of live or formalin-fixed R. salmoninarum into rainbow trout have been examined in detail (Bruno, 1986b). Within 45 min of injection, bacterial cells were within phagocytes of the kidney and spleen. After 4-6 days, disseminated extracellular R. salmoninarum were observed throughout these organs and, by 6-10 days, large numbers of R. salmoninarum were in blood monocytes and macrophages, where they appeared to have multiplied. At 14 days, phagocytic cells containing R. salmoninarum were between myocardial bundles in the heart. After 28 days, R. salmoninarum were found intracellularly in endothelial cells lining the glomerular blood-vessels and lumen of collecting ducts but not within proximal tubules of the kidney. Colonization of kidney tissues and cell-mediated immunity to R. salmoninarum results in development of granulomatous lesions. The involvement of the CNS has been described and appears to be more common in farmed Atlantic salmon as compared to chinook salmon (Speare et al., 1993, Speare, 1997). The reason for the different pathological presentation between species is unknown. Thymic infection has also been observed in experimentally infected coho salmon (Flano et al., 1996a).

R. salmoninarum is considered a facultative intracellular pathogen. The bacterium appears to have an affinity for phagocytes, sinusoidal cells, reticular and barrier cells (Flano et al., 1996b). The bacterium binds the C3b component of the complement pathway which enhances phagocytic uptake (Rose and Levine, 1992). In addition, R. salmoninarum p57 binds leukocytes which may aid attachment and invasion (Wiens and Kaattari, 1991). Following infection of macrophages in vitro, R. salmoninarum stimulates the respiratory burst (Campos-Perez et al., 1997) which can be inhibitory for bacterial growth when the macrophage is activated by cytokine stimulation (Hardie et al., 1996). Numerous researchers have observed R. salmoninarum within phagocytic cells (Young and Chapman, 1978; Bruno, 1986b; Zhuo, 1990), although conclusive evidence of intracellular replication has been difficult to substantiate (Bandin et al., 1993). Transmission electron microscopy photomicrographs have identified R. salmoninarum which appear to escape from the macrophage phagosome into the cytoplasm of the cell (Gutenberger et al., 1997). The histological observations combined with TEM evidence suggest that R. salmoninarum is an invasive organism capable of causing a disseminated infection. Tissue culture models for studying the process of intracellular invasion have been developed (McIntosh et al., 1997; Gonzalez et al., 1999) and an R. salmoninarum DNA fragment has been isolated which can confer internalization of Escherichia coli into the chinook salmon embryo cell line (CHSE-214) (Maulen et al., 1996). Intracellular invasion is a common strategy of a number of pathogenic bacteria thereby facilitating access to nutrients and evasion from the immune system.

Epidemiology

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Transmission

The natural route of infection is not fully understood. There is evidence that the disease is transmitted both horizontally and vertically. Horizontal transmission by co-habitation has been demonstrated under laboratory conditions (Bell et al., 1984; Murray et al., 1992), however, the route of entry is not clear. Infection via contaminated water may be one route of transmission. Viable R. salmoninarum have been demonstrated in fresh and salt water using FAT and bacterial culture (Austin and Rayment, 1985; McKibben and Pascho, 1999). The disease can also be transmitted via feeding infected adult viscera to juvenile fish (Wood and Wallis, 1955) leading to the discontinuance of feeding raw salmon products to juvenile fish (Fryer and Sanders, 1981). A natural source of infective material may be faeces of clinically infected or carrier fish. Oral intubation of infected faecal material, but not autoclaved material, transmits the microorganism and results in clinical disease (Balfry et al., 1996). The ingestion of faecal material may contribute significantly to the horizontal transmission of R. salmoninarum among salmonids reared in sea-water net-pens (Balfry et al., 1996). In an in vitro gill culture, bacterial attachment and uptake was not observed suggesting this may not be a route of infection (McIntosh et al., 2000). However, bath challenge induces iNOS expression in gill tissue indicating Renibacterium-responsive cells are present in gill tissue (Campos-Perez et al., 2000).

Renibacterium salmoninarum is unusual among bacterial pathogens as it is transmitted to offspring via the egg. Allison (1958) and Bullock et al. (1978) were the first to report circumstantial evidence that gametes from infected adults, transferred to historically disease-free locations, resulted in clinically infected progeny. The bacterium has been identified on both the surface and the inside of eggs of a coho salmon naturally infected with 4 x 109 colony-forming units (CFU) R. salmoninarum ml-1 of ovarian fluid (Evelyn et al., 1984). R. salmoninarum was cultured from 15.1% of surface-disinfected eggs and also observed within the yolk of sectioned eggs. Under laboratory conditions, infection of unfertilized steelhead (Oncorhynchus mykiss), coho (Oncorhynchus kisutch) and chinook (O. tshawytscha) salmon eggs by immersion challenge has only been accomplished using high numbers of R. salmoninarum (1.4 x 109, 1.3 x 1012 and 1.7 x 105, respectively) (Evelyn et al., 1986a). Under these conditions, only 1-5.5% of the experimentally exposed eggs contained viable R. salmoninarum (Evelyn et al., 1986a; Lee and Evelyn, 1989). It is not clear why only low percentages were infected or how the bacterium enters eggs. Evelyn et al. (1986a) postulated that eggs in nature became infected after ovulation while they were in contact with coelomic fluid. However, Bruno and Munro (1986b) observed the presence of R. salmoninarum in tissue sections of maturing oogonia of experimentally infected trout. This suggests that egg infection may result directly from ovarian tissue prior to ovulation. Current evidence suggests that intraovum infection results in progeny with low levels of infection. Lee and Evelyn (1989) demonstrated that smolts reared from externally disinfected eggs from infected adults, but not from uninfected adults, had subclinical levels of R. salmoninarum. Low levels of R. salmoninarum (23-113 cfu ml-1 in adult ovarian fluid) were associated with a 1-2% infection in smolts and subclinical disease. Unfortunately, bacterial cells from FAT-positive smolts were not cultured or enumerated. The mechanism(s) of intraovum transmission and the contribution of vertically transmitted bacteria to overt disease warrants further investigation as this is an uncommon mode of bacterial transmission.

Impact Summary

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CategoryImpact
Fisheries / aquaculture Negative
Rare/protected species Negative

Impact: Economic

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The precise magnitude of the worldwide economic losses due to BKD is unknown, but is likely to be considerable. In the Pacific Northwest, BKD is generally regarded as one of the most devastating bacterial diseases affecting wild and propagated anadromous salmonid stocks. It is especially problematic for culturists, as salmon are afflicted with the disease in both freshwater and salt-water life stages (Earp et al., 1953; Fryer and Sanders, 1981; Banner et al., 1983). BKD is considered to be one of the most significant causes of mortality for farmed chinook and coho salmon (Kent, 1992) and has been implicated in the decline of the Lake Michigan chinook salmon fishery in the late 1980’s (Holey et al., 1998). BKD is also regarded as a high priority to the Atlantic salmon industry on the east coast of Canada (Griffiths et al., 1998), although these fish are thought to be more resistant than Pacific salmon species (Kent, 1992). In Norway, the mortality caused by BKD is usually low, but cumulative losses as high as 70% have been reported for some locations (Dale et al., 1997).

Impact: Environmental

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Much research has focused on the impact of BKD on hatchery and wild spring/summer chinook salmon (Oncorhynchus tshawytscha) in the Columbia River system (Bullock and Wolf, 1986; Warren, 1991; Williams, 2001). Extensive surveys of out-migrating smolts from the Columbia River system identified high percentages of R. salmoninarum antigen positive fish (Maule et al., 1996; Elliott et al., 1997; Vanderkooi and Maule, 1999). Although most fish have low levels of infection, in some tributary systems the majority of fish had medium to high infection levels (Maule et al., 1996; Elliott et al., 1997). Under experimental conditions, fish with medium to high levels of infection displayed increased vulnerability to predation (Mesa et al., 1998), increased susceptibility to the effects of dissolved gas supersaturation (Weiland et al., 1999), reduced feeding (Pirhonen et al., 2000), and impaired saltwater adaptation (Moles, 1997; Mesa et al., 1999). These findings in conjunction with the high prevalence of R. salmoninarum suggest that BKD may be an important factor contributing to poor smolt survival and low percentages of returning adult fish (Raymond, 1988; Williams, 2001). In addition to impacts on salmonid stocks in the U.S., BKD is thought to have been transferred in the early 1970s to Japan with imports of coho salmon eggs and subsequently spread to the indigenous, and highly valued, Cherry salmon (O. masou) (Scientific Committee on Animal Health and Welfare, 1999; http://europa.eu.int/comm/food/fs/sc/scah/out36_en.pdf). Efforts to eradicate this disease in Japan have not yet proven successful. The pathogen has also been spread to Chile with the importation of Pacific salmonid species (Sanders and Barros, 1986)

The effects of BKD on endangered salmonid stocks are a significant concern. The disease has emerged as the major factor limiting successful captive rearing of endangered salmon species in western North America (Flagg et al., 1995).

Zoonoses and Food Safety

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This species is not a zoonosis.

References

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Links to Websites

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WebsiteURLComment
J.P.Euzeby: Dictionnaire de Bacteriologie Veterinairehttp://www.bacterio.cict.fr/bacdico
OIE Manual of Diagnostic Tests for Aquatic Animalshttp://www.oie.intManual accessible from homepage
Renibacterium Genome Projecthttp://micro.nwfsc.noaa.gov/rs-genome/
Scientific Committee on Animal Health and Welfare, 1999. Bacterial kidney diseasehttp://europa.eu.int/comm/food/fs/sc/scah/out36_en.pdf

Contributors

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Main Author
Greg Wiens
National Center for Cool and Cold Water Aquaculture, USDA/ARS, 11861 Leetown Road, Kerneysville, WV 25430, USA

Joint Author
Kaattari, SL

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