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INTRODUCTION
Aeromonas salmonicida has been recognized as a pathogen of fish for over 100
years. Emmerich and Weibel (1894) made the first authentic report of its
isolation during a disease outbreak at a Bavarian brown trout hatchery, the
manifestations of the disease including furuncle-like swelling and, at a later
stage, ulcerative lesions on infected trout. Since that time a number of
subspecies of A. salmonicida have been recognized, although the taxonomy of
the species is far from settled. Aeromonas salmonicida is one of the most studied
fish pathogens, because of its widespread distribution, diverse host range and
economically devastating impact on cultivated fish, particularly the salmonids
(Austin and Austin, 1993). The continued importance of salmonids to rod
fishermen, commercial fisheries and fish farmers and the extent of the impact
that A. salmonicida has on these various methods of exploitation have served to
maintain the status of A. salmonicida as an important fish pathogen. A number of
excellent reviews of A. salmonicida have been published (McCarthy and
Roberts, 1980; Austin and Austin, 1993; Bernoth et al., 1997). This chapter
attempts to summarize the current information, both research and anecdotal,
available on A. salmonicida and its associated pathologies in a manner
accessible to all those for whom this organism is of concern.
Susceptibility to furunculosis
Most fish species would appear to be susceptible to infections by A. salmonicida,
but the level of susceptibility is variable. For example, among salmonids,
susceptibility to infection is reported to be low in rainbow trout (Cipriano and
Heartwell, 1986; Pérez et al., 1996), while brook trout, brown trout and many
other salmon species appear to have a high susceptibility (McCraw, 1952;
Evelyn, 1971; Klontz and Wood, 1972; Miyazaki and Kubota, 1975; McCarthy,
1977a; Cipriano and Heartwell, 1986; Austin and McIntosh, 1988). In addition,
susceptibility may vary within the same fish species raised from different
genetic lines (Dahle et al., 1996; Marsden et al., 1996) or with different histories
of exposure to A. salmonicida (St Jean, 1992). Because of the potentially
inheritable nature of some disease resistance, directed breeding programmes
aimed at raising stocks inherently resistant to furunculosis have been
investigated as a possible disease-control strategy in salmonids (Gjedrem et al.,
1991; Lund et al., 1995; Gjedrem, 1997) and non-salmonids (Sövényi et al.,
1988; Hjeltnes et al., 1995). However, the multifactorial nature of inheritable
characteristics complicates selective breeding programmes and much work
remains to be done in this area (Gjedrem, 1997). Species susceptibility to
infection by atypical A. salmonicida is discussed in a later section.
Among salmonids, susceptibility to furunculosis may also be age-related.
Many early workers in furunculosis research believed that, in wild salmonid
populations, furunculosis was mainly a disease of older fish (Plehn, 1911;
Mettam, 1915; McCraw, 1952). Although this perception may, in part, have been
due to the easier observation of large carcasses in rivers, experimental evidence
did suggest that young fish (under 1 year old) are relatively resistant to A.
salmonicida infections (Blake and Clarke, 1931; Mackie and Menzies, 1938;
Scallan, 1983). The mechanisms of resistance in young fish are essentially
unknown but are probably non-specific (Krantz and Heist, 1970). Furthermore,
not all workers agree that age plays a significant part in susceptibility to
furunculosis (McCarthy, 1977a; Inglis et al., 1993). McCarthy and Roberts
(1980), referring to the disease in fingerlings, observed that fish of this size
contract an acute form of the disease, which results in rapid death with little
more than slight exophthalmos. Mortalities in infected fish in the 0+ age group
Table 10.1. Salmonid fish species from which typical Aeromonas salmonicida has been isolated (after Bernoth, 1997a).
Fish species
History of
Common name Scientific name Habitat isolation Reference
Atlantic salmon Salmo salar Fresh water Covert* McCarthy (1977a)
Clinical Bernoth (1997a)
Sea water Clinical Smith (1997)
Amago salmon Oncorhynchus rhodurus Sea water Clinical† Miyazaki and Kubota (1975)
Brook trout Salvelinus fontinalis Fresh water Covert Bullock and Stuckey (1975)
Clinical Jensen (1977)
Brown trout Salmo trutta m. lacustris Fresh water Covert Jensen and Larsen (1980)
Clinical Mackie et al. (1930)
Chinook salmon Oncorhynchus tshawytscha Not specified Not specified† Fuller et al. (1977)
Chum salmon Oncorhynchus keta Fresh water Covert Nomura et al. (1993)
Clinical Sakai and Kimura (1985)
Sea water Clinical Wiklund et al. (1992)
Coho salmon Oncorhynchus kisutch Fresh water Clinical Wiklund et al. (1992)
Cutthroat trout Salmo clarki Fresh water Not specified McCarthy (1975)
Dolly Varden Salvelinus malma Fresh water Not specified McCarthy (1975)
Furunculosis
Japanese char Salvelinus leucomaenis Not specified Clinical Sakai and Kimura (1985)
Lake trout Salvelinus namaycush Fresh water Covert Daly and Stevenson (1985)
Clinical McCarthy (1975)
Masu salmon Oncorhynchus masou Fresh water Covert Nomura et al. (1993)
Pink salmon Oncorhynchus gorbuscha Fresh water Covert* Nomura et al. (1993)
Clinical Sakai and Kimura (1985)
Pollan Coregonus pollan Fresh water Not specified† McCarthy (1975)
Rainbow trout Oncorhynchus mykiss Fresh water Covert Bullock and Stuckey (1975)
Clinical Jensen (1977)
Sea water Clinical Fernández et al. (1995)
Sea trout Salmo trutta m. trutta Fresh water Covert Hirvelä-Koski et al. (1988)
Clinical Mackie et al. (1935)
Sockeye salmon Oncorhynchus nerka Not specified Clinical Sakai and Kimura (1985)
*Apparently healthy fish not showing signs of infection (see Hiney et al., 1997b).
†Not specified whether typical or atypical A. salmonicida but assumed to be typical.
343
344
Table 10.2. Freshwater non-salmonid species from which typical Aeromonas salmonicida have been isolated (after Bernoth,
1997a).
Fish species
History of
Common name Scientific name isolation Reference
American eel Anguilla rostrata Clinical Noga and Berkhoff (1990)
Brassy minnow Hybognathus hankinsoni Unclear* McFadden (1970)
Brook stickleback Culaea inconstans Unclear McFadden (1970)
Carp Cyprinus carpio Clinical Mackie et al. (1930)
Unclear Bernoth (1997b)
Catfish Silurus glanis Clinical Mackie et al. (1930)
Chestnut lamprey Ichthyomyzon castaneum Unclear Bernoth (1997a)
Common shiner Notropis cornutus Clinical Ostland et al. (1987)
Creek chub Semotilus atromaculatus Clinical Ostland et al. (1987)
Unclear McFadden (1970)
European eel Anguilla anguilla Incidental Slack (1937)
Fathead minnow Pimephales promelas Clinical McFadden (1970)
Goby Cottus gobio Clinical Bernoth (1997b)
Golden shiner Notemigonus crysoleucas Clinical Ostland et al. (1987)
M. Hiney and G. Olivier
Fish species
History of
Common name Scientific name isolation Reference
Atlantic cod Gadus morhua Incidental Willumsen (1990)
Coalfish Pollachius virens Incidental Willumsen (1990)
Cuckoo wrasse Labrus bimaculatus Clinical Treasurer and Cox
(1991)
Goldsinny wrasse Ctenolabrus rupestris Clinical Treasurer and
Laidler (1994)
Rock cook Centrolabrus exoletus Clinical Treasurer and
Laidler (1994)
Sea bream Sparus aurata Clinical Real et al. (1994)
Striped trumpeter Latris lineata Incidental Bernoth (1997a)
Surf smelt Thallichthys pacificus Unclear* Schiewe et al.
(1988)
Turbot Psetta maxima Clinical Nougayrede et al.
(1990)
Scophthalmus maximus Clinical Toranzo and Barja
(1992)
Wrasse Labridae Clinical Treasurer and Cox
(1991)
*Unclear from history whether isolation was from a clinical case or was an
incidental finding.
can be high and have been reported to reach 93% to 40% during the egg to smolt
stages (St Jean, 1992). In the experience of these authors, furunculosis can occur
in Atlantic salmon alevin whose yolk sacs are still attached.
Geographical distribution
At present, the geographical distribution of A. salmonicida subsp. salmonicida is
almost worldwide, including Japan (Miyazaki and Kubota, 1975) and the
mainland of Asia (Inglis et al., 1993), from where it was previously considered
to be absent (Fryer et al., 1988). The possible exceptions to this distribution are
South America and New Zealand, from which reports of the isolation of A.
salmonicida have yet to be made (Bernoth, 1997a). The introduction of
furunculosis into Sweden in 1951 was reported by Wichardt et al. (1989) and it
was recognized in Norway in 1964 (Lunder and Håstein, 1990; Johnsen and
Jensen, 1994). This more recent identification of A. salmonicida in Scandinavian
countries has been tentatively traced to importation of live fish stocks, initially
from other European countries (Egidius, 1987; Wichardt et al., 1989) and then
within Scandinavia (Rintamäki and Valtonen, 1991). To date, there have been no
reports of ‘typical’ furunculosis in salmonids in Australia, despite many attempts
to isolate the organism (Bernoth, 1997a). Atypical A. salmonicida was, however,
348 M. Hiney and G. Olivier
identified from diseased goldfish (Trust et al., 1980b). In South Africa, the first
incident of infection of rainbow trout by an atypical A. salmonicida was noted by
Boomker et al. (1984).
The widespread distribution of furunculosis is reflected by the fact that the
disease has never been listed by the Office Internationale des Epizooties (OIE)
as one that merits special attention, being considered endemic in most countries
and capable of control (C. Michel, personal communication). Likewise, the
European Community assigned furunculosis to its List 3 diseases, that is,
diseases which are endemic in many member states (Council Directive 91/67/
EEC). Individual member states may enforce control strategies on importation
of stocks only with the approval of the Standing Veterinary Committee, which
must ensure that valid reasons exist for the proposed controls, such as a disease-
free status, and that they are not a concealed trade barrier (McLoughlin, 1993).
The disease
Clinical infection by typical Aeromonas salmonicida
Classical furunculosis derives its name from the boil-like lesions observed by
Emmerich and Weibel (1894) on the skin and in the musculature of infected fish.
However, development of ‘furuncles’ on the dorsal body are the exception rather
than the rule (Bernoth, 1997b) and, in the experience of this author, only occur in
older fish suffering from the chronic form of the disease. The clinical
manifestations of furunculosis are often divided into peracute, acute and
subacute or chronic forms and will be discussed here under these headings
(Table 10.4). It should be noted that clinical manifestations of more than one
form of the disease may be present in individual fish within a population
(Bernoth, 1997b). Reviews on the macro- and microscopic features of
furunculosis are given in Ferguson (1977), McCarthy and Roberts (1980),
Frerichs and Roberts (1989), Armstrong (1992), Austin and Austin (1993) and
Bernoth (1997b).
PERACUTE FURUNCULOSIS
Because the peracute form of furunculosis is usually restricted to young fish,
whose defences against a severe bacterial septicaemia will be poor, this form of
the disease normally results in rapid death with little more that slight
exophthalmus (McCarthy and Roberts, 1980; Frerichs and Roberts, 1989). The
gross pathology of peracute furunculosis is typical of a peracute septicaemia.
Microcolonies can be observed histologically in a number of organs, with no
inflammatory infiltration and only limited necrosis. McCarthy and Roberts
(1980) considered that cardiac damage was the most likely cause of death in
young fish.
ACUTE FURUNCULOSIS
In growing fish, furunculosis tends to occur in an acute form, which is
manifested clinically by a generalized bacterial septicaemia displaying the
‘standard’ features (Table 10.4). As its name implies acute furunculosis is often
Furunculosis 349
fatal in 2–3 days and, because of the short duration of the disease, furuncle
development is unusual. Fish with an acute infection show signs of a
haemorrhagic septicaemia, including bloody anal vents. Skin lesions may be
haemorrhagic patches or blotches along the side or on the dorsal body surface,
or, more typically, raised furuncles, which usually develop in the dermis rather
than the hypodermis (Bernoth, 1997b).
SUBACUTE/CHRONIC FURUNCULOSIS
The chronic form of furunculosis is common in older fish and is probably the
form first observed by Emmerich and Weibel (1894). In chronic cases, fish may
show a lesser degree of skin darkening and inappetence than in the acute form.
Other signs are summarized in Table 10.4. Furuncles are likely to be observed
during the progress of a chronic infection and, where they do occur, more than
one lesion may be present. These furuncles may be large and, when ruptured, the
viscous fluid may contain more necrotic material than furuncles found in acute
cases (Bernoth, 1997b). For a description of the histopathology of furuncles, see
the reviews of McCarthy and Roberts (1980) and Frerichs and Roberts (1989).
INTESTINAL FURUNCULOSIS
A fourth form of furunculosis associated with low mortality, intestinal
furunculosis, has been described by Amlacher (1961) (cited in Austin and
Austin, 1993). The only clinical sign of this form of the disease was prolapse
of the anus, although examination revealed haemorrhage and intestinal
inflammation.
Table 10.4. Diagnosis of clinical infections of typical A. salmonicida aetiology (documented signs).
Age of fish Very young fish Growing fish Usually older fish
Clinical signs Darkening of skin; tachybranchia Darkening of skin; inappetence; Slight darkening of skin; inappetence;
(rapid breathing); slight lethargy (sluggishness); lethargy; congested blood vessels at
exophthalmus (pop-eyed) tachybranchia; small base of fins; slight exophthalmus;
haemorrhages at base of fins expression of serosanguineous fluid
from nares and vent; may have
pale or congested gills; may have
furuncles on flank or dorsal surface
Gross pathology Dilated blood-vessels; punctate Hyperaemia of serosal General visceral congestion and
haemorrhages in parietal and surfaces; punctate peritonitis; multiple haemorrhages in
visceral peritoneum and over haemorrhages scattered over muscle and liver; splenomegaly;
myocardium; may have focal abdominal walls, viscera and necrotic kidney; adhesions between
haemorrhages in gills heart; soft and friable or viscera and between viscus and
liquefied kidney; enlarged, abdominal cavity; intestinal
cherry-red spleen with round inflammation; septic, necrotic and
M. Hiney and G. Olivier
detect covert infections. In addition, the variety of names that have been applied
to these infections makes it extremely difficult to present comparisons of these
studies (Hiney et al., 1997b). In an effort to clear up some of the confusion that
surrounds both the nomenclature and the exact nature of what is being studied,
Hiney et al. (1997b) proposed a number of alternative definitions of clinically
unapparent infections, strictly related to the diagnostic methods that have been
used. Using this approach, three categories could be identified and all studies on
covert infections were reassigned to one of these three categories:
1. Covert infection: demonstration of A. salmonicida, its antigens or its
deoxyribonucleic acid (DNA) in, or on, a fish that manifests no clinical signs of
furunculosis.
2. (Covert) carrier infection: demonstration of the shedding of A. salmonicida
or its antigens or DNA into the environment by a fish that manifests no clinical
signs of furunculosis; demonstration of the ability of fish that manifest no
clinical signs of disease to transmit, in cohabitation experiments, furunculosis to
fish free of this disease.
3. (Covert) stress-inducible infection (stress-inducible furunculosis (SIF)):
demonstration of clinical disease following the stressing of a fish that manifests
no clinical signs of furunculosis.
It is apparent from these definitions that information on the location of A.
salmonicida in covertly infected fish would do much to improve our
understanding about the mechanisms of these types of infections. When
considering covert infections, these clinically unapparent infections of fish
may, in fact, represent a number of infection types, which are mediated by
fundamentally different processes (Hiney et al., 1997b). However, the under-
lying processes of covert infection will remain unknown until we develop
methods that can distinguish between different infection types in individual fish.
The microorganism
Taxonomic classification of Aeromonas salmonicida subsp. salmonicida
Aeromonas salmonicida is a member of the genus Aeromonas, which also
includes the mesophilic aeromonads (Popoff, 1984). The species has been
described by a number of different names since its original isolation, being
variously called Bacillus der Forellenseuche, or bacillus of contagious trout
disease (Emmerich and Weibel, 1894), Bacillus truttae (Marsh, 1902), ‘pigment-
forming Bacillus’ (Arkwright, 1912), Necromonas salmonicida (Smith, 1963)
and Bacterium salmonicida (Griffin et al., 1953). Griffin and coworkers
recommended the reclassification of B. salmonicida to the newly created genus
Aeromonas, as A. salmonicida, a taxonomic position the species has held to this
day. Since Aeromonas species share common properties with members of the
Enterobacteriaceae, the Vibrionaceae and the Pseudomonadaceae, there has
been some controversy over the classification of these species within the family
Vibrionaceae. Molecular genetic studies have led to proposals that the genus
Aeromonas be placed in a new family, Aeromonadaceae (Colwell et al., 1986).
There is agreement that A. salmonicida is the only psychrophilic member of
the genus Aeromonas. Within this group, a number of subspecies have been
proposed, which have been traditionally referred to as ‘typical’ and ‘atypical’ A.
salmonicida. There has been much confusion about what actually constitutes a
typical or atypical A. salmonicida strain. McCarthy (1978) proposed a functional
split of A. salmonicida subspecies based on the host from which they were
isolated and the associated pathology. Other workers, reviewed in a later section,
have favoured separating A. salmonicida subspecies by phenotypic/genotypic
Furunculosis 355
DIAGNOSTIC METHODS
Biological characteristics of Aeromonas salmonicida
The traditional description of A. salmonicida subsp. salmonicida is of a non-
motile, non-sporulating, fermentative, Gram-negative, aerobic bacillus (Popoff,
1984), which reduces nitrate, liquefies gelatine, hydrolyses starch (Popoff and
Lallier, 1984) and produces cytochrome oxidase, although isolation of an
oxidase-negative ‘typical’ A. salmonicida has been reported from coho salmon
(Teska et al., 1992). Staining has a tendency to be bipolar, and the organism
measures approximately 1.0 µm × 2.0 µm, varying morphologically from an
almost coccoid form in freshly isolated cultures to distinct rods in cultures
maintained on artificial media, the latter often proving avirulent. Aeromonas
356
Table 10.5. General guidelines for subspecies separation of the psychrophilic aeromonads.
Properties
Dominant Genetic Biochemical
Designation Subspecies Distribution host Pathology profile* profile*
Typical salmonicida Almost worldwide Salmonids Classical furunculosis Homogeneous Homogeneous
Atypical masoucida Japan Salmonids Other pathologies – –
achromogenes Extensive Non-salmonids/
salmonids Other pathologies Heterogeneous Heterogeneous
nova Extensive Non-salmonids/
M. Hiney and G. Olivier
Phage typing Surface receptors allow Useful for differentiation Popoff (1971); Rodgers et al.
binding by specific viruses of geographically distinct (1981); Olivier (1992)
and selective infection and strains
lysis of cells
Pseudomonas Differential inhibition of Results difficult to Rudden and Smith (1995)
inhibitory assay A. salmonicida on solid interpret. Not
medium by a panel of discriminatory enough
Pseudomonas spp.
Continued over
357
358
Immunological LPS antibody binding Antigenic variation in Not sufficient LPS Ezura et al. (1980); Chart et al.
A. salmonicida LPS antigenic heterogeneity (1984); Rockey et al. (1991)
detected by binding of in A. salmonicida to be
specific monoclonal useful
antibodies
Serotyping Microscopic slide Few serological Paterson et al. (1980); Hahnel
agglutination and differences found et al. (1983); Bernoth and
cross-absorption of Böhm (1988); Dalsgaard
A. salmonicida with et al. (1994)
antiserum raised against
different strains
Genetic Plasmid profiling Generation of profile by Plasmid profiles of Toranzo et al. (1983); Bast et al.
plasmid extraction and A. salmonicida too (1988); Nielsen et al. (1993);
sizing of plasmids by gel uniform to be Hänninen et al. (1995)
M. Hiney and G. Olivier
electrophoresis discriminatory
RAPD analysis PCR amplification of Results difficult to Hänninen et al. (1995); Miyata
polymorphic DNA with a interpret. May have et al. (1995)
panel of random primers, promise but very
sizing of products by gel unreproducible at present
electrophoresis
REF analysis Enzyme restriction of total Usefulness limited by McCormick et al. (1990)
cellular DNA sizing of genetic homogeneity of
fragments by gel A. salmonicida
electrophoresis
RFLP/DNA probe
analysis Enzyme restriction of Could detect no Hennigan et al. (1989)
polymorphic DNA and polymorphisms
hybridization with a panel
of probes targeted against
A. salmonicida
Ribotyping Enzyme restriction of Low discriminatory power Nielsen et al. (1994); Hänninen
rRNA genes and for A. salmonicida and Hirvelä-Koski (1995);
hybridization with a Hänninen et al. (1995)
suitable probe
LPS, lipopolysaccharide; RAPD, randomly amplified polymorphic DNA; rRNA, ribosomal ribonucleic acid; RFLP, restriction
fragment length polymorphism.
Furunculosis
359
360 M. Hiney and G. Olivier
salmonicida is normally isolated from the kidney of infected fish, although it can
also be isolated from lesions, blood and other organs (Daly and Stevenson,
1985). The size of colonies is variable, ranging from 0.5 to 3.0 mm in diameter
after 72 h incubation (Drinan, 1985). Temperatures of 18–22°C are optimal for
growth. A. salmonicida grows poorly at 4°C and does not grow at 37°C
(Snieszko, 1957), although isolation of an atypical strain capable of growth at
37°C has been reported (Austin, 1993).
On agar media containing tryptone, typical A. salmonicida normally, though
not always (Wiklund et al., 1993), produces a brown, melanin-like, water-
soluble pigment. This pigment and its production pathway have been described
in detail by Donlon et al. (1983). Virulent colonies are small and friable and, on
initial isolation from fish, autoagglutinate in 0.85% physiological saline
(Evenberg and Lugtenberg, 1982; Evenberg et al., 1982; Drinan, 1985). Pro-
longed subculture on laboratory media, or incubation of strains above optimum
temperatures, produces non-aggregating variants with altered cell morphology
(Ishiguro et al., 1981). The hydrophobic nature of A. salmonicida is due to the
possession of an additional surface-protein layer (A-layer), first described by
Udey and Fryer (1978). The A-layer has been well characterized (Kay and Trust,
1997), because of its association with the pathogenesis of A. salmonicida and its
role in resistance to host defence mechanisms (Sakai and Kimura, 1985;
Secombes and Olivier, 1997). However, the detection of an intact A-layer in
laboratory culture cannot be used, in isolation, as a predictor of the virulence of
an isolate (Ellis et al., 1988; Olivier, 1990).
organs, rather than kidney alone, has been demonstrated to increase the detection
rate in fish populations where the incidence of infection is low (Bernoth, 1997b).
The recommended diagnosis of the presence of A. salmonicida in clinically
diseased fish is based on isolation of the organism from the kidney on either
tryptone soya agar (TSA) or brain–heart infusion agar (BHIA) (Department of
Fisheries and Oceans, 1984; Shotts, 1984). The brown water-soluble pigment
produced by typical A. salmonicida on TSA after 2–4 days’ incubation at
20–25°C is used as a presumptive identification. However, caution must be
exercised when pigmentation on TSA is used as a presumptive identification of
A. salmonicida. Other bacteria have also been found to produce a brown
diffusible pigment on TSA, such as mesophilic aeromonads and Pseudomonas
fluorescens (McCarthy, 1975; Frerichs and Holliman, 1991). Neither TSA nor
BHIA is selective for A. salmonicida, allowing the growth of competing
organisms, which may inhibit pigmentation or the growth of A. salmonicida
(Austin and Austin, 1993). Inhibition of growth may result from the ability of
faster-growing organisms to sequester the available nutrients in the medium
(particularly iron) or from the production of inhibitory substances by these
competing organisms (Cornick et al., 1969; Michel and Dubois-Darnaudpeys,
1980; Smith and Davey, 1993). Supplementing TSA with 0.01% (w/v)
Coomassie brilliant blue (CBB) (CBB agar) (CBBA) has been found by these
and other authors to aid in the preliminary differentiation of A. salmonicida from
competing bacteria (Cipriano and Bertolini, 1988; Markwardt et al., 1989;
Cipriano et al., 1992). On this medium, A-layer-positive A. salmonicida colonies
stain deep blue to navy and can be easily distinguished, the intensity of staining
being dependent on the source of the dye and the batch of TSA. However, CBBA
cannot be totally relied upon, because bacteria other than A. salmonicida can
produce dark blue colonies on CBBA (Teska and Cipriano, 1993). None the less,
the use of CBBA as a primary plating medium reduces the numbers of bacteria
that need to be screened to ensure definitive identification.
Occasional failure to isolate typical A. salmonicida from diseased fish with
macroscopic and histological signs of furunculosis has been noted. There are a
number of reasons why A. salmonicida may fail to yield colonies on solid media,
even in the absence of competing bacteria. Firstly, the number of detectable cells
present in the original sample may be below the lower detection limit of cultural
isolation (Bernoth, 1997b). Attempts to overcome this limitation, by incorpor-
ating a pre-enrichment step, carried out in liquid media, prior to plating on solid
media, were reported by Daly and Stevenson (1985). Pre-enrichment of kidney
samples in tryptone soya broth (TSB) for 48 h more than doubled the A.
salmonicida detection rate in a fish population undergoing a furunculosis
epizootic. A second reason for lack of growth might be the unsuitability of
laboratory media, such as TSA and BHIA, to support the growth of A.
salmonicida. Little is known about the specific nutrient requirements of typical
A. salmonicida, other than that it requires methionine and arginine (Nerland et
al., 1993). Most artificial media have been formulated for the isolation of
medically important bacteria and do not, therefore, present an ideal environment
for terrestrial and aquatic organisms. Another potential problem with the use of
TSA as the primary isolation medium is that some batches may occasionally fail
362 M. Hiney and G. Olivier
Cytochrome oxidase Oxidase positive Oxidase negative isolates have been described
production (Chapman et al., 1991); test colonies from TSA
only
Catalase production Catalase-positive Test colonies from TSA only
Oxidation/fermentation Fermentative Incubate at 25°C; check after 48 h and 5 days
test (Hugh/Leifson)
0/129 sensitivity Resistant on blood agar Incubate at 25°C; check after 48 h and 5 days
Serological tests
Latex agglutination Clumping (souring) of antibody coated latex Always include positive and negative control
in solution with A. salmonicida suspension strains because of possible autoagglutination of
A. salmonicida
A. salmonicida-targeted Colour response from binding of conjugated Always include positive and negative control
ELISA antibody to A. salmonicida antigen(s) strains
(PCR)/DNA probe assays (Gustafson et al., 1992; O’Brien et al., 1994). Culture-
based detection of A. salmonicida in the environment has traditionally been seen
as problematic (Cornick et al., 1969). However, the success of Ford (1994) and
Cipriano et al. (1996a), using dilution filtration and CBBA, in isolating typical
A. salmonicida from hatchery water suggests that these methods are promising
in routine monitoring of hatchery water-supplies (Cipriano, 1998). None the
less, the use of culture-based techniques are complicated, as A. salmonicida may
enter a non-culturable-but-viable state once it has been shed into the
environment (Roszak and Colwell, 1987; Enger, 1997). Therefore, techniques
that do not rely on culture, such as PCR and enzyme-linked immunosorbent
assay (ELISA), have received considerable attention and may have the potential
to overcome the problems encountered by culture-based techniques. However,
these non-culture-based detection techniques present significant problems of
interpretation when applied in the environment (Hiney, 1994; Hiney et al.,
1997a; Hiney and Smith, 1998) and will be discussed later in this section.
(1977), Scallan (1983) and Scallan and Smith (1985). Scallan (1983) described a
method for the bath administration of the corticosteroid prednisolone acetate to
fish of 5 g or less, which might not survive injection administration. She also
demonstrated that the amount of corticosteroid injected into fish was not a
critical factor in precipitating overt disease, with concentrations over a fourfold
range being effective. In a recent comparison of the stress test and culture-based
assays, Cipriano et al. (1997) found that the probability of detecting A.
salmonicida in apparently healthy salmon and trout, where the prevalence of
covert infection was assumed to be low, was 17 times greater using stress testing
than direct culture of either external mucus or kidney. When a 24 h pre-
enrichment of kidney and mucus was included prior to plating, the probability of
detecting A. salmonicida was 10 and 27 times greater for stress testing, as
compared with kidney and mucus culture, respectively (Cipriano et al., 1997). It
should be noted that infections detected by the stress test of Bullock and Stuckey
(1975) or its later modifications should properly be termed SIF, and the
relationship between SIF and covert infections detected by other methods should
be considered unknown (Hiney et al., 1994).
isolated from the kidney of Atlantic salmon and brown trout that had been
vaccinated against furunculosis. Attempts to culture A. salmonicida from the
kidneys of parallel, unstressed, groups of salmon and trout were unsuccessful,
confirming that the infection was of a stress-inducible nature. The results of
Hiney (1995) suggested that vaccinated fish could become or remain covertly
infected following vaccination and that the extent of protection provided by
vaccination was not sufficient to prevent SIF. Hiney et al. (1997b) and Smith
(1997) have suggested that covertly infected fish with increased systemic
immunity (i.e. vaccinated) might act as ‘immune carriers’. If such fish exist, then
they will present a number of important questions for managers of fish farms and
wild fisheries. For example, should potential ‘immune carriers’ be treated with
antimicrobial agents immediately prior to transfer to a sea site or restocking into
fresh water in order to eliminate any carried A. salmonicida? In theory, ‘immune
carriers’, while remaining disease-free, could act as a source of infection for
unvaccinated fish. However, at present not enough is known about the
interactions of covert infection, vaccination and immunity to address the issue of
‘immune carriers’.
Table 10.8. Non-culture-based techniques developed for, or applied to, the detection of typical Aeromonas salmonicida since
1990.
Target sample
Detection principle Assay type Clinical Environmental Reference
Immunological ELISA Kidney Adams and Thompson (1990)
ELISA Kidney Bernoth (1990b)
IFAT, ELISA Kidney, liver Lallier et al. (1990)
Immunofluorescence Water, sediment Enger and Thorsen (1992)
ELISA Blood Yoshimizu et al. (1992)
ELISA Kidney, intestine Hiney et al. (1994)
Immunodot-blot Various tissues Banneke and Bernoth (1994)
ELISA Kidney Sediment Gilroy and Smith (1995)
ELISA Kidney, intestine Sediment Hiney et al. (1997a)
ELISA Kidney, mucus, gills, spleen Bullock et al. (1998)
Genetic 16S rDNA-PCR Pure culture Barry et al. (1990)
DNA-PCR Spleen, kidney Fish faeces, tank effluent Gustafson et al. (1992)
DNA-PCR Pure culture Hiney et al. (1992)
DNA-PCR Freshwater microcosm Morgan et al. (1993)
M. Hiney and G. Olivier
the introduction of monoclonal antibodies and ELISAs, which allow for more
specific assays that can be semiautomated. As a result, a number of ELISAs have
been developed for screening of clinical samples for signs of A. salmonicida
(Bernoth, 1997b; Table 10.8). In a comparison of ELISA and an indirect
fluorescent antibody test (IFAT), similar to the fluorescent antibody microscopy
(FAM) technique, Lallier et al. (1990) found that ELISA was more sensitive than
IFAT when tested on pure cultures of A. salmonicida and both methods were
found to be more sensitive than bacteriological culture. However, it has been
argued that the use of IFAT coupled with experience overcomes the problems of
lesser specificity of this technique, making it comparable to ELISA (E.-M.
Bernoth, CSIRO, 1995, personal communication). Perhaps the most useful
application of immunological assays would be in the detection of covert A.
salmonicida infections, and a number of ELISA tests have been applied for this
purpose. Good correlation was found between detection of covert A.
salmonicida infection by stress testing and ELISA of kidney material from non-
stress-tested fish (Scallan, 1983; Rose et al., 1989). These findings were
supported by Hiney et al. (1994), who found that ELISA examination of the
kidney, mucus and intestine of covertly infected fish detected A. salmonicida
antigens in 45% of fish as compared with culture of the organism from the
kidney of 24% of a parallel group of stress tested fish. On the other hand,
Bullock et al. 1997 found that culture of mucus, gills, kidney and spleen was
more sensitive than ELISA for identification of A. salmonicida in fish that had
been stress-tested. Enger and Thorsen (1992) have reported on an IFAT which
was applied to detection of A. salmonicida antigens in the environment of a fish
farm. None of these tests have, however, been applied successfully under true
field conditions, nor are they recommended with any conviction in diagnostic
manuals (Crane and Bernoth, 1996).
There are a number of problems to resolve when attempting to detect
bacterial antigens in situ in tissue or environmental samples. Many of the
immunological assays developed do not appear to offer any greater sensitivity or
reliability than conventional bacteriological methods (Inglis et al., 1993). The
lower limit of detection has been found to be 103 cells ml–1 or greater in both
clinical and environmental samples (Sakai et al., 1986; Adams and Thompson,
1990; Bernoth, 1997b). A major problem with cross-reactivity of antisera or
antibodies to epitopes expressed by ubiquitous motile Aeromonas species or
other aquatic microorganisms has been reported, which requires that
confirmatory bacteriological isolation of the pathogen be performed (Bernoth,
1990b). Importantly, immunological assays do not differentiate between live and
dead cells (Rose et al., 1989) and A. salmonicida-targeted ELISA has been
shown to generate positive results from the spleen and intestine of fish which
were immunized with a killed whole-cell furunculosis vaccine (Gilroy and
Smith, 1997). Another problem with immunological assays is that the antiserum
raised against bacteria grown in vitro must detect the organism as it occurs in situ
in a clinical or environmental sample. For A. salmonicida, at least, cells grown in
vivo have been found to express novel antigens, including an antigenically new
form of lipopolysaccharide (LPS), which were not induced under in vitro growth
conditions (Garduño et al., 1993; Thornton et al., 1993). The question of how
372 M. Hiney and G. Olivier
fish health studies, it is probable that this will, in fact, be the meaning attributed
to any data generated by a non-culture-based detection technique. The use of
proxy measurements does not preclude the generation of meaningful data but
does present difficulties about the interpretation of results (Wildavsky, 1995).
Hiney (1997) and Hiney and Smith (1998) have argued strongly that these
difficulties can only be overcome by validation, that is, demonstration that a
technique does what it is supposed to do (Thrusfield, 1986). Although important
for all techniques, validation is essential for techniques that involve proxy
measurements.
Validation is not the property of a technique but, rather, is a property of its
application. It establishes that a technique can be correctly and properly used for
a particular purpose. A formal structure for the validation of non-culture-based
detection techniques in laboratory studies was presented by Hiney and Smith
(1998). However, no amount of laboratory studies can validate the application of
such techniques under field conditions. Comparative and predictive validation
represent the only two available strategies for the validation of such
applications. In comparative methods, the technique under test can be compared
either with a technique that has been previously validated or with one which is,
itself, also unvalidated. This second approach, mutual covalidation, is the more
frequently encountered but is, however, limited in power.
Predictive validation requires that the application intended for the technique
must be clearly defined in terms which are empirically meaningful. For example,
the intended application might be the prediction of the disease incidence, for
hatchery smolts covertly infected with A. salmonicida following transfer to a sea
site. An empirically meaningful measure of ‘disease’ could, in this case, be the
frequency of the isolation of A. salmonicida from the kidney of moribund fish,
following transfer. The process of predictive validation would then involve
measuring the degree of correlation between the results generated by the non-
culture-based technique and the incidence of positive isolation of A.
salmonicida. Such a correlation, if it is to be useful, must be determined over a
number of years and in a variety of environmental contexts. When, and only
when, it has been established that a satisfactory correlation exists can the
technique be used alone and the data it generates be interpreted as a proxy
measurement of disease. Simply stated, techniques can only be used to predict an
event if they have been shown to predict it.
Despite the importance of validation, few papers that have reported on the
development of non-culture-based assays for A. salmonicida have paid adequate
attention to demonstrating that these techniques have field validity. Hiney et al.
(1997a) illustrated the danger of assuming that a positive response, generated by
a non-culture-based assay for A. salmonicida in a field sample, was indicative of
the presence of the organism in a form capable of causing disease. In their study,
positive responses generated from hatchery inflow sediment by A. salmonicida-
targeted ELISA and a DNA/PCR assay showed no correlation with the health
status of fish at that hatchery over a 2-year period, as assessed by routine
bacteriological analysis and stress testing. These results suggested that
interpretation of the results generated by either of these techniques as indicators
of the presence of a disease risk would be absolutely invalid. More seriously, the
374 M. Hiney and G. Olivier
use of this type of data by regulators would be both unwarranted and dangerous.
There is little doubt that the current developments in non-culture-based
techniques for the detection of A. salmonicida have the potential to improve our
understanding of, and limit the impact of, diseases caused by this pathogen.
However, as discussed, adequate validation of the development and utilization
of these techniques is critical to their correct interpretation, especially if that
interpretation will be used to impose regulatory limitations on aquaculture
activities.
TREATMENT REGIMES
Standard treatment regimes are available in a number of texts and are frequently
supplied by the manufacturers. Table 10.9 presents a summary of these data. As
a general rule, it can be assumed that sea water interferes with oral uptake and
therefore higher doses should be employed if treatment is of fish in the marine
environment.
one of the aims is to protect the fish in the environment to which the infected fish
are being moved. Thus, in this situation, the attempt is to control both the carrier
and the latent dimensions of covert infections, and therefore the extra effort and
risks associated with therapeutic treatments may be justified.
Scallan and Smith (1985) reported the successful therapeutic treatment of
covert infections with a protocol that involved a standard oral administration of
flumequine followed, 2 days before transport, by the i.p. injection of all fish with
30 µg kg–1 flumequine. Protocols involving the bath administration of flume-
quine have also been reported (O’Grady et al., 1988; O’Grady and Smith, 1992;
Cazabon et al., 1994; Hiney et al., 1995). The uptake by fish of flumequine and
oxolinic acid from their water can be extremely efficient and rapid. Almost
uniquely in treatments of fish with antimicrobial agents, bath administration of
these agents runs a significant risk of administering a lethal overdose (Coyne et
al., 1994). The rate of uptake of these agents is strongly dependent on water
quality and is favoured by soft acid conditions (O’Grady et al., 1988). In hard
alkaline waters, uptake is greatly reduced and in sea water it is practically
negligible. Rates of uptake are also dependent on the condition of the fish and
are frequently greater during commercial-scale treatments than in small-scale
laboratory trials (Cazabon et al., 1994). It is recommended that, before any new
treatment of fish is performed, trials of the safety of the procedure are made
before a full treatment of a commercial population is initiated. When bath
administration of quinolones is contemplated, this precautionary requirement
becomes absolutely essential. The aim of bath treatments is to produce serum
levels of 30 µg ml–1 and this should be monitored by appropriate analytical
procedures. Serum concentration in excess of approximately 60 µg ml–1, which
can easily be achieved by bath administration, is lethal for salmonids (Cazabon
et al., 1994).
In any treatment aimed at protecting fish during a movement from fresh
water to sea water, the influence of the change in the environment of the fish on
the internal concentrations of antimicrobial agents must be taken into account.
Movement to the sea has been shown to result in a very rapid excretion of
flumequine from fish (Hiney et al., 1995). It is probable that this is associated
with the rapid excretion of Mg2+ ions that fish perform on entering the sea. If this
explanation is correct, then it is probable that any antimicrobial agents that form
complexes with Mg2+ (oxytetracycline and the quinolones) will also be rapidly
excreted under these conditions. As a consequence, it is probably unwise to
assume that any agent introduced into fish in fresh water will persist in
meaningful concentrations after their entry into the sea.
Vaccination
Vaccination strategies designed to control furunculosis were reported as early as
the 1940s (Duff, 1941). Midtlyng (1997) has reviewed the attempts that were
made, over the next 50 years, to produce vaccines and vaccine administration
methods that would provide adequate control of this disease. Succinctly, and
possibly rather unfairly, these attempts can be summarized, at least from the
perspective of commercial salmon farmers, as failures. Oral, immersion and
injection administrations of a variety of bacterins were developed and a few
Furunculosis 379
Cell-surface structures
In common with many pathogenic bacteria A. salmonicida can manifest an
additional surface protein microcapsule (Kay and Trust, 1997). These crystalline
surface protein arrays are generally referred to as S-layers, but, for historical
reasons, in A. salmonicida the term A-layer is more commonly used (Udey and
Fryer, 1978). SDS-polyacylamide gel electrophoresis (PAGE) analysis and X-
ray diffraction studies have shown the A-layer of A. salmonicida to be a protein
of approximately 50 kDa, with a tetragonal structural arrangement (Trust et al.,
1980a; Kay et al., 1981; Evenberg et al., 1982; Garduño and Kay, 1992a; Kay
and Trust, 1997). A-layers isolated from a wide variety of A. salmonicida strains
were shown to be immunologically conserved (Kay et al., 1984). The evidence
implicating the A-layer of A. salmonicida as a primary virulence factor is very
strong. Authors have demonstrated that typical strains possessing the A-layer are
both virulent for susceptible fish species and autoaggregating, while A-layer-
negative variants are non-virulent and non-aggregating (Udey and Fryer, 1978;
Ishiguro et al., 1981; Kay et al., 1984; Cipriano and Bertolini, 1988; Cipriano
and Blanch, 1989; Noonan and Trust, 1995). Kay and Trust (1997) have
suggested that the ability of the A-layer to bind immunoglobulins and other
extracellular proteins may result in the masking of bacterial immunogenic
receptors, thus allowing A. salmonicida to evade the host’s immune response.
The A-layer has been reported to promote bacterial penetration and adhesion and
Furunculosis 381
to inhibit complement-mediated lysis in host serum (Trust et al., 1983; Sakai and
Kimura, 1985; Garduño and Kay, 1992b; Garduño et al., 1995). It was also
reported that the net negative charge of A-layer-containing A. salmonicida cells
plays a crucial role in their long-term survival in a freshwater microcosm (Sakai,
1986) and that the production of exopolysaccharides under low nutrient
conditions, which may protect the cell from desiccation, was greater in an A-
layer-containing strain than in an A-layer-deficient mutant (Bonet et al., 1993).
Udey (1982) demonstrated that the incorporation of the protein-specific dye
CBB into common growth media could provide a differentiation between strains
containing an intact A-layer (A+), which grew as blue colonies, and those lacking
it (A–), which grew as white colonies. Using CBB-containing media, Cipriano
and Bertolini (1988) demonstrated a correlation between the A+ phenotype on
this medium and virulence for brook trout, although Bernoth (1990a) reported
that colony colour on CBB-containing media did not always correlate with
virulence of A. salmonicida. Anomalies have been reported which might
challenge the importance of the A-layer as a virulence factor. Specifically, one
A– A. salmonicida strain was reported to be virulent for rainbow trout (Bernoth,
1990a), while Olivier (1990) reported that the presence of an intact A-layer did
not always correlate with virulence. It must be remembered, however, that A.
salmonicida strains lacking an A-layer are laboratory artefacts. To our
knowledge, no strain lacking an intact A-layer has ever been isolated from a
natural furunculosis infection.
The other major component of the cell surface of A. salmonicida, in
common with all Gram-negative cells, is LPS. In A. salmonicida, LPS is
normally composed of two types, a low-molecular-weight lipo-oligosaccharide
(LOS), situated beneath the A-layer, and a high-molecular-weight LPS,
containing attached O-polysaccharide chains, some of which traverse the A-
layer (Ishiguro et al., 1983; Chart et al., 1984; Evenberg et al., 1985). The role of
LPS in the structure of the A-layer and the virulence of A. salmonicida has been
elusive. Observations of O-polysaccharide-deficient mutants have indicated that
they play a role in securing the A-layer to the cell surface (Belland and Trust,
1985; Griffiths and Lynch, 1990), and Cipriano and Blanch (1989) reported that
only A. salmonicida strains containing both an intact A-layer and LPS were
virulent for brook trout.
It must be remembered, however, that virtually all of the studies cited above
were carried out on A. salmonicida strains grown in vitro on artificial laboratory
media. It is unlikely that these conditions will reflect the behaviour of A.
salmonicida occurring naturally either in a fish host or in the environment.
Garduño et al. (1993) found that cells grown in vivo in diffusion chambers which
had been implanted in rainbow trout peritoneal cavities displayed enhanced
resistance to host-mediated serum and oxidative killing, and expressed a
polysaccharide capsular layer, which they suggested might act as a mechanism
of phagocytosis resistance or evasion of the host immune system. Thornton et al.
(1993) have also reported that A. salmonicida cells grown in vivo expressed
novel surface antigens. It is reasonable to postulate that these additional cell-
surface components play an important role in the virulence of A. salmonicida,
although this has not, as yet, been adequately demonstrated. Further in vivo
382 M. Hiney and G. Olivier
studies will be required to clarify the exact role and importance of cell surface
components in the pathogenicity of A. salmonicida.
Extracellular products
In common with other pathogenic organisms, A. salmonicida has been found to
produce a number of ECPs many of which have enzyme activity. Injection of
crude extracellular material of A. salmonicida has been clearly demonstrated to
kill susceptible fish (Munro et al., 1980; Ellis et al., 1981; Ellis, 1991) and a
considerable amount of work has been performed to analyse the constituents of
ECPs and to understand their role in virulence and pathogenesis. Ellis (1997a)
has provided an excellent review of the known ECPs of A. salmonicida, which
together make for a long list. These are grouped by Ellis (1997a) into three types,
namely proteases, membrane-damaging toxins and other toxins, including H-
lysin, which have not yet been fully investigated.
Tajima et al. (1983) were the first to report a lethal 70 kDa protease in A.
salmonicida ECP, which was found to have an LD50 of 2.4 µg g–1 when injected
into young salmon and to produce haemorrhaging and muscle liquefaction (Lee
and Ellis, 1989). These effects were not as severe as when total ECP was used,
but equivalent lesions were produced when the protease was combined with a
haemolytic factor in the ECP, suggesting an interaction between these
components (Lee and Ellis, 1991b). It is believed that the protease toxin is
produced by A. salmonicida to digest host proteins as a nutrient source, although
it has been found to have only limited specificity, mainly towards proteins with a
relatively open structure (Price et al., 1990). The 70 kDa protease has also been
shown to reduce the clotting time of trout blood, which may account for the
presence of microthrombi in fish tissue in cases of clinical furunculosis and
following injection of crude toxins (Ellis et al., 1988). There is, however,
conflicting evidence for the exact role of the 70 kDa protease in virulence. A lack
of correlation between the amount of protease in ECP and the killing ability of
that ECP has been reported (Drinan et al., 1989), and Sakai (1985) reported that
a protease-deficient mutant of A. salmonicida was avirulent, while other workers
have identified virulent isolates which do not produce any protease under
standard culture conditions (Hackett et al., 1984; Ellis et al., 1988). However,
this confusion may be an artefact of in vitro studies, because apparent protease-
deficient strains have been found to produce protease in vivo (Ellis, 1991). A
second protease, whose preferred substrates are gelatine and collagen, has been
identified (Sheeran and Smith, 1981), but its physiochemical properties have not
yet been determined in detail.
A number of membrane-damaging activities of ECP have been described.
Present evidence suggests that, of these membrane-damaging toxins,
glycerophospholipid–cholesterol acyltransferase (GCAT) complexed with LPS,
so called GCAT/LPS, is the most important factor in the lethal toxicity and
pathology of the ECP (Ellis, 1997a). The LD50 of purified GCAT/LPS for
Atlantic salmon has been reported to be 45 ng g–1 body weight (Lee and Ellis,
1990). In vitro studies of GCAT/LPS have shown it to be leucocytolytic,
cytolytic and highly haemolytic for salmonid erythrocytes, although this
haemolysis was incomplete in the absence of the 70 kDa protease (Lee and Ellis,
Furunculosis 383
1990), which has been suggested by Ellis (1997b) to be necessary for activation
of GCAT activity. However, there is no evidence for in vivo haemolysis in
clinical furunculosis (Lee and Ellis, 1991a) and Ellis (1997a) has suggested that
in vivo GCAT/LPS may function in destabilization of host red cell membranes
rather than active haemolysis. The histopathological effects of GCAT/LPS are
not extensive and cannot account for the death, within 20 h, of fish injected with
purified product, and it has been suggested that in vivo toxicity may be due to
metabolic effects, although this has yet to be confirmed (Ellis, 1997a). Although
GCAT/LPS is clearly an important ECP, it has recently been demonstrated that a
GCAT-deficient A. salmonicida mutant could produce the manifestations of
classical furunculosis (A.E. Ellis, Aberdeen, 1997, personal communication).
Therefore, it has become clear, with respect to the pathogenesis of furunculosis
and the lethal toxicity of the exotoxins, that, instead of one toxin being critical
for pathogenesis, a combination of ECPs act in concert to produce disease.
killed whole cells or ECP. High serum antibody titres can be routinely elicited in
salmonids by both i.p and i.m. injection of A. salmonicida (Secombes and
Olivier, 1997). At permissible temperatures (10–15°C) antibody titres rise
within 2–3 weeks and peak within 8–12 weeks, although water temperature is
critical to this response and at lower temperatures responses may be slower or
absent (Ellis et al., 1992; Eggset et al., 1997). The sites of antibody production in
salmonids would primarily seem to be spleen and head kidney (Reitan and
Thuvander, 1991; Davidson et al., 1993). Antibody-secreting cells have also
been found in mucosal sites, such as the gut, but these can take up to 7 weeks
post-vaccination to appear (Davidson et al., 1993). Antibody responses have
also been elicited following administration of A. salmonicida by subcutaneous
injection (Anderson, 1969), by bath (Anderson et al., 1979) and orally
(Davidson et al., 1993). The inclusion of an adjuvant, such as an oil or glucan, in
vaccines administered by a parental route has been shown to enhance the
antibody titre and may act as a depot of antigen, allowing vaccination at low
temperatures (Cipriano and Pyle, 1985; Anderson et al., 1997; Ellis, 1997b;
Midtlyng, 1997; Secombes and Olivier, 1997). It should be noted, however, that
high antibody titres do not necessarily correlate with protection and it is the
specificity of the antibodies that appears to be important (Hirst and Ellis, 1994;
Ellis, 1997b). In older fish, proliferation studies suggest that specific B-cell
memory can be established to A. salmonicida and that a second exposure to the
organism results in a faster and stronger antibody response (Secombes and
Olivier, 1997). However, in younger fish, it is possible that a primary exposure
to A. salmonicida may induce tolerance rather than specific immunological
memory (Manning et al., 1982).
Unlike higher vertebrates, antibody response to A. salmonicida in salmonids
is essentially independent of T cells. In fish the regulatory role of T cells in the
immune response is thought to be mediated by released cytokines, analogous to
interleukin-2, chemokines and macrophage-activating factor (MAF), following
exposure to specific antigens (Secombes, 1994b). Although little is generally
known about cytokine release in response to A. salmonicida, release of MAF has
been demonstrated in vitro from cultured cells, removed from fish
immunologically primed with killed whole A. salmonicida or ECP, 2–3 weeks
post-exposure, peaking 4–5 weeks post-exposure (Marsden et al., 1994). In vitro
studies have also shown that MAF-treated macrophages acquired the ability to
kill A. salmonicida (Graham et al., 1988). In fish, MAF production has also been
demonstrated to correlate with both lymphocyte proliferation and antibody
production following vaccination with whole cells (Secombes and Olivier,
1997). Therefore, unlike antibody production, any epitope on A. salmonicida can
potentially induce a cell-mediated response, such as MAF release. In common
with antibody production, however, cytokine release is temperature dependent
and has been shown to be absent in fish cells kept at 7°C or less (Hardie et al.,
1994).
386 M. Hiney and G. Olivier
Fish species
History of
Common name Scientific name Habitat isolation Reference
Atlantic salmon Salmo salar Fresh water Clinical Groman et al. (1992)
Incidental Benediktsdóttir and Helgason (1990)
Brackish water Clinical Harmon et al. (1991)
Sea water Clinical Olivier (1992)
Incidental Benediktsdóttir and Helgason (1990)
Arctic char Salvelinus alpinus Fresh water Clinical Wichardt et al. (1989)
Sea water Clinical Olivier (1992)
Brook trout Salvelinus fontinalis Fresh water Clinical Ljungberg and Johansson (1977)
Brown trout Salmo trutta m. lacustris Fresh water Clinical Rintamäki and Valtonen (1991)
Furunculosis
Fish species
History of
Common name Scientific name isolation Reference
American eel Anguilla rostrata Clinical Olivier (1992)
Bream Abramis brama Clinical McCarthy and Roberts
(1980)
Bighead Aristichthys nobilis Clinical Csaba and Szakolczai
(1991)
Carp Cyprinus carpio Clinical Csaba et al. (1984)
Unclear Bernoth (1997b)
Chub Leuciscus cephalus Clinical Wilson and Holliman (1994)
European carp Not given Not Chart et al. (1984)
specified
Goldfish Carassius auratus Clinical Elliot and Shotts (1980)
Japanese eel Anguilla japonica Clinical Kitao et al. (1984)
Minnow Phoxinus phoxinus Clinical Håstein et al. (1978)
Northern pike Esox lucius Clinical Wiklund (1990)
Unclear Wichardt et al. (1989)
Ornamental Unclear Bernoth (1997a)
cyprinids
Perch Perca fluviatilis Clinical Bernoth (1997a)
Unclear Wichardt et al. (1989)
River bleak Alburnus alburnus Clinical Bernoth (1997a)
Roach Rutilus rutilus Clinical Austin (1993)
Unclear* Wichardt et al. (1989)
Rudd Scardinius Unclear Barker and Kehoe (1995)
erythrophthalmus
Silver carp Hypophthalmichthys Clinical Csaba and Szakolczai
molitrix (1991)
Silver bream Blicca bjoerkna Clinical McCarthy (1975)
Silver perch Bidyanus bidyanus Clinical Whittington et al. (1995)
*Unclear from history whether isolation was from a clinical case or was an
incidental finding.
Fish species
History of
Common name Scientific name isolation Reference
Atlantic cod Gadus morhua Clinical Cornick et al. (1984)
Incidental Oliver (1992)
American plaice Hippoglossoides Clinical Olivier (1992)
platessoides
Black rockfish Sebastes schlegeli Clinical Izumikawa and Ueki
(1997)
Common wolffish Anarhichas lupus Clinical Hellberg et al. (1996)
Dab Limanda limanda Clinical Wiklund and
Dalsgaard (1995)
Flounder Platichthys flesus Clinical Wiklund et al. (1994)
Goldsinny wrasse Ctenolabrus rupestris Incidental Frerichs et al. (1992)
Greenback Rhombosolea tapirina Clinical Whittington et al.
flounder (1995)
Incidental Bernoth (1997a)
Greenling Hexogrammos otakii Clinical Iida et al. (1997)
Haddock Melanogrammus Clinical Olivier (1992)
aeglefinus
Japanese flounder Paralichthys olivaceus Clinical Iida et al. (1997)
Pacific herring Clupea harengus Clinical Traxler and Bell
pallasi (1988)
Plaice Pleuronectes platessa Clinical Wiklund and
Dalsgaard (1995)
Sablefish Anoplopoma fimbria Clinical Evelyn (1971)
Sand eels Ammodytes lancea Clinical Dalsgaard and
Paulsen (1986)
Hyperoplus Clinical Dalsgaard and
lanceolatus Paulsen (1986)
Shotted halibut Eopsetta grigorjewi Clinical Nakatsugawa (1994)
Tom cod Gadus microgadus Clinical Olivier (1992)
Turbot Scophthalmus Clinical Pedersen et al. (1994)
maximus
Wrasse Ctenolabrus rupestris Covert* Frerichs et al. (1992)
*Apparently healthy fish not showing signs of infection (see Hiney et al.,
1997b)
elongatus, in 1986 and from sable fish in 1987 and 1990 (Bell et al., 1990;
McCormick et al., 1990; T.P.T. Evelyn, Nanaimo, 1990 personal com-
munication). Atypical strains have been isolated from other marine species,
some of which are now in the process of being developed for aquaculture
purposes, and, according to some authors, disease conditions of atypical A.
salmonicida aetiology may become a restricting factor for the mass culture of
these species (Pedersen et al., 1994). In several cases, the disease has been
diagnosed in wild fish transferred and maintained in aquarium facilities,
reinforcing the hypothesis that there could be strains of A. salmonicida of marine
origin (Cornick et al., 1984: Dalsgaard and Paulsen, 1986; Harmon et al., 1991;
Olivier, 1992; Whittington et al., 1995). Furthermore, these findings indicate
390 M. Hiney and G. Olivier
that marine species may act as carriers or reservoirs of some atypical strains and
the disease condition will be expressed when animals are stressed.
The microorganisms
Biological characteristics of atypical Aeromonas salmonicida
The description and characterization of several atypical isolates over the last few
years confirm the close relationship between typical and atypical strains.
Although their growth characteristics and their biochemical profiles are quite
distinctive, almost all atypical isolates investigated so far possess phenotypic
properties similar to those of typical strains. They are all strongly
autoaggregating, forming small convex raised colonies on agar, which are
compact, are strongly adherent and slide on the surface of agar media when
pushed with a loop (friable). Their cell-surface structure is similar to typical
strains. Most strains investigated have an A-layer and LPS similar to those of the
typical strains, but their mobility under electrophoresis is slightly different
(Evenberg and Lugtenberg, 1982; Evenberg et al., 1982, 1985; Kay et al., 1986;
Griffiths and Lynch, 1990). In addition, the structural gene for the A-protein
appears to be conserved in both typical and atypical isolates, as demonstrated by
Chu et al. (1991), using Southern blot analysis: a 2.5 kb portion of the gene was
detected in typical and atypical strains alike, but not in A. hydrophila isolates
tested. Atypical isolates grown above 30°C can give rise to the phenotypic
variants A–LPS+, A–LPS- and A+LPS–, which are similar to the recognized
phenotypes of typical strains (Evenberg et al., 1985; Griffiths and Lynch, 1990;
Rockey et al., 1991). Using a preparation of A-protein obtained from a typical
isolate, Griffiths and Lynch (1990) demonstrated that the A layer could be
reconstituted on the surface of A–LPS+ phenotypes of both typical and atypical
strains.
Typical and atypical isolates are serologically cross-reactive, using mono-
clonal or polyclonal antibodies (Paterson et al., 1980; Sövényi et al., 1984;
Evenberg et al., 1985; Kitao et al., 1985; Austin et al., 1986; Böhm et al., 1986;
Adams and Thompson, 1990). Rockey et al. (1991), using monoclonal
antibodies, have demonstrated different epitopes on the LPS of typical and
atypical isolates. In addition, using restriction endonuclease fingerprinting and
plasmid profiles, strong homology between typical and atypical strains has also
been demonstrated (Bast et al., 1988; McCormick et al., 1990).
Disease transmission
The transmission of atypical A. salmonicida has not been thoroughly
investigated, due to a lack of understanding of the ecology of the organisms.
Only a few reports describe some ecological aspects of atypical A. salmonicida.
Evelyn (1971) reported that the atypical strain isolated from sable fish survived
better in salt water compared with fresh water. In another study, Wiklund
(1995a) investigated the survival of atypical strains isolated from flounders in
microcosms. Best survival was observed in the presence of sediments and at
high water temperature and the highest survival was observed in brackish water
compared with fresh and salt water. Additional studies on the ecology of these
strains is required if the epizootiology of these isolates is to be better understood.
Furunculosis 393
In several cases, transmission of the disease seems to have been linked to the
transfer of infected fish (Wichardt et al., 1989; Håstein and Lindstad, 1991). The
best evidence has been provided by the example of the goldfish ulcer disease
agent, which was introduced into Australia through the importation of live
infected or subclinically infected goldfish in 1974. Following this introduction,
the agent has been recovered from wild goldfish and the disease is now thought
to be endemic in several areas of Australia (Whittington et al., 1987; Humphrey
and Ashburner, 1993).
Biochemical identification
Similar to typical isolates, the presumptive identification of atypical isolates is
based on a few characteristics, i.e. the isolates are Gram-negative coccobacilli,
oxidase-positive and fermentative and do not grow at 37°C. It is, however,
important to note that some atypical strains are oxidase-negative, including
isolates recovered from flounder (Wiklund and Bylund, 1991), herring (one out
of four isolates) (Traxler and Bell, 1988), tom cod (Olivier, 1992) and turbot
(Scophthalmus maximus) (Pedersen et al., 1994). These results strongly suggest
that care is needed to properly identify some of these atypical isolates, and that
even discrepancies in basic characteristics, such as the oxidase reaction, should
not necessarily cause a tentative identification to be rejected. Additional tests are
needed to ensure that atypical strains are not implicated in the condition under
investigation. A schematic representation of the various steps that should be
taken for the culture and identification of atypical strains of A. salmonicida is
presented in Table 10.13.
Differentiation between typical and atypical isolates is achieved based on
the following properties; atypical isolates are generally achromogenic, lack the
capacity to produce gas from glucose, utilize sucrose, produce indole and are
gelatinase-negative (Popoff, 1984). All of these characteristics are useful for the
differentiation of atypical isolates, although it must be borne in mind that
exceptions to the above have been reported. A more comprehensive list of
biochemical characteristics is presented in Table 10.14, although this level of
investigation may not be necessary for routine diagnosis.
Pedersen et al., 1994), although there are reports that, in some instances,
chemotherapy may be difficult or ineffective (Dalsgaard and Paulsen, 1986;
Whittington and Cullis, 1988; Groman et al., 1992). New antibiotics are being
evaluated; for example, Heo and Seo (1996) have found that ciprofloxacin can
be effective in protecting carp against infection with an atypical carp isolate. As
with typical strains, development of antibiotic resistance has been recognized in
atypical A. salmonicida. Hirvelä-Koski et al. (1994) reported resistance to
sulphonamides, and strains isolated from turbot (Pedersen et al., 1994) were
found to be resistant to trimethoprim. In addition, Sandaa and Enger (1996)
found that a plasmid encoding for multiple antibiotic resistance could be
transferred from atypical strains to several marine bacteria.
Vaccination
There is a paucity of studies on vaccine development against diseases of atypical
A. salmonicida aetiology. Protection of carp against carp erythrodermatitis has
been investigated by Evenberg et al. (1986, 1988). These authors developed a
reproducible experimental challenge in carp, using the subcutaneous route. They
found that the i.m. route of immunization was superior to the i.p. route and that
whole-cell vaccines or purified antigens conferred only marginal protection. The
best protection was observed using a vaccine containing concentrated, formalin-
inactivated, culture supernatants. Interestingly, carp sublethally infected with A.
salmonicida were not protected against a subsequent challenge, and immunity in
these fish was thought to be related to humoral immunity (antitoxoid antibodies)
and not cellular immunity. A subsequent study (Daly et al., 1994) demonstrated
that carp could be successfully challenged by bath administration, and animals
exhibited classical signs of carp erythrodermatitis. During these studies, carp
that received sublethal infections were able to withstand subsequent lethal
infections and recover, regardless of the route of infection. Sublethally bath-
exposed carp were protected from subsequent lethal challenges of A.
salmonicida subsp. nova for at least 5 months. The authors only speculated on
the mechanism of protection, but their results suggest that carp vaccinated with a
live vaccine (sublethal infection with virulent bacteria) can be protected,
indicating that cellular immunity might be important in providing protection in
this fish species.
Host resistance
As an alternative to chemotherapy and vaccination, disease resistant fish have
been investigated. The susceptibility to carp erythrodermatitis of a strain of
Hungarian carp and F1 crosses with Japanese coloured carp was investigated by
Sövényi et al. (1988), who found that the morbidity, calculated by lesion size, of
the hybrid fish was only half that of the homozygous group. A subsequent study
by Houghton et al. (1991), on the resistance of carp to erythrodermatitis,
confirmed that some strains of carp were more resistant to this disease than
others, the Hungarian line was more resistant than the Polish line and there were
differences within each strain. These results indicate that breeding carp for
disease resistance could improve the health of fish stocks.
396
Table 10.13. Diagnostic procedure to culture and identify atypical Aeromonas salmonicida.
Growth on blood agar Fastidious stains require this medium; small, Incubate for at least 7 days
creamy colonies strongly adhering to the
medium
Growth temperature Growth at 18–22°C; no growth at 37°C Strains which grow at 37°C have been reported
(Austin, 1993)
Sedimentation test Cells autoagglutinate in 0.85% PBS
Gram stain Short Gram-negative rods
Hanging drop test Non-motile
M. Hiney and G. Olivier
Serological tests
Slide agglutination Clumping (souring) of antibody-coated latex in Always include positive and negative control strains
(presumptive solution with cell suspension because of possible autoagglutination of A.
identification only) salmonicida
Biochemical tests
Cytochrome oxidase Oxidase-positive Oxidase-negative isolates have been described
production (Traxler & Bell, 1988; Olivier, 1992; Pederson et al.,
1994; Wiklund et al., 1994)
Catalase production Catalase-positive Catalase-negative isolates have been described
(Csaba et al., 1984; Böhm et al., 1986)
Indole production Indole-positive Indole-negative isolates have been described
(Austin et al., 1989; Wiklund, 1990; Olivier, 1992)
Oxidation/fermentation Fermentative Incubate at 25°C; check after 5 and 7 days
test (Hugh/Leifson)
Substrate utilization Sucrose-positive; do not produce gas from Not all strains will be positive for these tests
glucose
Resistance to Resistant to ampicillin (25 mg) and Incubate at 25°C; check after 5 and 7 days; not
antimicrobial agents cephaloridin (15 mg) all strains will be positive for these tests
Furunculosis
397
398 M. Hiney and G. Olivier
isolate was virulent for trout, roach and rudd, while the cyprinid isolate was only
virulent for brown trout and the goldfish isolate was virulent for all the species
tested.
In some cases, atypical strains isolated from salmonids can be almost as
virulent as typical isolates. The atypical strain isolated from Atlantic salmon in
Newfoundland, Canada, has an LD50 of less than 50 bacteria for juvenile Atlantic
salmon, following an i.p. challenge (Olivier et al., 1990; Olivier, 1992). Other
salmonid isolates from Sweden, Norway and Iceland were also virulent for
salmonids, with LD50 values of 1 × 102–3 for Atlantic salmon (Olivier et al.,
1990), confirming the results of Gudmundsdóttir et al. (1997) with similar
isolates from Iceland.
Several atypical strains of A. salmonicida are virulent in their host of origin.
The carp isolate (V234/81) produces 100% mortality in carp inoculated between
dorsal scales with 1 × 106 cfu (Evenberg et al., 1988). The LD50 of an eel isolate
was approximately 1 × 103 cfu by i.m. injection into healthy eels (Ohtsuka et al.,
1984); similarly, herring isolates were highly virulent for their host, killing 50%
of fish injected with fewer than 100 cfu (Traxler and Bell, 1988). A goldfish
isolate is highly virulent for goldfish, with LD50 less than 1 × 104 cfu (Trust
et al., 1980b), while a wrasse isolate had a low LD50 (5 × 102) when determined
in wrasse by i.p. injection. Other isolates were not found to be as virulent for
their host: isolates from cod, flounder, turbot and minnow had LD50 values
higher than 1 × 106 (Håstein et al., 1978; Cornick et al., 1984; Wiklund, 1995b).
As salmonid culture represents an important asset in several countries,
several non-salmonid isolates have been tested for their possible virulence in
salmonids. There is no doubt that one goldfish isolate can be highly virulent for
salmonids after i.p. injection, with LD50 values ranging from 1 × 102 to 1 × 103
injected cells. This has been confirmed by other studies, in which goldfish
isolates were found to be virulent for Atlantic salmon, brook trout and rainbow
trout under different challenge conditions, including i.p. injection and bath
challenge, with or without prior skin abrasion, and by cohabitation (Carson and
Handlinger, 1988; Whittington and Cullis, 1988). In addition, a sand eel isolate
killed 50% of rainbow trout injected i.p. with 8 × 106 cfu (Dalsgaard and
Paulsen, 1986). Isolates from other species, including flounder, turbot, wrasse
and cod, were not found to be virulent for salmonids (Pedersen et al., 1994).
Even if some atypical strains isolated from non-salmonids have been shown to
be virulent for salmonids under laboratory challenge conditions, there are few
reports of atypical strains of non-salmonid origin causing losses in salmonids in
the field. However, in one instance a sable fish isolate was found to infect
cultured Pacific salmon, but other virulent non-salmonid isolates, such as the
goldfish strain, have not been reported from salmonids to date. Furthermore,
there is little evidence of crossover of atypical strains from one non-salmonid
species to another, although one goldfish isolate has been found to infect silver
perch (Whittington et al., 1995) and the same isolate was found in two hosts,
sable fish and ling cod, on the west coast of Canada (G. Olivier, unpublished
results).
400 M. Hiney and G. Olivier
Virulence factors
The virulence factors of atypical A. salmonicida strains are not as well
characterized as those of typical strains (Trust, 1986). Atypical strains are able to
sequester iron under conditions of iron limitation, but their ability is less than
that reported for typical strains and some atypical strains are able to utilize
siderophores produced by typical isolates (Chart and Trust, 1983). According to
Hirst et al., (1991, 1994), the iron-uptake mechanism of atypical strains is
siderophore-independent, in contrast to typical strains. These results were
recently confirmed when Hirst and Ellis (1996) demonstrated that typical and
atypical strains differed in their mechanism of utilization of non-haem protein-
bound sources of iron. Typical strains utilize tranferrin via a siderophore-
mediated mechanism and are also able to digest transferrin with the extracellular
serine protease. Atypical strains utilize transferrin by a siderophore-independent
mechanism, probably involving the proteolytic degradation of transferrin by the
extracellular metalloprotease.
As stated previously, most atypical strains examined so far possess surface
structures (A-layer and LPS) similar to those of typical isolates. Small
differences have been noted in the electrophoretic mobility of both the A-layer
and LPS of atypical strains (Kay et al., 1984; Evenberg et al., 1985; Griffiths and
Lynch, 1990). In the only report where a correlation between surface structure
and virulence was tested, Trust et al. (1980a) demonstrated that an i.m. injection
of 1 × 104 A-layer-positive cells in goldfish killed all test animals (8/8), whereas
a similar injection of 108 cfu of an A-layer-negative isogenic strain killed only
one goldfish. Except for these results, the role of surface structure in the
pathogenicity of atypical strains has not been fully assessed, but it would not be
surprising if the important role of the A-layer and additional surface structures as
virulence factors were confirmed in atypical isolates.
Extracellular virulence factors produced by atypical strains are poorly
understood. Pol et al. (1981) have reported that the ECP of one atypical strain
was lethal for carp and similar results were obtained by Evenberg et al. (1988).
Filter-sterilized culture supernatants of a carp isolate were lethal for carp by i.p.
injection; interestingly, toxicity of the supernatants was dependent on growth
conditions. Hastings and Ellis (1985) noted differences in the production of
haemolysin and protease between typical and atypical strains, and Gudmunds-
dóttir et al. (1990) have purified and characterized a new toxic protease from an
atypical strain of A. salmonicida. The enzyme was caseinolytic and gelanolytic,
possessed properties of a metalloprotease and had a molecular mass of 20 kDa.
This protease was only identified in atypical strains (5/9 strains tested) and not in
typical isolates and thus may be specific for atypical strains. A recent study of 25
atypical strains isolated from a variety of hosts indicates that the proteases
produced by atypical strains are variable, and the author was able to differentiate
five groups, based on protease properties (Gudmundsdóttir, 1996). Compared
with typical strains which were homogeneous in producing protease and
gelatinase, atypical strains were heterogeneous.
The possibility that atypical strains can cause immunosuppression in carp
was investigated by Evenberg et al. (1986). Infected carp given a sublethal i.m.
injection showed a progressive decrease in total serum protein and immuno-
Furunculosis 401
globulin (Ig) levels but differential blood counts did not differ. In carp immunized
with sheep erythrocytes, a sublethal infection produced a reduction of plaque-
forming cells and serum antibodies against sheep erythrocytes, but the cellular
response, tested by skin allograft rejection, was enhanced as the disease progressed
(Pourreau et al.,1986). Further studies by Pourreau et al. (1987) indicated that
crude supernatants of a virulent atypical strain modulated the carp immune
response, when tested by mitogen stimulation of carp leucocytes. Stimulation of
leucocytes was enhanced by the supernatant from a young culture (20 h) of the
virulent strain but was severely inhibited by supernatants from older cultures (96
h). This inhibitory activity was lost after heat treatment, suggesting it was due to a
proteinaceous structure, but the substance was not characterized further.
With respect to atypical strains causing ‘furunculosis’ in salmonids, Gud-
mundsdóttir et al. (1995) have provided evidence that bacterins prepared from
an atypical strain isolated from salmonids in Iceland can provide protection
against the disease. Further work by Gudmundsdóttir et al. (1997) showed that
Atlantic salmon vaccinated with detoxified ECP provided better protection than
formalin-killed cells or a mixture of both bacterins. Since passive immunization
with rainbow trout or rabbit antiprotease (AsaP1) was demonstrated to be
efficacious and since the same antisera, but containing anti-A-layer antibodies,
were not protective, the authors conclude that the protection of Atlantic salmon
against atypical A. salmonicida is probably due to humoral immunity, with
antibodies directed against bacterial toxins contained in the ECP.
FUTURE RESEARCH
There is still much to be learned about A. salmonicida and its associated
pathologies. However, as long as there is continued availability of oil-based
vaccines, furunculosis will no longer be perceived as a major problem for
commercial marine salmonid farmers. Therefore, future research must focus on
the appropriate and sustainable management of freshwater fisheries, in
particular restoration programmes and native fisheries. Many of the disease
control strategies available to commercial farmers such as vaccination and
chemotherapy may not be appropriate in these situations. For example, the
advisability of vaccinating fish with oil-based preparations prior to their release
into river systems, from which they may be removed for consumption by anglers
must be questioned. Oil based adjuvants are highly immunogenic, not only for
fish but for consumers of those fish. Likewise, the presence of chemotherapeutic
residues in the flesh of fish which are available for consumption is undesirable.
Therefore, the control strategies that can be adopted by fresh water fisheries
managers will be affected by different concerns than the more controlled
commercial sector.
A number of important questions can be identified for fresh water fisheries
in relation to furunculosis. These may be summarized as follows:
• do fresh water hatcheries act as amplifiers of disease for the water body on
which they are situated and how can this effect be minimized or eliminated
402 M. Hiney and G. Olivier
• what impact will the release of covertly infected fish into a water body have
on their survival and on resident fish populations, and how can this impact
be minimized or eliminated
• does vaccination of covertly infected hatchery stocks prior to release
constitute the creation of ‘immune carriers’, what impact are these ‘immune
carriers’ likely to have on non-vaccinated fish and how can this impact be
minimized or eliminated
• how can hatchery reared stocks be protected from the impact of anadromous
fish in the water body on which the hatchery is situated.
Many of these questions have yet to be answered. It is hoped, therefore, that
future research into furunculosis will address the fundamental concerns outlined
here. Only in this way will we limit both the commercial and biological impact
of furunculosis among valuable and endangered fish populations. Non-culture-
based methods may have a valuable contribution to make to the study of
furunculosis epizootiology but much work on the validation of these techniques
needs to be done before they can be applied to field studies. The presence of
atypical strains in wild marine fish with ulcerations is also of concern and their
impact on wild fish populations is still unknown. The importance of these
isolates originating from wild marine fish could represent a threat to the future
culture of these species in aquaculture conditions.
A second important problem associated with atypical A. salmonicida
isolates is that they are highly variable in their biochemical profile and cannot be
easily placed in known or accepted subspecies. Further work on their taxonomy
will be necessary to unravel this basic problem. Results so far indicate that
atypical strains are for the most part highly restricted to precise geographic
areas, however, some strains including goldfish and carp isolates have a wide
distribution probably due to the international trade of these species. The limited
work carried out to date would indicate that atypical strains are restricted to their
host of origin, this is especially true for atypicals isolated from non-salmonid
fish. Most of the strains isolated from these various hosts have so far been
biochemically different. More work on this issue will, however, be necessary to
gain a fuller epizootiological picture of atypical A. salmonicida and its
associated pathologies.
ACKNOWLEDGEMENTS
We would like to thank Dr Peter Smith for his input into this manuscript,
especially the section on control and treatment, and for his many helpful
comments and editorial advice.
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