Bacterial Kidney Disease (Renibacterium Salmoninarum) : G.D. Wiens and S.L. Kaattari

269
CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
7
Bacterial Kidney Disease
(Renibacterium salmoninarum)
G.D. Wiens
1
and S.L. Kaattari
2
Department of Microbiology and Laboratory for Fish Disease Research
Oregon State University, Corvallis, Oregon 97331-3804, USA; Present
addresses:
1
Department of Molecular Microbiology and Immunology, Oregon
Health Sciences University, 3181 SW Sam Jackson Park Road, Portland,
Oregon 97201, USA;
2
Department of Environmental Sciences, School of
Marine Science, Virginia Institute of Marine Science, College of William and
Mary, Gloucester Point, Virginia 23062, USA.
INTRODUCTION
Bacterial kidney disease (BKD) is a prevalent disease of cultured and wild
salmonids (Fryer and Sanders, 1981). Salmon, trout and char are the natural
hosts of the aetiological agent Renibacterium salmoninarum, which causes a
chronic bacteraemia with focal lesions in the viscera, particularly the kidney
(Fryer and Sanders, 1981). The precise magnitude of the worldwide economic
losses due to BKD is unknown, but is likely to be considerable. In the Pacific
North-West, BKD is generally regarded as one of the most important 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). It is estimated that, in British Columbia coastal
waters, 2060% of farmed salmon may succumb to BKD prior to commercial
harvest (Albright et al., 1988). The effects of BKD on spring/summer chinook
salmon (Oncorhynchus tshawytscha) are of particular concern (Bullock and
Wolf, 1986; Warren, 1991). A survey of out-migrating spring/summer chinook
smolts on the lower Columbia River, one of the major salmonid production areas
of the world, identified detectable levels of R. salmoninarum antigen in 97
100% of the fish (Elliott, and Pascho, 1991). Thus the presence of R.
salmoninarum may be a major factor contributing to poor smolt survival and low
percentages of returning adult fish (Raymond, 1988).
In spite of the high prevalence of BKD, techniques for its control are
limited, in both in number and efficacy (Elliott et al., 1989; Warren, 1991).
Management strategies designed to control BKD have included reducing stress
to fish, quarantining infected stocks, dietary modification, chemotherapy,
culling of infected brood stock and/or total hatchery depopulation and
sterilization. There is no efficacious vaccine available for widespread use
270
G.D. Wiens and S.L. Kaattari
(Kaattari et al., 1988; Munro and Bruno, 1988). Control strategies are hampered
primarily due to a limited knowledge of the physiology of R. salmoninarum, its
pathogenic mechanisms and the hosts immune response to the infection. One
reason why research on R. salmoninarum has been limited is due to technical
difficulties related to the culture and growth of the organism. These difficulties
include a very slow generation time of 24 h (Fryer and Sanders, 1981) and
difficult primary isolation (Evelyn, 1977). Experimental challenges are also
time-consuming due to the chronic progression of disease, ranging from 1 to 12
months depending on the method of challenge and dose of inoculum (McCarthy
et al., 1984; Murray et al., 1992).
In this chapter, we shall review recent findings on R. salmoninarum and
propose future avenues of study. For additional information concerning the
epizootiology of BKD, vaccine development, disease control and pathogenesis,
the reader is referred to reviews by Earp et al. (1953), Fryer and Sanders (1981),
Klontz (1983), Bullock and Herman (1988), Kaattari et al. (1988), Munro and
Bruno (1988), Elliott et al. (1989) and Evenden et al. (1993).
EPIDEMIOLOGY, PATHOLOGY AND TAXONOMY
Geographical distribution
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; Gudmundsdttir 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., 1986; Elliott and Pascho,
1991; Sanders et al., 1992, Meyers et al., 1993).
Host range
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) and Thymallus thymallus (grayling) (Kettler et al.,
1986). Experimental infection and mortality have been induced in a
non-salmonid species Anoplopoma fimbria (sablefish) (Bell et al., 1990) but
not in Cyprinus carpio (carp) (Sakai et al., 1989c) 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)
271 Bacterial Kidney Disease
and non-salmonid fin-fish which reside in and around sea-water net pens in the
Pacific North-West have not been successful (Paclibare et al., 1988).
Pathology
The gross external pathology of BKD is variable and ranges from a complete
lack of clinical signs to a general darkening of the body, distended abdomen,
exophthalmia, petechial haemorrhaging and haemorrhaging near the fins
(Belding and Merrill, 1935). The infection is characterized by a chronic,
systemic infiltration of the viscera by the bacteria (Ferguson, 1989).
Pathological changes in the kidney are the most obvious signs and include small
to large granulomatous lesions (Fig. 7.1a) and a general enlargement of the
kidney (Wood and Wallis, 1955; Smith, 1964). Focal granulomas also occur in
the spleen and liver (Fig. 7.1b) as well as in the musculature, heart, swim-
bladder, pancreas and subcutaneous tissue behind the eye. Less affected but
reported sites of infection include the lamina propria of the gut and meninges
of the brain (Ferguson, 1989). Pseudo-membranes surrounding the kidney,
liver and spleen, composed of thin layers of fibrin and collagen with
fibroblasts, histiocytes, degenerating leucocytes and R. salmoninarum, have
also been described (Smith, 1964). Bruno (1986b) concluded that death could
be attributed to the obliteration of normal kidney and liver tissue by bacterial
lesions leading to a generalized organ dysfunction, possibly in conjunction
with heart failure.
Atypical BKD has been reported in which ocular and skin lesions are the
predominant pathological signs. Hoffman et al. (1984), using fluorescent
antibody staining, identified R. salmoninarum frequently in these lesions in trout
fingerlings, but less frequently in the kidney. Spawning coho salmon have
demonstrated external pustules, with few lesions in the kidney (Hoskins and
Stone, 1980). This pathology may be related to spawning rash, which is
peculiar to rainbow trout in southern Ontario, Canada (Ferguson, 1989). Small
intradermal vesicles and granulomas were detected at spawning time but were
not apparent at other times of the year. These external pathologies may be the
result of eye and skin abrasions, which may serve as primary entry points of R.
salmoninarum (Hendricks and Leek, 1975).
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). Belding and Merrill (1935)
observed a Gram-positive bacterium associated with necrotic kidney lesions of
salmonids in the USA, but they were unable to culture the disease agent.
Cultivation of the organism on cysteineblood-enriched medium made possible
the completion of Kochs postulates and identification of the aetiological agent
272
Fig. 7.1. (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 through-
out 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.
(a)
(b)
as a slow-growing Gram-positive bacterium (Rucker et al., 1954; Ordal and
Earp, 1956).
Current classification
The classification of the aetiological agent of BKD was unresolved until
recently. 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., 1991b) and
recent 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, no additional species have been included in this genus, nor
have subspecies been identified.
Phenotypic characteristics
Renibacterium salmoninarum is a small (0.31.5 m 0.11.0 m), non-motile,
non-acid-fast, non-sporeforming, strongly Gram-positive rod that usually occurs
in pairs (Sanders and Fryer, 1980). The biochemical properties and cell wall
composition of R. salmoninarum isolates appear to be remarkably conserved
(Bruno and Munro, 1986c; Fiedler and Draxl, 1986). Biochemical character-
istics include an inability to produce acid from sugars (Sanders and Fryer, 1980),
a cysteine requirement for growth (Ordal and Earp, 1956; Daly and Stevenson,
1985), the presence of catalase (Ordal and Earp, 1956) and a lack of oxidase.
Protease (Ordal and Earp, 1956; Smith, 1964, Rockey et al., 1991b),
deoxyribonuclease (DNAse) and -haemolytic activities against salmonid
erythrocytes have also been described (Bruno and Munro, 1986c).
Renibacterium salmoninarum possesses a unique peptidoglycan and an
unusual cell wall polysaccharide containing N-acetylfucosamine in addition to
galactose, rhamnose and N-acetylglucosamine (Kusser and Fiedler, 1983). This
polysaccharide comprises more than 60% of the dry weight of trypsinized cell-
wall preparations (Fiedler and Draxl, 1986) and may be a primary constituent of
the capsule (Dubreuil et al., 1990b). Growth is aerobic and optimal at 1518C.
The generation time during the log phase of growth is approximately 24 h.
274
Antigenic characteristics
Renibacterium salmoninarum isolates appear antigenically homogenous by
analysis with polyclonal rabbit antisera. Bullock et al. (1974) demonstrated
serological homogeneity using agglutination and precipitin reactions with sonic
extracts of whole R. salmoninarum from ten US isolates. Fiedler and Draxl
(1986) identified a homogeneous, polymeric polysaccharide, possessing an
approximate subunit weight of 10 kDa, which was immunogenic to rabbits. The
polysaccharide was similar among 13 R. salmoninarum isolates from the USA,
Canada and Europe. In addition, they identified a predominant, trypsin-sensitive
protein present on the cell surface with an approximate molecular mass of 70
kDa. Getchell et al. (1985) characterized R. salmoninarum antigens, using a
variety of techniques, including immunodiffusion, immunoelectrophoresis
rocket immunoelectrophoresis and two-dimensional immunoelectrophoresis.
Seven antigens (AG) were identified as being shared by seven isolates of R.
salmoninarum. The major extracellular component is a 57 kDa protein (named
antigen F), which is heat-stable (100C for 0.5 h). Cross-adsorption experiments
with other R. salmoninarum isolates demonstrated that this antigen is also the
predominant cell-surface antigen. Analysis of antigen F using immunoblotting
has also revealed that, in addition to the major 57 kDa band, there is a related
minor 58 kDa band (Wiens and Kaattari 1989; Daly and Stevenson, 1990). The
production of monoclonal antibodies (MAbs) to the 57/58 kDa protein (also
referred to as p57) (Rockey et al., 1991b; Wiens and Kaattari, 1991) has
confirmed that p57 contains at least two conserved epitopes, common to ten
isolates from geographically disperse locations (Wiens and Kaattari, 1989).
Monoclonal antibodies have also been produced which recognize antigens
unique to particular R. salmoninarum isolates (Arakawa et al., 1987). However,
the composition of these unique antigens was not determined.
DIAGNOSTIC METHODS
The accurate and rapid identification of R. salmoninarum is increasingly
important in efforts to control BKD. Two general strategies for diagnosis of
infection have been pursued: (i) direct detection of R. salmoninarum, using
culture methods or fluorescent antibody staining, or detection of R.
salmoninarum-specific antigens or deoxyribonucleic acid (DNA) sequences;
and (ii) detection of specific host 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 10
79
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. Bruno and
Munro (1982) used a periodic acid-Schiff stain to locate R. salmoninarum in
tissue sections; the non-specificity of this method was noted.
Culture
Culture of R. salmoninarum from fish tissues, followed by serological
identification, is considered the definitive test (Fryer and Sanders, 1981).
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 (KDM1)). 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 5% spent media to culture plates (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.
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 (Benediktsdttir 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 anti-
microbials (0.005% w/v cycloheximide, 0.00125% w/v D-cycloserine, 0.0025%
polymyxin B sulphate 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
tissue, in vitro, is inhibitory to 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
276
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 10
2
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
decreased accuracy 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 has more sensitivity than that observed
with 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
coagglutination 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; Gudmundsdttir 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 220 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 (Fig. 7.2a,b). 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 with high agglutinin titres of 1 : 128 or greater
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 in detecting active
infections (Banowetz, 1974; Bruno, 1987). However, since recovering fish
produce a humoral response to R. salmoninarum, detection of seropositive fish
using agglutination, ELISA (Bartholomew et al., 1991) or electroimmuno-
transfer blot assays (Olivier et al., 1992) may be useful methods to identify
populations which have been exposed to R. salmoninarum.
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; Gudmundsdttir 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). 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, 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. In our opinion, Western blotting is an ideal
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
278
(a)
antigenic identity (Turaga et al., 1987b; Sakai et al., 1990; Wiens et al., 1990). A
limitation of the Western blot has been the sensitivity (Rockey et al., 1991a;
Griffiths et al., 1991; Olivier et al., 1992), however, we have increased the
sensitivity 50100-fold using a chemiluminescent development system (Wiens,
1992). The presence of p57 in samples was detectable at levels as low as
13 ng ml
1
of tissue homogenate and 10 ng ml
1
in ovarian fluid. Disadvantages
of this technique are that it is still three- to fourfold less sensitive than the
monoclonal-based ELISA and, secondly, even if antigen 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 and 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).
(b)
Fig. 7.2. (and opposite) (a) 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 avidinenzyme conjugate. (5) A chromogenic
substrate is added and the colorimetric reaction is measured on an ELISA reader. (b) 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 AE (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 J ohn Reddington, Diaxotics, Wilton, Connecticut.
280
Molecular probes
In addition to detection of antigen, DNA hybridization and the polymerase chain
reaction (PCR) have been recently exploited for the rapid and sensitive detection
of R. salmoninarum. Etchegaray et al. (1991) isolated and used a 149 bp DNA
probe for dot-blotting. This probe did not cross-react with six species of Gram-
negative bacteria; however, the utility of the sequence may be limited, due to its
cross-reactivity with Corynebacterium striatum. PCR may offer a more sensitive
method of detecting R. salmoninarum using either primers to the 16S ribosomal
RNA (Magnusson et al., 1994) or the p57 gene (Brown et al., 1995). Magnusson
et al. (1994) found that nested reverse-transcription PCR of 16S R.
salmoninarum ribosomal RNA from ovarian fluid was as sensitive as
conventional culture techniques. However, the assay was unreliable when it was
used to examine kidney tissue, due to inhibition of the Taq polymerase by an
unidentified tissue component.
CONTROL AND TREATMENT
Transmission
While the transportation of cultured salmonids is believed to have facilitated the
dissemination of R. salmoninarum (Rohovec and Fryer, 1988), the natural route
of infection in salmonids is not fully understood. There is evidence that the
disease is transmitted both horizontally and vertically. Circumstantial evidence
for natural horizontal transmission was obtained by Mitchum and Sherman
(1981). They stocked rainbow, brook and brown trout that were negative in a
river system enzootic with BKD. When stocked trout were examined, R.
salmoninarum was identified in 3245% of the fish, using the IFAT. Mortalities
attributed to BKD were observed within 9 months of stocking. Unfortunately, a
group of stocked fish was not kept as controls to determine if they had
undetectable levels of R. salmoninarum prior to release. Horizontal transmission
has been demonstrated under laboratory conditions (Bell et al., 1984; Murray
et al., 1992); R. salmoninarum-inoculated fish were placed in the same tank as
uninoculated fish and the uninoculated fish became infected.
The mechanism through which horizontal transmission occurs is not
clear. Infection through contaminated water may be one route. Viable R.
salmoninarum have been demonstrated in fresh and salt water using FAT and
bacterial culture (Austin and Rayment, 1985; Elliott and Pascho, 1991), and
infection is probably via ingestion of infected water. Wood and Wallis (1955)
infected 100% of salmon by feeding them infected adult viscera. A natural
source of infective material may be faeces of clinically or subclinically infected
or carrier fish. Balfry et al. (1996) showed that oral intubation of infected faecal
material, but not autoclaved material, resulted in IFAT-positive fish and
subsequent mortality. They suggested that this mechanism may contribute
significantly to the horizontal transmission of R. salmoninarum among
salmonids reared in sea-water net-pens.
In addition, R. salmoninarum may be 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. Renibacterium
salmoninarum has been identified on both the surface and the inside of eggs
from a naturally infected female coho salmon. The fish harboured 4 10
9
colony-forming units (CFU) R. salmoninarum ml
1
of ovarian fluid (Evelyn et
al., 1984). The bacterium was cultured from 15.1% of surface-disinfected eggs
and observed also 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 10
9
, 1.3 10
12
and 1.7 10
5
, respectively) (Evelyn et al.,
1986a). Under these conditions, only 15.5% of the experimentally exposed
eggs contained viable R. salmoninarum (Evelyn et al., 1986a; Lee and Evelyn,
1989). It is not clear why only a low percentage of eggs were infected or when
they became infected. 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 infection may result directly from ovarian tissue prior to ovulation.
Apparently, intraovum infection results in infected progeny. Lee and Evelyn
(1989) demonstrated that smolts reared from the eggs of naturally infected
adults, but not from uninfected adults, had subclinical levels of R. salmon-
inarum, detectable using FAT. Low levels of R. salmoninarum (23113 cfu ml
1
in ovarian fluid) were associated with a 1-2% infections in smolts with
subclinical disease. Experimental immersion challenges of eggs also correlated
with the prevalence of subclinical infections in smolts. Interestingly, no
mortality was attributed to BKD in subclinically infected offspring, even though
the prevalence was 44% in the highest challenge group. Unfortunately, bacterial
cells from FAT-positive fish were not cultured or enumerated. Certainly, the
mechanism(s) of intraovum transmission need to be further studied as this
represents a novel mode of transmission of bacterial pathogens in vertebrates.
Chemotherapy
Chemotherapeutic control of BKD is widely recognized as a difficult problem
for fish culturists (Elliott et al., 1989). Currently, the primary antibiotic used is
erythromycin, which is either injected into prespawning adult broodfish or
incorporated into the feed of juvenile fish. Erythromycin phosphate, when
injected (11 mg kg
1
) at 2130-day intervals until spawning, reduces mortality in
infected adult chinook (Groman and Klontz, 1983). Antibiotic injection also
reduces levels of R. salmoninarum within the egg (Evelyn et al., 1986b; Brown
et al., 1990). Feeding of erythromycin reduces mortality of infected eastern
brook trout (Wolf and Dunbar, 1959), rainbow trout (Austin, 1985) and chinook
salmon experimentally challenged with R. salmoninarum (Moffitt and Bjornn,
282
1989). A dose of 200 mg kg
1
body weight for 21 days appears to be the most
effective treatment (Moffitt, 1992). Use of erythromycin in the USA is
somewhat limited, since it has not been registered as a therapeutic drug for use in
fish culture. It is only available as an investigational new animal drug (INAD)
through the Food and Drug Administration (Moffitt, 1992). Even if erythro-
mycin is approved, chemotherapy is still problematic as erythromycin is not
completely effective in curing infected fish or preventing vertical transmission
in fish (Austin, 1985; Brown et al., 1990). Additionally, the demonstration of
bacterial resistance in vitro to erythromycin (Bell et al., 1988), the strictly
bacteriostatic mechanism of action and the widespread prophylactic treatment of
juvenile fish and adult brood stock increase the probability that the emergence of
resistant strains may be a future concern.
Adult segregation
The lack of an efficacious vaccine (Kaattari et al., 1988; Munro and Bruno,
1988) and potential problems with chemotherapy have stimulated research to
develop novel methods for the control of BKD. One novel approach is to refrain
from using gametes from infected adults (Armstrong et al., 1989; Elliott et al.,
1989). This control method is based on the premise that progenies from
disease-free adults or, perhaps, those with low levels of infection have decreased
prevalence of BKD if intraovum transmission is the predominant route of
infection. Reports on this approach appear promising. Pascho et al. (1991b)
studied the effects of adult segregation and rearing of spring chinook salmon
returning to Dworshak National Fish Hatchery in Idaho. Gametes were
segregated into two groups and offspring were reared separately, based on
whether they came from spawners with high or low levels of R. salmoninarum as
determined by ELISA and FAT. Offspring from highly infected adults had a
higher cumulative pond mortality (17% vs. 5%) and higher prevalence of
infection at the time of release (85% vs. 62%) than offspring from the adults with
low antigen levels. The authors suggested that segregation may be a useful
method to control BKD, even in situations where untreated hatchery water is
used. However, the significant difference in mortality and prevalence of
infection in the progenies also suggest that horizontal transmission may be
important under hatchery conditions or that fish with even marginal levels of R.
salmoninarum infection are capable of promulgating transmission. In a separate
experiment, Warren (1991) found that segregation resulted in an increased
return rate of progeny from parents with low or undetectable levels of R.
salmoninarum. Segregation experiments in a number of hatcheries are under
way to confirm these results.
PATHOGENESIS AND IMMUNITY
Process of infection
Bruno (1986b) characterized the histopathological changes that occur after
intraperitoneal injection (i.p.) of live or formalin-fixed R. salmoninarum into
rainbow trout. Within 45 min of injection, bacterial cells were in phagocytes of
the kidney and spleen. After 46 days, disseminated extracellular R. salmon-
inarum were observed throughout these organs and, by 610 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. Colonization of kidney tissues and cell-mediated reactions to R.
salmoninarum probably result in development of granulomatous lesions, a
common reaction to intracellular pathogens. 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. Preliminary evidence suggests that
R. salmoninarum is able to escape from the macrophage phagosome into the
cytoplasm of the cell (Gutenberger et al., 1991a). This suggests that R.
salmoninarum is an invasive organism capable of causing a disseminated
infection. Consistent with this is the identification of an R. salmoninarum DNA
fragment 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 numerous pathogenic bacteria and is thought to
facilitate access to nutrients and evasion from the immune system.
Infection changes a number of haematological and serum parameters in both
experimentally and naturally infected fish. Bruno (1986a,b) found that
circulating erythrocytes decreased 5966% compared with uninfected fish.
Correlated with this decrease is a reduction in erythrocyte diameter from
16.6 m to 14.5 m and an increase in the erythrocyte sedimentation rate. A
decrease in haematocrit, cholesterol, sodium and electrophoretically faster-
migrating serum proteins has been correlated with increased progression of
infection, as well as an increase in serum bilirubin, blood urea nitrogen and
potassium (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).
Correlated with a progression of disease is an increase in the levels of p57
antigen, proteolytic degradation products and a decrease in haematocrit (Turaga
et al., 1987b). Since p57 is found in large concentrations in sera of
experimentally and naturally infected fish (Turaga et al., 1987b; Rockey et al.,
1991b), it is possible that humoral immunity to p57 may result in immune
complex formation and subsequent hypersensitivity reactions in the glomeruli of
the kidney, as was postulated by Turaga (1989). In support of this hypothesis,
284
Young and Chapman (1978) and Sami et al. (1992) saw electron-dense
subendothelial deposits that resemble immune complexes in experimentally and
naturally infected fish. Also, staining of alternate tissue sections with a MAb
specific for fish immunoglobulin or p57 revealed similar immunofluorescent
profiles, which is suggestive evidence of immune complex deposition (Turaga,
1989). However, double-labelling with MAbs as well as the isolation of immune
complexes, is required before this hypothesis can be confirmed.
Humoral and cell-mediated immunity
Little is known about the induction of humoral and cell mediated immunity to R.
salmoninarum. There is some circumstantial evidence to suggest that infected
salmonids 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% had agglutinin response and
demonstrated a resolution of granulomatous lesions. These facts suggest that a
protective immune response occurred in the survivors. Agglutinating antibodies
can be induced experimentally by i.p. injection of killed R. salmoninarum;
however, the response is very slow (Evelyn, 1971). In general, the protection
provided by vaccination with heat- or formalin-killed R. salmoninarum is
limited in efficacy (Munro and Bruno, 1988; Sakai et al., 1989c). Turaga (1989)
found that the best protection afforded by vaccination of coho salmon was with
Freunds complete adjuvant or killed Mycobacterium cheloni without inclusion
of R. salmoninarum, both of which significantly delayed the mean time to death.
It is uncertain whether a specific immune response to a cross-reactive
determinant or a non-specific cell-mediated response resulted in the limited
protection observed.
Virulence factors
A number of enzymatic activities of R. salmoninarum have been identified and
these may contribute to virulence. These include haemolytic, proteolytic,
exotoxin, catalase, DNAse and iron reductase activities (Bruno and Munro,
1982; Shieh, 1988b; Grayson et al., 1995b). In addition, a 50100 nm capsule, a
common virulence factor of other bacteria, has been observed using electron
microscopy (Dubreuil et al., 1990b). However, conclusive proof of any
correlation of these enzymes or the capsule with isolate virulence has not been
demonstrated. In efforts to characterize the molecular nature of these putative
virulence factors, an R. salmoninarum haemolysin has been cloned (Evenden et
al., 1990). The cloned DNA is approximately 1.6 kb in length and produces a
haemolysin that causes lysis of rainbow trout erythrocytes. The cloned insert
hybridized with four isolates of R. salmoninarum genomic DNA, including the
type strain ATCC 33209. Antisera have been made against a soluble fusion
protein produced in E. coli, and Western blotting indicates that the protein is cell-
associated and not secreted in broth culture (Grayson et al., 1995a). Recently,
Grayson et al., (1995d) cloned a second gene encoding a predicted 66.7 kDa
iron-regulated protein with haemolytic activity for erythrocytes from a number
of mammalian species and also fish. Other investigators (Bandin et al., 1991)
have been unable to detect expression of haemolytic activity in ten R.
salmoninarum isolates. This discrepancy may be explained by the low amount of
haemolysin produced, or by Bruno and Munros (1986c) observation that
haemolytic activity decreases with continued passage.
Proteases are important virulence factors in a number of pathogenic
microorganisms (Trust, 1986). Proteolytic activities of R. salmoninarum have
been described by a number of investigators (Smith, 1964; Bruno and Munro,
1986c; Rockey et al., 1991b). Rockey et al. (1991b) identified two proteolytic
bands using substrate gel electrophoresis, one larger than 100 kDa and the other
less than 20 kDa. The higher-molecular-weight protease has activity against p57
and against denatured bovine serum albumin and hen ovalbumin substrates. The
direct role of the protease(s) in virulence has yet to be established.
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 that developed meningitis suggest the
presence of a toxin (Bell et al., 1990). Shieh (1988b) has identified a putative
exotoxin isolated from R. salmoninarum culture supernatant, which at a dose of
160 g was lethal for Atlantic salmon fingerlings (912 g). Unfortunately, a
control extract was not prepared to rule out the possibility that the exotoxin was
a component of the semidefined medium used (Shieh, 1988a). In contrast to the
work of Shieh, Bandin et al. (1991) were unable to demonstrate any toxicity with
extracellular product (ECP) by i.p. injection of rainbow trout fingerlings or by
addition of the ECP to fish cell lines. We have not found R. salmoninarum ECP
to exhibit cytotoxic effects when added to in vitro cultures of salmonid
leucocytes using trypan blue staining (Turaga et al., 1987a; Wiens and Kaattari,
1991). However, these experiments did not rule out the possibility of toxicity to
a small set of leucocytes. Further purification of extracellular components
to homogeneity, as demonstrated by sodium docedyl sulphate (SDS)-poly-
acrylamide gel electrophoresis (PAGE)) is needed to identify exotoxin(s)
produced by R. salmoninarum.
Isolation of mutants that have a reduced virulence, followed by
identification of the characteristic(s) which the mutants lack, is a common
method of identifying virulence factors (Smith, 1989). Three R. salmoninarum
isolates have been identified which have reduced virulence by i.p. challenge
(Bruno, 1988). Low-virulence isolates produced 818% total mortality with the
final recorded mortality, after 2838 days, while virulent isolates produced 73
81% mortality, with a final mortality after 1425 days. Gross lesions were
observed in fish injected with virulent or low-virulence isolates. The low-
286
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 and had reduced
cell-surface hydrophobicity. The loss of autoaggregation and cell surface
hydrophobicity phenotypes was thought to be the result of routine subculturing.
Renibacterium salmoninarum reisolated from reduced-virulence isolates did not
revert to the original phenotype and were stable during the 18 months of
experiments. Interestingly, the absence of a saline-extractable 57 kDa protein
was correlated with the reduction of virulence, suggesting that the 57 kDa
protein may be a virulence factor (Bruno, 1990).
Characterization of the 57/58-kilodalton protein
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
recently been cloned and sequenced and it encodes a protein of 557 amino acids
with a calculated M
r
of 57,190 (Chien et al., 1992). Amino acids 126 encode a
putative leader peptide sequence, which, after processing, results in a mature
54,505 kDa protein. The amino-terminal sequence derived from micro-
sequencing 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 if 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 disulphide
bonding, as the addition of -mercaptoethanol does not change the
electrophoretic migration (Daly and Stevenson, 1990). There does not appear to
be carbohydrate modification of p57, as Schiff staining has been negative
(Dubreuil et al., 1990a). Based on the gene sequence analysis, the theoretical
isoelectric point (pI) of p57 should be 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 under electron microscopy of R.
salmoninarum cells may be composed of p57 (Dubreuil et al., 1990b).
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, as evidenced by the appearance of a 53 kDa band concomitant upon the
decrease in p57 (Griffiths and Lynch, 1991). Also, more lower-molecular-weight
bands can be found in aged culture supernatants. This proteolysis can be
inhibited by the addition of the serine protease inhibitor, phenylethylsulphonyl
fluoride (Griffiths and Lynch, 1991; Rockey et al., 1991b). In an attempt to
identify this 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 observed in the 57 and 3337 kDa bands,
suggesting that the protein might be autolytic or possibly comigrated with a
protease. Rockey et al. (1991b) identified a high-molecular-weight protease
which has low activity against p57 at 17C and is highly active at 37C. This
might explain some of the proteolysis observed by Griffiths and Lynch (1991).
The protease was inhibited by phenylethylsulphonyl fluoride, methanol, ethanol
and 10 min incubation at temperatures greater than 65C. No degradation was
observed in purified p57 preparations, which suggested that the protein was not
autolytic or that the autolytic activity was destroyed during purification.
Addition of the R. salmoninarum protease to purified p57 at 17C yielded a
spectrum of its breakdown products similar in molecular mass and antigenicity
to those seen in the extracellular protein. However, p57 was completely
degraded into numerous smaller-molecular-weight bands when enzymatic
reactions were performed at 37C. This suggests that either the protease is highly
active at higher temperatures or p57 is more susceptible to degradation due to
denaturation and exposure of more proteolytic sites at higher temperatures. The
physiological function of the protease is unknown, but one possibility may be
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). These culture conditions may mimic the iron-limited
conditions inside fish and facilitate expression or stability of p57.
The in vivo function of p57 is uncertain, but the concentration in fish tissues
and sera 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 pathology that is observed. Among these activities is
mammalian erythrocyte agglutination (Daly and Stevenson, 1987), salmonid
spermatocyte and leucocyte agglutination (Daly and Stevenson, 1989; Wiens
and Kaattari, 1991) and non-specific suppression of salmonid antibody
responses (Rockey et al., 1991b). Daly and Stevenson (1990) demonstrated that
the 57/58 kDa protein has haemagglutinating activity for rabbit and a number of
other mammalian erythrocytes. Interestingly, one out of nine strains does not
have the 58 kDa protein and yet still has haemagglutinating activity. This
suggests that only the 57 kDa protein is required for haemagglutinating activity.
Strain MT239, originally described by Bruno (1988), lacked both the 5758 kDa
bands and haemagglutinating activities (Daly and Stevenson, 1990). The 57 kDa
protein also contributes to bacterial surface hydrophobicity, as the hydro-
phobicity is restored to water-washed cells after incubation with gel-purified
57 kDa protein. Relative cell-surface hydrophobicity is resistant to trypsin and
protease-K treatment; however, purified haemagglutinin is sensitive to 20 min
incubation in either enzyme. Daly and Stevenson (1990) hypothesize that, when
288
the protein is present on the bacterial cell surface, proteolytic sites are not
available, but, when the protein is removed from the cell surface, the proteolytic
sites are exposed.
Further research on the proteolytic products has helped define the structure
and function of p57 (Wiens and Kaattari, 1991). Digestion of p57 at 17C
typically produces predominant breakdown products with approximate
molecular weights of 45, 36, 34, 25 and 20 kDa. Analysis of the pI, N-terminal
sequences and differential recognition, using MAbs, has led us to the proposed
structural model of p57 depicted in Fig. 7.3. Monoclonal antibodies were
categorized into three groups, based on recognition patterns, determined using
Western blot. Group I MAbs recognize a region proximal to the amino terminus
of the protein and, since two of these antibodies block the leucoagglutination
activity, it suggests that the leucocyte-binding domain may be located near the
amino terminal portion of the protein. The carboxy terminus of p57 appears to be
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;
this suggests that the epitope(s) recognized 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 presence of these fragments suggest that they contain a
region(s) of the protein which remain attached to the bacterial cell surface, even
after proteolysis. Confirmation of the epitopes recognized by the MAbs and the
localization of the functional regions on p57 will be facilitated with the
availability of a recombinant p57.
Fig. 7.3. 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).
SUMMARY AND FUTURE RESEARCH
Bacterial kidney disease has been one of the most intractable diseases of
salmonid fish. The disease has severely affected aquaculture in both the northern
and southern hemispheres; however, fully efficacious prophylactic or
therapeutic treatments have not been developed. Control of the disease is of
utmost practical importance, but, before this can be adequately achieved, more
research is required to understand R. salmoninarum virulence factors,
transmission and the salmonid immune response to the bacteria.
Although various potential virulence factors have been identified, there is
no conclusive evidence that one or more of these are important in the disease.
Intensified study on the pathogenesis of BKD in salmonids is greatly needed.
These studies will be facilitated by the development of genetic systems for
creating isogenic mutants and the purification of the specific protein products
from the virulence genes. It is likely that virulence will be multifactorial and
research on the coordinate regulation of virulence factors will be a productive
area of future study. One future target of research is p57 and resolution of its role
in pathogenesis. The hydrophobic composition, the filamentous structure and
the ability to bind to fish leucocytes suggests that p57 may function in causing
the bacteria to adhere to specific cell types, thereby promoting intracellular
invasion (Fig. 7.4). It is unlikely that p57 functions solely in adhesion, as such a
large quantity of p57 is secreted into fish tissues. Interestingly, recent data of
Brown et al. (1996) suggest that injection of unfertilized eggs with 100 ng of a
p57-enriched bacterial cell extract resulted in long-term immunosuppression
Fig. 7.4. 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.
290
and decreased ability to resist subsequent challenges with R. salmoninarum. The
presence of proteolytic breakdown components in the extract and the possibility
of other R. salmoninarum products indicate the need to purify p57 protein away
from its breakdown products for further definitive functional studies. The recent
availability of a cloned p57 gene and expression in E. coli may help solve these
technical limitations (Grayson et al., 1995c). Understanding the genetic
regulation of the p57 gene and other antigens will facilitate the designing of
optimal immunodiagnostic assays to identify infected or recovering fish.
Specific and sensitive ELISAs have been developed for detecting antigen in
juvenile and adult fish; however, the ELISA cannot detect R. salmoninarum
antigen inside eggs (Winton et al., 1990). New assay systems, such as PCR may
be useful for identifying minute amounts of R. salmoninarum DNA in tissues,
ovarian fluid and eggs (Brown et al., 1994; Leon et al., 1994; Magnusson et al.,
1994). Technical limitations, such as the sample preparation and mass screening
techniques of PCR, still need to be overcome before such an assay will be useful
for widespread practical applications.
Sensitive and specific assays for the detection of R. salmoninarum are
critical, as segregation of brood stock appears to be a promising strategy for
reducing mortality among offspring and increasing adult returns. Research is
still required to determine the level of infection permissible for optimal
segregation. Since the prevalence of infection is extremely high in many
hatcheries on the west coast of the US, large numbers of gametes would have to
be eliminated to significantly reduce vertical transmission. This may have
unintended impacts on the gene pool of hatchery stocks. Future research must be
performed to determine the correlation between natural BKD infection and
genetic traits of fish. Since prevalence of R. salmoninarum is extremely high in
some locations (Maule et al., 1996), it is possible that susceptibility to BKD may
confer some selective advantage to fish. Therefore, the culling of segregated fish
in hatcheries should be approached with caution. These concerns may not be as
important for commercial net pen culture, as the fish are artificially reared
throughout their life cycle. An alternative approach to destruction of gametes
from adults with R. salmoninarum is to raise the infected fish separately with
intensive chemotherapy.
Analysis of the mechanisms of transmission and the role of virulence factors
in the disease process will enhance efforts to develop novel vaccines. Thus far,
induction of immunity to BKD has not been consistent. The primary reasons
have been the lack of a standardized and repeatable challenge procedure which
mimics natural exposure but does not take prohibitively long periods of time to
lead to mortality. Measuring antigen levels in tissues after immersion challenge
with R. salmoninarum may be a novel and effective method for evaluating
disease progression (Elliott et al., 1991). Secondly, the status of immunological
assessments is still in its infancy with respect to salmonids. Although techniques
exist for the sensitive assessment of specific antibodies, there are a limited
number of approaches available for measuring specific cellular immunity.
Overcoming this deficiency is likely to be critical for the assessment of salmonid
immunity to BKD, since protection against other intracellular pathogens is
mediated through the cellular component of the immune response.
ACKNOWLEDGEMENTS
The authors thank Dr B. Wiens for critically reading this review. Support for
research on BKD has been provided by contracts DE-A179-84BP16480,
DE-FG79-89BP95906 from the Bonneville Power Administration (DOE) and a
Sigma Xi Grant-in-Aid of Research. This is Oregon State technical paper no.
9969 and Virginia Institute of Marine Science (VIMS) contribution no. 2146.
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