341

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

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
10
Furunculosis (Aeromonas salmonicida)
M. Hiney
1
and G. Olivier
2
1
Fish Disease Group, Department of Microbiology, National University of
Ireland Galway, Galway City, Ireland;
2
Department of Fisheries and Oceans,
PO Box 550, Halifax, Nova Scotia, Canada B3J 2S7.
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.
THE DISEASE AND THE AGENT
Species of fish affected
Aeromonas salmonicida was traditionally thought of as a pathogen of salmonids,
although this may be due in part to the volume of research carried out on this
group. Salmonids are usually the most valuable species in developed countries,
where good diagnostic bacteriological facilities are available (McCarthy and
342
M. Hiney and G. Olivier
Roberts, 1980). It is now recognized that the host range of A. salmonicida is
wide and that furunculosis is only one of several clinical diseases associated
with A. salmonicida. Furthermore, infections of fish with A. salmonicida are not
necessarily associated with clinical manifestations and may remain covert
(Hiney et al., 1997b). Typical A. salmonicida have been associated with clinical
or covert disease in a variety of salmonid and non-salmonid species in fresh
water, brackish water and sea water. Reports of fish species that have been
documented as suffering from diseases of typical A. salmonicida aetiology are
outlined in Tables 10.110.3. These tables do not represent all available reports
or all species but are intended to demonstrate the taxonomic breadth of fish
species that are susceptible to infection with this organism. For a list of species
from which atypical A. salmonicida have been isolated, readers are referred to
Tables 10.11 to 10.13.
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; Prez 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 (Svnyi 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
343 Furunculosis
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M. Hiney and G. Olivier
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345 Furunculosis
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346
M. Hiney and G. Olivier
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.
Seasonal variation in the incidence of infection
There are predisposing factors, other than age and inherent susceptibility, that
are associated with the precipitation of clinical furunculosis in hatchery stocks
throughout the year. These include physical damage, poor water quality,
presence of ectoparasites and other diseases, diet and physical and psychological
stresses, such as grading, tagging, injection and netting (Olivier, 1997;
Pickering, 1997). However, a marked seasonality in the incidence of both
clinical and covert furunculosis infections has been observed in hatchery stocks
and wild populations. In hatchery stocks, both smolting and high water
temperatures have been implicated in this apparent seasonality. The period of
smolting is associated with major physiological changes, including chronic
cortisol elevation, which can bring about severe depression of the fishs defence
system and increased susceptibility to bacterial infections (Maule et al., 1986;
Pickering, 1997). In addition, high water temperatures (1215C) in late spring
early summer increase the likelihood of furunculosis outbreaks in both fresh
water and sea water (Klontz and Wood, 1972; Johansson, 1977; Novotny, 1978).
In fact, Malnar et al. (1988) would contend that high temperature is the major
factor influencing the development of furunculosis. High water temperature not
only influences the stress response of fish but may act at the level of the
pathogen (Groberg et al., 1978), and it has been demonstrated that the growth of
Table 10.3. Marine non-salmonid species from which typical Aeromonas
salmonicida have been isolated (after Bernoth, 1997a).
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.
Fish species
347 Furunculosis
A. salmonicida in the blood of cherry salmon (Oncorhynchus masou) correlated
positively with water temperatures in the range 520C (Sako and Hara, 1981).
Not surprisingly, the seasonal nature of clinical infection by A. salmonicida is
not confined to hatchery stocks. As early as 1926, Horne observed that the
incidence of furunculosis in a riverine population of brown trout first appeared
towards the end of May and declined in October (Horne, 1928). Blake and
Clarke (1931) observed that spawning in salmon rendered them susceptible to
furunculosis. There is no reason to suspect that the temperature effects observed
in hatchery reared stocks will not also apply to wild stocks. The influence of
spawning on increased susceptibility to furunculosis would appear to be similar
in a wide range of salmonids (Nomura et al., 1993) and includes chronic cortisol
elevation and associated lymphocytopaenia (Pickering, 1986), decline in anti-
body production (Yamaguchi et al., 1980) and immunosupression associated
with gonadal steroids (Slater and Schreck, 1993). The presence of wild
spawning fish in the vicinity of freshwater hatcheries may also have an impact
on the seasonality of furunculosis in stocks contained within those hatcheries.
The occurrence of covert A. salmonicida infections in hatchery populations
has also been observed to be seasonal (Jensen and Larsen, 1980; Scallan and
Smith, 1984, 1993; Hiney, 1994). However, neither smolting, spawning nor high
water temperatures can explain other peaks in the incidence of clinical and
covert infections observed by these authors at a number of freshwater hatcheries
and supported by anecdotal evidence from the industry. Scallan (1983)
suggested that these peaks may result from the stress induced in fish by both high
water temperatures and rapidly changing water temperature.
As a general rule, both clinical and covert furunculosis are more likely to
occur in smolting and spawning fish with the onset of higher water temperatures
in spring or during periods of rapid temperature change. However, it is important
to keep in mind that furunculosis outbreaks can also occur in very young fish
(alevin and fry) and at temperatures as low as 24C (Drinan, 1985).
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 Hstein, 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 (Rintamki 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
349 Furunculosis
fatal in 23 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.
Covert infection by typical Aeromonas salmonicida
The existence of covert furunculosis, that is, clinically unapparent infections,
has been recognized almost as long as the disease itself (Plehn, 1911). The
epizootiological importance of fish with covert furunculosis in the maintenance
and spread of the disease within and between susceptible fish populations was
understood by early workers. In 1935 the Scottish Furunculosis Committee
concluded that clinically unapparent infections by A. salmonicida could persist
in fish populations, that these infections could be latent and that clinical
furunculosis could be precipitated in covertly infected fish populations by stress.
In addition, fish with covert infections were capable of acting as carriers and
could shed sufficient bacteria to transmit the infection to other fish (Mackie et
al., 1930, 1933, 1935). However, despite the large body of work that exists on
covert furunculosis, a number of important questions on the nature, persistence,
location, lack of clinical signs, host defence mechanisms and transmission of
covert infections remain unanswered or have not been answered satisfactorily.
NATURE OF COVERT INFECTIONS
There is a paucity of information available on the nature of covert furunculosis,
despite more than 100 years of research on A. salmonicida. The studies that have
been performed have employed a variety of methods, of differing efficiency, to
350
M. Hiney and G. Olivier
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351 Furunculosis
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352
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.
LOCATION OF AEROMONAS SALMONICIDA IN COVERT INFECTIONS
Despite almost 80 years of speculation, we have no certainty as to the location of
A. salmonicida subsp. salmonicida in covertly infected fish. Convincing
evidence exists for an external location of A. salmonicida on the mucous layer,
on the gills and in the intestine during a covert infection (Klontz, 1968;
Markwardt and Klontz, 1989b; Cipriano et al., 1992; Hiney et al., 1994).
Cipriano et al. (1996b) successfully eliminated SIF by the topical administration
of chloramine T and have isolated typical A. salmonicida from the mucus
of apparently healthy fish (Cipriano et al., 1992, 1994, 1996a,c). The
pathogenicity of mucus-isolated A. salmonicida has been demonstrated by
Hiney et al. (1994), who could induce clinical furunculosis in disease-free
brown trout by injection with a mixture of mucus and gill scrapings collected
from Atlantic salmon with SIF.
An internal primary location of A. salmonicida in covertly infected fish has
been proposed by a number of authors (cited by Hiney et al., 1997b). However,
the experimental protocols employed in many of these studies make meaningful
interpretation of the results difficult. In particular, experiments that involved the
transport of fish would have been stressful and, when examined in the
353 Furunculosis
laboratory, fish that originally had a commensal infection might be experiencing
the early phase of a clinical infection. Preliminary work carried out in Galway,
Ireland, on the salmon intestinal mucosa suggests that bacteria resident in the
intestine may breach this barrier following a brief transport stress. Thus,
arguments for an internal location of A. salmonicida in fish must be viewed with
caution in the absence of detailed descriptions of the protocols employed for
handling and transport. Nomura and his coworkers in Japan have, however,
presented evidence suggesting an internal location for A. salmonicida in covertly
infected adult fish (Nomura et al., 1991a,b, 1992).
LACK OF CLINICAL SIGNS IN COVERT INFECTIONS
The lack of clinical signs of disease during a covert furunculosis infection is
relatively simple to understand if the location of A. salmonicida is external to the
fish. The clinical signs of furunculosis are those of a systemic bacteraemia and
are unlikely to be present where the bacterium is confined to the gills, mucus or
intestine. If, however, the location is internal, then the absence of disease
becomes much more difficult to explain. A number of studies have demonstrated
that the median lethal dose (LD
50
)

of virulent A. salmonicida subsp. salmonicida
strains for salmonids is between 10
2
and >10 colony-forming units (cfu), when
injected either intramuscularly (i.m.) or intraperitoneally (i.p.) (McCarthy,
1977a; Cipriano et al., 1981; Cipriano and Starliper, 1982; Drinan and Smith,
1985; Olivier et al., 1985; Shieh, 1985; Bernoth and Krting, 1992). However,
Scallan (1983) could isolate over 10
6
cfu of A. salmonicida from the kidneys of
salmonid mortalities within 4 days of their being stressed. Knowledge of the
possible mechanisms whereby A. salmonicida might avoid the fishs internal
defence systems during a covert infection would facilitate an understanding of
the processes underlying these infections. In practically the only attempt to offer
any explanation for the temporary reduction of virulence that would appear to be
necessary if an internal location were accepted, McIntosh and Austin (1991)
hypothesized that A. salmonicida might reside in fish as L-forms (spheroplasts)
(McIntosh et al., 1991). They argued that the renal medulla of fish may provide
a favourable niche for L-forms, because of the high electrolyte concentration in
this tissue. This environment inhibits complement activity, and McGee (1986)
has suggested that it may protect L-forms from the lethal effects of the antibody
complement system. However, infectivity studies with these naturally occurring
A. salmonicida L-forms failed to produce disease, even after stressing
experimentally infected fish (McIntosh and Austin, 1990).
PERSISTENCE OF COVERT INFECTIONS
Although persistence of covert infections has frequently been suggested, there is
no definitive evidence that covert infections of individual fish can persist for
extended periods. Early furunculosis research demonstrated the existence of
persistent covert infections (Plehn, 1911), with no doubt that the duration of
these infections had major epizootiological significance (Mackie et al., 1935). A
study by Scallan (1983), involving the regular sampling of a population, would
suggest that high frequencies of stress-inducible infections may persist in such
populations for at least 1 year. However, studies of populations cannot provide
354
M. Hiney and G. Olivier
information on the persistence of covert infections in individual fish over time.
Where the parental population is maintained in a natural water body, the
possibility of continual reinfections by A. salmonicida derived from the water
cannot be eliminated. Persistence in a population may also result from repeated,
overlapping, short-term infections in individual members of the population
(Scallan, 1983).
In summary, a number of observations can be made about covert furun-
culosis infections. It is likely that covert infections are heterogeneous in nature
and that there is, in theory, more than one form of covert infection, whose
underlying processes differ fundamentally. Covert infections can be seasonal
(Scallan and Smith, 1984, 1993) and transitory in occurrence (Andrews, 1981;
Scallan et al., 1993). It is possible that they may persist for long periods in a
population without obvious harm to the fish. Scallan (1983) also demonstrated,
using a quantitative stress test, that the level of covert infection in a population
varied throughout the year. Cipriano (1997) also reported that the numbers of A.
salmonicida that could be isolated from the mucus of salmonids were variable
over the growing season. Covertly infected fish may act as carriers of disease or
be latently infected. Both internal and external locations have been proposed for
covert infections, but, to date, the locations of A. salmonicida in fish suffering
from commensal covert infections have not been resolved.
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
355 Furunculosis
characteristics. Guidelines for subspecies separation of the psychrophilic
aeromonads are presented in Table 10.5. Within these guidelines, A. salmonicida
subsp. salmonicida, associated with classical furunculosis, is termed typical,
while all isolates that do not fit this description are termed atypical. The
taxonomy of typical A. salmonicida will be discussed in this section, while a
later section presents a discussion of the taxonomy of atypical strains.
The most striking taxonomic feature of A. salmonicida subsp. salmonicida
strains is their homology. Taxonomic studies conclude that this subspecies is a
compact phenotypic and genotypic group, with a relationship to the mesophilic
aeromonads at the genetic level, unlike atypical A. salmonicida, which are a
highly heterogeneous group (Table 10.5) (Griffin et al., 1953; Ewing et al.,
1961; Liu, 1961; McCarthy, 978; MacInnes et al., 1979; Paterson et al.., 1980;
Belland and Trust, 1988; OhIci et al., 1998). The homogeneity of typical A.
salmonicida strains creates problems for substrain identification, which could
prove useful for improving our understanding of both the ecology of typical A.
salmonicida and the epizootiology of classical furunculosis. Techniques based
on phenotypic or immunological expression, which have found application in
epidemiological studies of other organisms, have not proved useful for
differentiating between strains of A. salmonicida subsp. salmonicida (Table
10.6). A number of newer techniques, based on the heterogeneity of the genetic
material contained within bacterial cells, have also been applied to the
differentiation of typical A. salmonicida strains with little success (Table 10.6).
Phage typing is the only typing system that has so far proved useful (Popoff,
1971; Rodgers et al., 1981). This technique has been employed in epizo-
otiological studies of A. salmonicida isolates in east-coast Canada since 1984
(Olivier, 1992). The study by Olivier (1992) has demonstrated that subtyping
techniques can provide valuable information about the origin of an epizootic of
furunculosis in a region, the impact of fish movements between freshwater and
sea-water sites and the relative success of disease control strategies employed.
Because so much is still unknown about the epizootiology of furunculosis and
the ecology of typical A. salmonicida, the search will continue for other
subtyping techniques that are inexpensive, reliable and user-friendly.
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 m 2.0 m, 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
M. Hiney and G. Olivier
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358
M. Hiney and G. Olivier
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359 Furunculosis
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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 1822C are optimal for
growth. A. salmonicida grows poorly at 4C and does not grow at 37C
(Snieszko, 1957), although isolation of an atypical strain capable of growth at
37C 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).
Diagnosis in clinically infected fish
The boil-like lesions observed by Emmerich and Weibel (1894), from which
classical furunculosis derives its name, are the exception in clinical cases, rather
than the rule (Bernoth, 1997b) and are normally only observed during chronic
infections in older fish. Table 10.4 summarizes the main clinical signs, gross
pathology and histopathological features that have been described for the
different forms of typical furunculosis (peracute, acute, subacute/chronic). The
gross pathological signs of peracute and acute furunculosis in young fish are
often indistinguishable from other bacterial septicaemias on preliminary
examination. The inexperienced diagnostician may also have difficulty in
differentiating the morphology and Gram-staining behaviour of A. salmonicida
in fish tissue, as seen under a microscope, from other Gram-negative bacteria
occurring in fish (Bernoth, 1997b). Thus, a firm diagnosis of clinical furun-
culosis requires isolation of the dominant infecting organism on agar media and
identification by morphology, combined with either biochemical or serological
tests, as A. salmonicida.
Detection of typical Aeromonas salmonicida during clinical infections
Under ordinary circumstances, typical A. salmonicida can be recovered from
clinically diseased fish, especially from the kidney and surface lesions, where
present (Austin and Austin, 1993). However, culturing samples from several
361 Furunculosis
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 brainheart infusion agar (BHIA) (Department of
Fisheries and Oceans, 1984; Shotts, 1984). The brown water-soluble pigment
produced by typical A. salmonicida on TSA after 24 days incubation at
2025C 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
to support the growth of typical A. salmonicida which will grow on BHIA
(Power et al., 1987) or blood agar (Bernoth and Artz, 1989). To exclude this
possibility, the Galway laboratory now routinely checks the ability of each TSA
batch to support the growth of a positive control A. salmonicida.
Biochemical identification of typical Aeromonas salmonicida
Presumptive identification of typical A. salmonicida colonies by pigmentation
on TSA or dark blue to navy staining on CBBA after 24 days incubation and
growth at 2025C but not 37C is not sufficient to make a firm diagnosis of
furunculosis, and the identity of the isolate should be confirmed by other tests.
Gram-staining behaviour and cell morphology of a pure culture (for example,
friability of colonies on an agar surface) are essential preliminary criteria and, in
combination with a limited number of biochemical tests, should be sufficient to
confirm the identity of an isolate as typical A. salmonicida and to exclude other
members of the Vibrionaceae, Enterobacteriaceae and Pseudomonadaceae. A
diagnostic protocol to confirm the identity of typical A. salmonicida is presented
in Table 10.7. More extensive tables of biochemical tests for A. salmonicida
characterization are presented elsewhere (Austin and Austin, 1993; Munro and
Hastings, 1993), but performance of these tests is usually unnecessary, unless it
is suspected that the isolate is an atypical A. salmonicida. A number of
commercial rapid test kits for biochemical identification, such as API-
BioMerieux and Biolog, have become available. These test systems were
developed for bacteria of medical importance and function best at incubation
temperatures of 3037C. Their application to the biochemical characterization
of A. salmonicida has been reported to generate inconsistent results following
incubation at lower temperatures (Bernoth, 1997b). However, incubation of A.
salmonicida at 30C, although above its optimal growth temperature (Snieszko,
1957), will generate consistent results.
Serological identification of typical Aeromonas salmonicida
Aeromonas salmonicida subsp. salmonicida requires at least 48 h incubation to
produce colonies suitable for morphological and biochemical identification and,
as a result, diagnosis of furunculosis may take up to 1 week. This often
represents an unacceptable time-lag for the fish farmer or veterinarian, who
needs to make rapid decisions on the treatment and fate of infected fish. Rapid
identification methods that could be applied directly to colonies after 48 h have
the potential to overcome the current delays in identifying presumptive A.
salmonicida. Bernoth and Artz (1989) suggested that serological tests may be
more sensitive than cultural isolation for the detection of A. salmonicida in fish
tissue. A comprehensive list of studies on the development of serological
identification tests for typical A. salmonicida has been presented by Bernoth
(1997b). She also provides a discussion of the technical difficulties that may be
encountered, such as heterogeneity of cell surface of the target species and cross-
reactivity with bacteria of other species or genera that may also be isolated.
While there remain considerable difficulties in applying these tests in situ in
infected tissue, they can facilitate more rapid confirmation of presumptive A.
salmonicida subsp. salmonicida colonies on agar plates (Bernoth, 1997b).
363 Furunculosis
Diagnosis of covertly infected fish
Diagnosis of covert A. salmonicida subsp. salmonicida infections poses a
number of important problems for the diagnostician, veterinarian, fish health
worker or regulator. As discussed by Bernoth (1997b), disease diagnosis in fish
must be understood as diagnosis in a population, rather than in an individual fish,
and therefore the method of sampling becomes an important diagnostic
consideration when covert furunculosis is suspected. The first sampling
consideration must be the number of fish that should be sampled in order to
reflect the health status of the population as a whole. Ossiander and Wedemeyer
(1973) have published sampling tables which specify statistically relevant
numbers of fish that should be sampled from populations of a given size. These
tables have been incorporated into regulations laying down the sampling plans
and diagnostic methods for the detection and confirmation of fish diseases by the
European Commission (Commission Decision 92/532/EEC), the USA
(Department of the Interior, 1993; Thoesen, 1994) and Canada (Department of
Fisheries and Oceans, 1984). However, Bernoth (1997b) was sceptical about the
reliability of such sampling tables in situations where the prevalence of covert
infection is low, but acknowledged that, in the absence of any detailed
epizootiological statistics, such recommendations will remain, for the moment,
our best guess. Even if statistically representative numbers of fish from a fish
population are sampled, detection of covert furunculosis remains problematic. A
number of approaches for the detection of covert infections have been presented
over the history of the disease. Hiney et al. (1997b) grouped these methods
according to what they demonstrated about the infection, i.e. the ability of
infected fish to shed A. salmonicida into their environment and transmit disease,
the presence of the organism or signs of the organism in/on covertly infected fish
or the precipitation of clinical furunculosis following the application of stress.
Demonstration of shedding and transmission of typical
Aeromonas salmonicida
The shedding of A. salmonicida by covertly infected fish can be demonstrated
indirectly, by cohabitation studies, or directly, by detection of the organism in
the environment of such fish. Cohabitation studies have been used by many
authors to demonstrate shedding and transmission of furunculosis. A number of
different transmission scenarios have been reported, including transmission of
clinical furunculosis from clinically infected fish to healthy fish, transmission of
clinical furunculosis from covertly infected fish to healthy fish, transmission of
covert furunculosis from clinically infected fish to healthy fish and transmission
of covert furunculosis from covertly infected fish to healthy fish so called
silent transmission (McCarthy, 1977a; Scallan, 1983; Hiney et al., 1997b).
Transmission experiments do not, however, represent a useful diagnostic
strategy for detection of covert infections.
Direct shedding of A. salmonicida into the environment of subclinically or
clinically infected fish has been demonstrated by culture methods (Scallan,
1983; Ford, 1994; Cipriano et al., 1996a), using immunological assays (Enger
and Thorsen, 1992; Gilroy and Smith, 1995) and polymerase chain reaction
364
M. Hiney and G. Olivier
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365 Furunculosis
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366
M. Hiney and G. Olivier
(PCR)/DNA probe assays (Gustafson et al., 1992; OBrien 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.
Detection of Aeromonas salmonicida subsp. salmonicida or
its components
The difficulty of isolating A. salmonicida or its components (antigens, DNA)
from covertly infected fish, in terms of not only the lack of selective
bacteriological methods (Cornick et al., 1969; Gustafson et al., 1992) but also
the reliability of current diagnostic techniques (Inglis et al., 1993; Crane and
Bernoth, 1996; Hiney and Smith, 1998) and the time-consuming nature of some
of these techniques, has been discussed (Austin and Austin, 1993). The problem
of deciding which organs of the fish should be examined is central to all methods
that fall into this group. However, as discussed, the location of A. salmonicida in
covertly infected fish remains uncertain.
Attempts to culture A. salmonicida from the internal organs of fish
suspected of covert infection constitute the method used by many authors.
However, there has been some disagreement about precisely which organ or
organs are most likely to yield the organism where no clinical signs are apparent.
Some workers have favoured the kidney as the organ of choice for detection of
covert infections (Mackie et al., 1935; Mackie and Menzies, 1938; McCarthy,
1977a), but others have demonstrated that examination of a number of organs is
more efficient (McDermott and Berst, 1968; Daly and Stevenson, 1985;
Sutherland and Inglis, 1992). In older salmonids, Nomura et al. (1991a,b, 1992,
1993) reported the isolation of A. salmonicida from coelomic fluid, as well as the
kidney.
The application of non-culture-based techniques to the detection of
components of A. salmonicida in tissues of covertly infected fish has not
resolved the issue of location in this infection type. In two studies which applied
an A. salmonicida-targeted ELISA to examination of the kidneys of similar
salmonid populations with SIF, A. salmonicida antigens were detected in one
study (Rose et al., 1989) but not in the other (Hiney et al., 1994). Similarly, using
PCR amplification and A. salmonicida-targeted DNA probes, positive responses
have been detected in a variety of salmonid organs, including spleen and kidney
(Gustafson et al., 1992; Hie et al., 1997) and blood (Mooney et al., 1995; Hie
367 Furunculosis
et al., 1997). However, in none of these studies was covert infection
demonstrated, and few successfully isolated the organism in primary culture
from the samples tested, making it difficult to interpret the meaning of these
data.
The use of external surfaces, including the intestine, as sampling sites when
attempting to detect typical A. salmonicida or its components has also been
reported. Although the intestine has been demonstrated as a site of colonization
of A. salmonicida in apparently healthy fish (Horne, 1928; Mackie et al., 1930)
and in fish that have been artificially challenged (Hodgkinson et al., 1987;
Markwardt and Klontz, 1989a,b), it has never been a popular sampling site. The
main reason for this is rapid overgrowth by concomitant flora, which
complicates isolation of A. salmonicida (Bernoth, 1997b). None the less, the
intestine is currently recommended as a sampling site for covertly infected fish
in the UK (Munro and Hastings, 1993) and USA (Shotts, 1984). In the opinion of
the present authors, this recommendation is misplaced. The difficulties
encountered with culture from the intestine make it unlikely that a covert
infection will be detected by this means. The use of non-culture-based detection
techniques for A. salmonicida may have the potential to overcome the
difficulties of bacteriological culture from the intestine. Intestinal contents have
been reported to yield positive results using both an A. salmonicida-targeted
ELISA assay (Rose et al., 1989; Hiney et al., 1994) and PCR-based assays
(Gustafson et al., 1992; Padley et al., 1997). However, until these assays have
been adequately validated for routine analysis they will remain research tools.
Perhaps the most extensive work on detection from external locations has
been carried out by Cipriano and coworkers. Using CBBA as a differential
medium (Cipriano and Bertolini, 1988; Markwardt et al., 1989), they
demonstrated that A. salmonicida could be isolated from the external mucus of
hatchery-reared salmon (Cipriano et al., 1996a), hatchery trout (Cipriano et al.,
1992, 1994) and feral salmon returning to spawn (Cipriano et al., 1996c) more
frequently that from the kidneys of the same fish. The method of Cipriano et al.
(1992) has the advantage of being non-lethal, which would be welcome in
restoration programmes and when assessing valuable brood stock (Cipriano,
1997).
Detection of covert furunculosis by the application of stress
Bacteriological culture from one or more organs may detect preclinical
infections or covert infections present at high levels in a fish population, but
culturing is unlikely to detect low levels of covert infections (McCarthy, 1977a;
Scallan, 1983; Hiney, 1995; Cipriano et al., 1997). Bullock and Stuckey (1975)
found that injection of the corticosteroid triamcinolone acetone (at 20 mg kg
1
body weight), combined with heat stressing at 18C for 14 days, activated latent
infections in covertly infected fish and facilitated their detection. The stress test
proposed by Bullock and Stuckey (1975) was demonstrated to be more efficient
at detecting covert infection than either heat stressing alone, as used by Plehn
(1911), or bacteriological culture alone (Blake and Clark, 1931; Mackie et al.,
1935; Mackie and Menzies, 1938). A number of modifications to the method of
Bullock and Stuckey (1975) were later published by McCarthy (1977a), Jensen
368
M. Hiney and G. Olivier
(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).
Limitation on the use of the stress-inducible furunculosis test for
detection of covert furunculosis infections
In Ireland, reliance on stress testing is widespread at the level of individual
companies and has been successful, when used, in limiting the spread of
furunculosis to marine farms (Smith, 1992; Scallan and Smith, 1993). Stress
testing of salmon smolts prior to their transport to sea is a regulatory requirement
in New Brunswick, Canada, and has also been found to be a successful measure
there (Olivier, 1992). However, although stress testing is valuable in the field, it
is not without its problems. In the experience of the present authors, stress
testing tends to result in the isolation of mixed cultures from the kidney, making
identification of A. salmonicida from this tissue difficult (Cornick et al., 1969).
Pigmentation of A. salmonicida strains, often used as a preliminary confirmation
of their presence, is inhibited in the presence of other organisms, requiring
careful examination and repeated subculture of kidney streaks to confirm
diagnosis (McCarthy, 1977a). Even so, the presence of A. salmonicida may be
missed (Scallan, 1983). No selective medium exists for the isolation of A.
salmonicida from mixed cultures, although CBBA has been found by the present
authors to simplify identification of putative A. salmonicida colonies isolated
from the kidneys of stress tested fish. Furthermore, the stress test is by definition
lethal. It requires 1416 days for a definitive diagnosis and uses large numbers
(150200) of fish to ensure detection of the infection where prevalence is low.
However, Bullock and Cipriano (1997) found, following sampling of the mucus
and gills of stress-tested fish, that, from day 5 of the test onwards, A. salmonicida
could be detected in these samples, thus eliminating the need to continue the test
to 14 days. This method may, therefore, represent a more rapid alternative to the
current stress test.
Prior vaccination of SIF-tested fish may complicate interpretation of the
results obtained from a stress test. Hiney (1995) demonstrated that, following
stress testing by the method of McCarthy (1997a), A. salmonicida could be
369 Furunculosis
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.
Rapid diagnostic methods
An important development in diagnostic microbiology over the last 25 years has
been the move to detect microorganisms directly in clinical samples or in the
environment of the host, without the need to culture. In line with this general
trend, non-culture-based microbial detection techniques are being increasingly
developed for the identification of typical A. salmonicida. Since 1990, in
particular, a number of genetic-based assays for A. salmonicida have been
described, and these are presented in Table 10.8, along with immunological
assays for the organism developed during the same period. For a more
exhaustive list of immunological assays developed prior to 1990 readers are
referred to Bernoth (1997b). Although many of the techniques presented in
Table 10.8 are intended for clinical diagnosis of furunculosis their potential for
detecting A. salmonicida in covertly infected fish and their environment has
also been investigated. In theory, non-culture-based techniques can be
designed to specifically detect low numbers of a target organism in a mixed
microbial flora. In addition, because culturing of the target organism is not a
requisite for its detection, they circumvent the numerous problems of loop-
and-plate microbiology (Bernoth, 1997b; Pace, 1997). Non-culture-based
techniques developed for the detection of A. salmonicida fall into two broad
categories, those based on immunological principles and those based on
genetic principles.
Immunological detection techniques
Immunological detection techniques offer many advantages over culture-based
techniques, particularly when attempting to detect organisms, such as A.
salmonicida, whose growth on laboratory media can be inhibited by the presence
of other bacteria. These techniques have been revolutionized in recent years by
370
M. Hiney and G. Olivier
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371 Furunculosis
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 10
3
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 (Garduo et al., 1993; Thornton et al., 1993). The question of how
372
M. Hiney and G. Olivier
relevant the current methods of antiserum generation from in vitro-grown A.
salmonicida components are for diagnosis in situ has to be addressed.
Genetic detection techniques
A second family of non-culture-based detection techniques that have been
investigated for the detection of components of A. salmonicida is based on
genetic principles. Typical A. salmonicida is homogeneous at the genetic level
(Vaughan, 1997) and presents, in theory, an ideal candidate for a genetic-based
detection technique. Since 1990, a number of assays targeted against 16S
ribosomal ribonucleic acid (rRNA) or DNA sequences of A. salmonicida have
been developed for this organism (Table 10.8). Most of these assays also utilize
the ability of PCR to amplify the number of copies of the target sequence in a
sample between several thousand- and 1 millionfold, making detection of
initially minute quantities of that sequence possible. However, it is difficult to
estimate the true lower detection limits of PCR-based assays for A. salmonicida
DNA sequences, because the kinetics of PCR amplification are essentially non-
linear and amplification is prone to variable inhibition by facets of both clinical
and environmental samples (Wilson, 1997).
Similar to immunological assays, the ultimate goal of genetic techniques is
to specifically detect DNA sequences of A. salmonicida in the tissues or
environment of both clinically and covertly infected fish. Clearly, then, the key
parameter of this type of technique is its specificity for the target organism.
Because of the relative newness of genetic detection techniques in comparison
with immunological techniques, there have been few reports to date about cross-
reactivity of these techniques. None the less, Hie (1995) found that his PCR-
based assay for an A. salmonicida DNA sequence cross-reacted with an
uncharacterized aquatic organism, and Hiney and Smith (1998) have expressed
concern about the relevance of laboratory studies of specificity for field
applications. In addition, PCR-based techniques are prone to false-positive
results through sample contamination, especially if a double cycle of PCR
amplification is employed, so-called nesting (Byers et al., 1997). As with
immunological assays, many PCR-based assays do not differentiate between
live and dead cells, and they have been demonstrated to detect DNA from killed
whole cells in the spleen and head kidney of fish vaccinated against furunculosis
(Hie et al., 1996), suggesting that this type of assay is unsuitable for application
to vaccinated stocks.
Validation of non-culture-based detection techniques
The biggest drawback to the use of non-culture-based detection techniques for A.
salmonicida is the difficulty of interpreting the results generated by these
techniques in field samples. Genetic techniques see a small segment of the
microbial genome, while immunological techniques see one or more epitopes
on the microbial surface. Therefore, the key characteristic of these techniques is
that they are proxy measurements of microbial presence, that is, indirect indices
that are presumed to signal the presence of the target organism in a sample
(Wildavsky, 1995). Where the target organism is a pathogen, the target sequence
or epitope(s) may also be used as a proxy measurement of disease potential. In
373 Furunculosis
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.
CONTROL AND TREATMENT
Transmission of the disease
The identification of the reservoir of the pathogen, the vector system it employs
to move from this reservoir to a new host and the mode of entry into the new host
are key questions in developing an understanding of the epizootiology of any
disease. Unfortunately, for A. salmonicida and its associated disease
furunculosis, none of these questions can be answered with certainty.
Standard textbooks of bacterial taxonomy refer to A. salmonicida as an
obligate fish pathogen (Bergey, 1984), suggesting that the bacterium is incapable
of sustained growth outside its piscine hosts. A number of laboratory microcosm
studies have suggested short survival times for the bacterium in aquatic
environments. It should, however, be noted that such experimental systems are
notoriously difficult to work with and the data they generate should only be
extrapolated to the real world with great caution (Enger, 1997). It is also true that
there are few reports of the isolation of the bacterium from the non-piscine
environment. However, colony formation by A. salmonicida can be inhibited by
a number of bacteria common in the aquatic environment, and Austin and Austin
(1993) has argued that the failure to culture A. salmonicida from water may be a
function of the inadequacy of our methods, rather than the absence of the
organism. A second factor that may have contributed to our failure is the extreme
hydrophobicity of the bacterium. This surface property would suggest that, in
nature, A. salmonicida is more likely to be isolated from particles (Sakai, 1986),
making traditional culture techniques unsuitable for the organism from the
environment. Michel and Dubois-Darnaudpeys (1980) demonstrated that A.
salmonicida can survive in a virulent form for many months in a sterile water
microcosm and there is some indirect evidence that, at least in marine sediment,
the organism can remain virulent for up to 6 months (Smith et al., 1982).
However, in a recent survey of a freshwater hatchery, in which they used both
ELISA and a PCR/DNA probe assay, Hiney et al. (1997a) found no correlation
between positive detection of the signs of A. salmonicida in hatchery sediment
and the disease status of hatchery stocks over a 2-year period, as assessed by
kidney culture and stress testing. These non-culture-based assays are, however,
capable of detecting non-culturable-but-viable cells and dead cells, and the
results generated by them cannot be used, therefore, as an indication of
virulence.
375 Furunculosis
Treatment and protection
Treatment of overt clinical epizootics
There are important differences between the treatment of large land-based
animals with antimicrobial agents and the treatment of populations of fish to
control furunculosis (Smith et al., 1994). In the former case, the primary aim
of therapy is to influence the outcome of an existing infection in an individual.
Orally administered treatments of furunculosis epizootics are, in contrast,
population-based and prophylactic in nature. Individual fish experiencing overt
furunculosis have seriously reduced appetite, and orally administered agents
are unlikely to achieve therapeutic levels in such fish. The primary aim of
treatments of fish populations is, therefore, to prevent the initiation of new
infections within the population. There are two major practical consequences
that flow from this prophylactic nature of fish treatment. The first is that all
infections initiated prior to the start of therapy can be expected to run their
course. Thus, significant mortalities will be experienced in a population for
some days after the commencement of therapy. The second is that the speed
with which therapy is initiated is critical. Laboratory confirmation of the nature
of an infection and the antimicrobial susceptibility of the isolate may take 45
days. Such a delay in the initiation of therapy will result in a significant
increase in mortalities. To achieve maximum reduction in mortalities, it is
essential that treatment is initiated at the first sign of mortalities which are
consistent with a septicaemia. With respect to Atlantic salmon smolts, the
occurrence, within any population, of mortalities that present evidence of
haemorrhage at the anus or the base of the fins (Bernoth, 1997b) should be
sufficient reason for initiating treatment. If such a rapid response is to be
achieved, the choice of agent to be employed will have to made on historical
data. Experience has shown that the sensitivity patterns of strains isolated at a
particular farm tend, in the absence of importation of foreign populations, to
remain reasonably consistent.
CHOICE OF ANTIMICROBIAL CHEMOTHERAPEUTANTS
In the laboratory, typical A. salmonicida can be shown to be sensitive to a wide
range of antimicrobial chemotherapeutants (Austin and Austin, 1993; Hastings,
1997). Its pattern of sensitivity is essentially similar to that of a typical Gram-
negative organism. Bacteria of this species do not manifest the widespread
resistance to ampicillin that is a property of Aeromonas hydrophila and other
mesophilic members of this genus (Sakazaki and Balows, 1981). The range of
agents available for the control of epizootics of furunculosis is limited by three
main factors. If the mode of administration is to be oral, then the agents must be
capable of achieving therapeutic levels when administered in this manner.
Secondly, the agents must be palatable. There have, for example, been
significant palatability problems associated with some preparations of
potentiated sulphonamides (Mitchell, 1992). It has been reported that
populations of fish may accept the first presentations of feed medicated with
these agents but that they will refuse subsequent presentations. The third factor
limiting the choice of agents is regulatory (Hastings, 1997). In many
376
M. Hiney and G. Olivier
jurisdictions the restrictive nature of the regulatory process will limit
significantly the range of agents available (Alderman et al., 1994).
RESISTANCE AND SENSITIVITY TESTING
Resistance to antimicrobial agents is common in A. salmonicida (Smith et al.,
1994) and places major constraints on the choice of agent. Sensitivity testing of
isolates is essential in every epizootic. There are considerable data that strains
manifesting more than one sensitivity pattern can be isolated from a single
epizootic and, on occasions, from a single fish (Scallan, 1983; Inglis et al.,
1991). It is, therefore, essential that, when sensitivity tests are being performed,
at least ten isolates from any case are tested. There are also data suggesting that
A. salmonicida can acquire resistance during therapy (Brazil et al., 1986),
suggesting that sensitivity testing should, particularly if mortalities continue, be
repeated during a treatment period. Even following a successful treatment, it is
common for a population to experience a second, normally less severe,
recurrence of the epizootic a few weeks later. Sensitivity testing of isolates from
such secondary epizootics is a wise precaution.
Many errors in therapy of furunculosis have resulted from the misinter-
pretation of data on the sensitivity of isolates of A. salmonicida. There are, at
present, no standard methods for measuring such sensitivities and therefore no
accepted means of interpreting the clinical significance of any sensitivity data
obtained (Smith et al., 1994). Serious errors can be made by employing
relationships between sensitivity and clinical resistance that have been
developed for humans or other land-based animals. These problems are most
acute with respect to the sensitivity to the quinolone group. Quinolone resistance
is mediated by reduced membrane permeability, is frequently low-level and
increases in a stepwise manner (Tsoumas et al., 1989). There are data to suggest
that strains with a minimum inhibitory concentration (MIC) as low as
0.75 g ml
1
and which give an inhibition zone of up to 18 mm when tested with
a disc containing 2 mg oxolinic acid are refractory to treatment (OGrady et al.,
1987). In practice, it is probably safer to treat an isolate showing any reduced
sensitivity to agents such as oxolinic acid or flumequine as clinically resistant.
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.
Treatments of covert infections
Hiney et al. (1997b) have presented a detailed review of the chemotherapeutic
treatment of covert furunculosis infection. Fish with covert infections can, with
good husbandry, be reared without any experience of overt disease (Scallan,
1983). Further, the presence of covert infections in a population are probably a
reflection of the presence of A. salmonicida in their water-supply. Therefore,
therapy of such covert infections is frequently neither justified nor liable, in the
377 Furunculosis
long run, to be effective. Under certain conditions, however, prudent health
management might suggest implementation of therapies to control such
infections.
Covert infections are latent and therefore, if it is known in advance that
populations with such infection are to be submitted to a stressful procedure,
therapy may be warranted. Examples of such predictable stressful events would,
ironically, include vaccination against furunculosis itself. Transport of fish
populations is of course a major and predictable stressful event but other
management procedures, such as grading or tagging, can also provide sufficient
stress to activate latent covert infections. In designing therapeutic interventions
to deal with these situations, it is necessary to decide whether the aim of the
intervention is prophylactic or therapeutic. In prophylactic treatments, the aim is
to provide adequate concentrations of the agent in the fish during the period of
the stress. In contrast, therapeutic treatments are aimed at eliminating the A.
salmonicida from the fish and ending the covert infection.
PROPHYLACTIC TREATMENT OF COVERT INFECTIONS
It is probable that any orally administered treatment regime that is successful in
controlling overt clinical epizootics of furunculosis (Table 10.9) would also
provide some degree of prophylactic cover against overt infections arising from
the activation of covert infections. There are, however, data that suggest that oral
administration of the quinolones oxolinic acid or flumequine would be the
treatment of choice. Standard practice would be to initiate a 10-day oral
treatment designed to end immediately prior to the predicted stressful event and
probably to follow this stressful event with a second oral treatment.
THERAPEUTIC TREATMENTS OF COVERT INFECTIONS
Only two treatment protocols have been developed that act in a therapeutic manner
and actually eliminate A. salmonicida from a population of covertly infected fish.
They are both significantly intrusive and stressful and therefore should only be
employed when the epizootiological situation demands them. When fish transport
is the primary reason for initiating a therapeutic treatment of a covert infection,
Table 10.9. Standard treatment regimes for orally administered chemo-
therapeutic agents in the treatment of furunculosis.
Antimicrobial Formulation Dosage
Environment agent (% active) (mg kg
1
) Regime*
Fresh water Oxytetracycline 50100 80 Orally for 10 days
Oxolinic acid 50100 10 Orally for 10 days
Romet 100 50 Orally for 5 days
Florfenicol 100 20 Orally for 10 days
Amoxycillin 100 4080 Orally for 1012 days
Sea water Oxytetracycline 50100 120 Orally for 1012 days
Oxolinic acid 50100 30 Orally for 10 days
Methasul 40 75 Orally for 10 days
Sulfatrim 50 60 Orally for 10 days
*Regime may be altered by response of fish to therapy.
378
M. Hiney and G. Olivier
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 g kg
1
flumequine. Protocols involving the bath administration of flume-
quine have also been reported (OGrady et al., 1988; OGrady 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 (OGrady 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 g ml
1
and this should be monitored by appropriate analytical
procedures. Serum concentration in excess of approximately 60 g 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 Mg
2+
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 Mg
2+
(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
379 Furunculosis
were commercially produced and marketed. Some, particularly those
administered by injection, were cost-effective in that the value of the fish
protected probably exceeded the cost of the vaccination. None, however, were
sufficiently effective to have a significant influence on the epizootiology of the
disease. This situation changed dramatically at the beginning of the 1990s. At
this time, oil-adjuvant injection vaccines became available (Bricknell and Ellis,
1993; Midtlyng, 1996; Midtlyng et al., 1996; Anderson et al., 1997).
Simply stated, the availability of oil-adjuvant injection vaccines has
transformed the significance of furunculosis in commercial salmon farming. In
the 1980s, prior to their introduction, furunculosis was, in Norway and Scotland,
one of the most important causes of mortality in sea-reared salmon. The use of
these vaccines is now almost universal throughout the European industry and
this has resulted in the perceived disappearance of furunculosis as a cause of
salmon mortality. Munro and Gauld (1996), for example, reported a reduction in
mortality of salmon in sea cages from 35% to approximately 10% that was
coincident with the introduction of these vaccines. Similarly Markestad et al.
(1995) related the 75% reduction in the use of antimicrobials in Norway that
occurred in 1994 due to the increased use of oil-adjuvant vaccines. The
development of these vaccines has been accompanied by the development of
machinery that allows the injection of a large number of fish in a short time. Not
only have these machines substantially minimized the logistical problems
associated with the administration of vaccines by injection, but they have also
significantly reduced the health risks to farm workers. Accidental self-injection
of furunculosis vaccines, particularly those that contain oil adjuvants, can result
in significant damage to workers. In any large-scale vaccination, significant
thought must be given to worker health issues, and clearly the use of machines to
perform the injection will significantly reduce the potential risks. Midtlyng
(1997) has also discussed the animal-welfare aspects of the use of oil-adjuvant
vaccines. While he accepts that there is evidence that such vaccines do result in
suffering for fish, he argues that any such suffering must be balanced against that
which would result from a devastating epizootic of furunculosis.
While the importance of oil-adjuvant vaccines for the salmon farming
industry is well attested (Markestad et al., 1995), the application of this
technology to salmonid farms whose primary function is restocking presents
problems. These problems are primarily associated with the persistence of the
adjuvant within the vaccinated fish. In restocking operations, there is inherently
less control over the time period between vaccination and consumption of fish.
In situations where fish are to be restocked into a put-and-take fishery or for the
purpose of competition fishing, considerations of the health risks that may be
associated with the consumption of highly immunogenic adjuvants may place
considerable constraints on the use of oil-adjuvant vaccines. Hiney (1995) has
demonstrated that covert infections can persist in oil-adjuvant-vaccinated fish.
Thus, there is a clear possibility that such fish can act as immune carriers. The
movement of such fish between water bodies may therefore be capable of acting
as a vector of the disease. As yet, there are no estimates of the size of this risk,
but its possible existence should be borne in mind in any movement of
vaccinated fish.
380
M. Hiney and G. Olivier
The success of oil-adjuvant vaccines for the control of furunculosis in
commercial fish farming has had a number of side-effects, which should also be
considered. Stress is the major precipitating factor associated with furunculosis.
In many situations, the fear of furunculosis has been a significant factor in the
introduction of husbandry practices that were aimed at reducing stress. In
particular, furunculosis has exerted a major constraint on stocking densities. The
effective removal, by vaccination, of the fear of furunculosis may result in the
relaxation of some farm practices, particularly when these place economic
constraints on the operation of a farm. It is axiomatic that any increase in stress
levels will ultimately result in reduction in fish health. The effective control of
furunculosis may, therefore, become the predisposing factor for another disease.
A second side-effect of the efficiency of modern vaccination is that funding for
research into furunculosis has been drastically reduced. Whether this will have
any significant effects outside the research community only time will tell.
PATHOGENESIS AND IMMUNITY
Pathogenesis of the organism
Despite the significant amount of work that has been undertaken on the
pathogenicity of A. salmonicida and the advances that have been made in this
area, the mechanisms whereby this organism produces disease are only partly
understood. Virulence mechanisms fall broadly into two categories, these being
cell-surface structures and extracellular products (ECPs) excreted by the cell.
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; Garduo 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 hosts immune response.
The A-layer has been reported to promote bacterial penetration and adhesion and
381 Furunculosis
to inhibit complement-mediated lysis in host serum (Trust et al., 1983; Sakai and
Kimura, 1985; Garduo and Kay, 1992b; Garduo 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.
Garduo 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 LD
50
of 2.4 g 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,
glycerophospholipidcholesterol 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 LD
50
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,
383 Furunculosis
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.
Host response to infection
Efforts over the last 10 years to improve the effectiveness of vaccines against
furunculosis have led to a much improved understanding of the salmonid
immune system, and a number of comprehensive reviews have been published
(Warr and Cohen, 1991; Secombes, 1994a; Secombes and Olivier, 1997). In
general, most components of the fish immune system are analogous to those of
higher vertebrates. These include physical barriers and chemical barriers to
prevent infection, inducible but non-specific humoral factors, phagocytes and
non-specific cytotoxic cells, which mediate an inflammatory response, and,
finally, specific immunity, effected by lymphocytes. It is this last type that is
responsible for immunological memory, ensuring that responses to a second
exposure are faster and stronger than the initial response, thus conferring
immunity (Secombes and Olivier, 1997). Specific immune responses include
both humoral immunity, based on the production of antibodies by B cells, and
cellular immunity, based on the production of activated macrophages with
enhanced bacterial activity, following release of cytokines from T cells
(Secombes and Olivier, 1997).
Non-specific humoral factors
Salmonids present physical barriers to infection by A. salmonicida in the
structure of their skin and intestinal mucosa and there is some evidence for
active immune cells on both of these surfaces (Ellis, 1985; Cipriano, 1986). Fish
also have a variety of non-specific humoral factors with which to counter
infection by A. salmonicida (Lund et al., 1991). These include enzyme systems
to lyse bacteria, such as lysozyme and complement, antiproteases to neutralize
bacterial proteases, proinflammatory molecules, which can attract leucocytes
and neutrophils to an infection site and increase local capillary permeability,
substances to sequester essential nutrients and thus limit bacterial growth, and
molecules which bind to the bacterial cell and trigger the complement system
and thus facilitate phagocyte uptake (Secombes and Olivier, 1997). In addition
384
M. Hiney and G. Olivier
to direct bactericidal effects, some components of the non-specific humoral
response serve to neutralize toxins excreted by A. salmonicida, for example,
2
-
macroglobulin, which has antiprotease activity (Salte et al., 1992). There is also
evidence that the complement system may have a role in neutralizing A.
salmonicida ECP (Sakai, 1984).
Non-specific cellular factors
Should A. salmonicida successfully breach the non-specific immune defences of
fish, it will encounter specialized cells of the host immune system, namely the
phagocytic cells. These include monocytes, or macrophages, and granulocytes
(neutrophils, eosinophils and basophils). The macrophages of fish are similar to
those of higher animals in both morphology and function and they act as antigen-
presenting cells, cytokine-secreting cells and effector cells, with the ability to
actively phagocytose bacterial cells (Secombes and Fletcher, 1992; Secombes
and Olivier, 1997). Less is known about the function of neutrophils in the
immune reaction of salmonids. Lamas and Ellis (1994a) demonstrated that
neutrophils isolated from Atlantic salmon migrated in fish serum in response to
the presence of A. salmonicida, although, strangely, A-layer-deficient strains
acted as stronger chemoattractants. Neutrophils were also found to be
phagocytic, but phagocytosis was low compared with macrophages (Lamas and
Ellis, 1994b). Eosinophils may play an important role in the host response
against infection by A. salmonicida, as they are widely distributed in connective
tissues, especially in the intestine and gills (Ellis, 1985). In the intestine,
eosinophilic granular cells (EGC) form a layer called the stratum granulosum
between the muscle layer and the stratum compactum. A number of studies have
suggested that fish EGC are the equivalent of mammalian mast cells (Ellis,
1982; Powell et al., 1991), because, in response to i.p. injection of A.
salmonicida ECP, there is a rapid and explosive degranulation of the intestinal
EGC, coincident with the appearance of histamine in the blood (Ellis, 1985;
Vallejo and Ellis, 1989).
Specific cellular responses
The specific immune response of salmonids includes three strategies, that is,
lymphocyte proliferation, antibody production and cytokine response. In higher
vertebrates, lymphocyte responses are characterized by their ability to
proliferate following contact with bacterial antigens, generating a clone of
antigen-specific cells. Some of these cells give rise to the primary response,
while others remain dormant as specific memory cells, able to proliferate in
response to subsequent contact with the same antigen (Secombes and Olivier,
1997). There are relatively few studies on lymphocyte proliferation in salmonids
in response to A. salmonicida infection. However, those that have been carried
out suggest that lymphocyte proliferation does occur in response to both
formalin-killed and live A. salmonicida cells (Reitan and Thuvander, 1991;
Vaughan et al., 1993), although this response is dependent on the dose and type
of antigen presented (Erdal and Reitan, 1992; Secombes and Olivier, 1997).
Antibody production to A. salmonicida, on the other hand, has been
extensively studied in salmonids, particularly following immunization with
385 Furunculosis
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 (1015C) antibody titres rise
within 23 weeks and peak within 812 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, 23 weeks
post-exposure, peaking 45 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 7C or less (Hardie et al.,
1994).
386
M. Hiney and G. Olivier
ATYPICAL AEROMONAS SALMONICIDA
Introduction
Aeromonas salmonicida subsp. salmonicida reviewed in the previous sections is
an important salmonid pathogen that has probably been studied more than any
other fish pathogen. However, the species A. salmonicida comprises a large
number of strains, routinely referred to as atypical strains, that is, strains that
do not conform to the general guidelines presented in Table 10.5 for typical A.
salmonicida. These atypical strains are responsible for several disease
conditions in both salmonids and non-salmonid species. Our knowledge of
atypical strains is growing rapidly and their importance is being increasingly
recognized. In the last decade, atypical isolates of A. salmonicida have become a
growing concern in several countries, because of their frequent association with
mortalities of both salmonids and non-salmonid species. Recent studies from
Canada and northern Europe indicate that cases of furunculosis in salmonids
caused by atypical isolates are increasing, probably because of better awareness
of these subspecies. This confirms the belief that atypical isolates are more
widely distributed than previously suspected (Wichardt et al., 1989; Rintamki
and Valtonen, 1991; Olivier, 1992; Pedersen et al., 1994; Wiklund et al., 1994).
The literature on atypical strains of A. salmonicida is expanding rapidly
but remains confusing, largely because of the problems associated with their
taxonomy and the variety of disease conditions they cause in different species of
fish. In previous reviews of A. salmonicida, atypical strains have always been
included with typical isolates (McCarthy and Roberts, 1980; Trust, 1986; Austin
and Austin, 1993). Although there are numerous similarities, we believe that
atypical strains are distinct subspecies and should be considered independently.
Diseases and disease agents
Species susceptibility to atypical Aeromonas salmonicida
The number of hosts from which atypical A. salmonicida isolates have been
cultured is rapidly increasing and includes several species of salmonids and non-
salmonids, wild or cultured, in fresh, brackish or salt water (Tables 10.10
10.12 ). Strains of atypical A. salmonicida have been reported in wild and farmed
populations of salmonids in northern Europe (Wichardt et al., 1989; Hstein and
Lindstad, 1991; Rintamki and Valtonen, 1991; Hirvel-Koski et al., 1994;
Pedersen et al., 1994; Gudmundsdttir et al., 1995). The most common clinical
sign in these infections is skin ulceration (Wichardt et al., 1989), but a number of
other clinical signs have been recognized some of which are similar to those seen
in cases of typical furunculosis (Rintamki and Valtonen, 1991).
Mortality of up to 60% has been reported from cultured sea trout in Sweden
(Wichardt et al., 1989) and 1520% in Finland and Iceland (Gudmundsdttir
et al., 1995: Rintamki and Valtonen, 1991). In North America, an ulcer disease
of trout, originally described by Sniezko et al. (1950), was associated with a
fastidious organism (Haemophilus piscium), which was subsequently
387 Furunculosis
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388
M. Hiney and G. Olivier
reclassified as an atypical strain of A. salmonicida (Paterson et al., 1980; Trust
et al., 1980b; Adams and Thompson, 1990). On the Atlantic coast of Canada,
fastidious atypical strains have been isolated from Atlantic salmon (Paterson et
al., 1980; Groman et al., 1992; Olivier, 1992) with mortality of up to 50% being
recorded in some instances (Groman et al., 1992). Thus, disease of atypical A.
salmonicida aetiology can have a significant economic impact, depending on the
fish species infected.
Some of the best-described conditions caused by strains of atypical A.
salmonicida in non-salmonid freshwater hosts are: carp erythrodermatitis
(Bootsma et al., 1977), the ulcer disease of goldfish Carassius auratus L. (Shotts
et al., 1980) and the head ulcer disease of cultured Japanese eels, Anguilla
japonica (Ohtsuka et al., 1984; Kitao et al., 1985). Atypical strains of A.
salmonicida have also been isolated from strictly marine hosts. Evelyn (1971)
isolated an atypical strain of A. salmonicida from a marine species, the sable
fish, Anopoploma fimbria. A similar strain was isolated from ling cod, Ophiodon
Table 10.11. Freshwater non-salmonid species from which atypical
Aeromonas salmonicida have been isolated (after Bernoth, 1997a).
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)
J apanese eel Anguilla japonica Clinical Kitao et al. (1984)
Minnow Phoxinus phoxinus Clinical Hstein 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
389 Furunculosis
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
Table 10.12. Marine non-salmonid species from which atypical Aeromonas
salmonicida have been isolated (after Bernoth, 1997a)
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
J apanese 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)
Fish species
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 30C 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; Svnyi et al., 1984;
Evenberg et al., 1985; Kitao et al., 1985; Austin et al., 1986; Bhm 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).
Taxonomy of atypical Aeromonas salmonicida
The classification of psychrophilic Aeromonas is not clear-cut. Table 10.5
attempted to provide guidelines for separating typical from atypical strains,
based on associated pathology and its correlation with genetic and phenotypic
characteristics. However, the atypical group still presents major difficulty when
attempting to assign isolates to a particular subspecies. In most cases, one must
resort to referring to the isolate as simply atypical A. salmonicida. Austin and
Austin (1993) have extensively reviewed the history of the taxonomy of
atypical A. salmonicida as they are currently classified in Bergeys Manual of
391 Furunculosis
Determinative Bacteriology (Holt et al., 1994), in which atypical isolates are
separated into three subspecies, masoucida, achromogenes and smithia. Strains
belonging to the subsp. masoucida were described by Kimura (1969). Because
subsp. masoucida is clearly restricted to Japan, its biochemical profile is quite
different from that of any other atypical isolates (Austin et al., 1989). Thus,
using the classification of Holt et al. (1994), all other atypical strains must be
placed in only two recognized subspecies, achromogenes or smithia. This
situation epitomizes the basic problem facing diagnosticians when atypical
isolates are recovered from diseased fish. Often these strains are biochemically
different from the reference strains of the subsp. achromogenes or smithia,
raising the difficult issue of how to classify them.
McCarthy (1978) proposed a different scheme to classify atypical A.
salmonicida. After an exhaustive numerical taxonomy study of 75 typical and 29
atypical isolates, he found that atypical strains could be separated into two
phenons (B1 and B2), distinguished by their biochemical and enzymatic
properties. He proposed to combine the two subspecies achromogenes and
masoucida (corresponding to his phenon B1) into a single subspecies,
achromogenes. Interestingly, all 18 strains of this phenon had been isolated from
salmonid hosts. His second suggestion was to create a new subsp., nova, to
accommodate the 11 isolates of phenon B2, ten of which had been isolated from
non-salmonid hosts. A subsequent DNA : DNA hybridization study of a series of
typical and atypical isolates carried out by Belland and Trust (1988) supported
this new classification, but these authors cautioned that there could be additional
subspecies present, because some strains could not be properly assigned to either
of the two proposed subspecies. The later study on A. salmonicida taxonomy, by
Austin et al. (1989), disagreed with the previous findings and these authors were
of the opinion that the subspp. achromogenes and masoucida should be retained
and that a new subspecies, smithia, should be created. It would incorporate a
cluster of strains closely related to the subsp. nova proposed earlier by McCarthy
(1978). Clearly, a consensus on the exact taxonomic position of atypical
isolates is yet to be achieved.
Over 60 atypical strains were biochemically analysed by Olivier et al.
(1990) and Olivier (1992) and, while not substantiated by DNA : DNA
hybridization, their results tend to agree with those of McCarthy (1977b, 1978)
and Belland and Trust (1988). Two major groups were identified according to
their biochemical and enzymatic profiles; these were similar to the subspp.
achromogenes and nova described by McCarthy (1978). In addition to this
primary separation into subspecies, different biotypes could be identified within
each of these two groups. Analysis of these results strongly suggests that there is
a definite correlation between biotypes and host species and also between
biotypes and their geographical origin. Interestingly, the proposal of Austin et al.
(1989) of a new subspecies, smithia, would fit into this classification scheme,
since most strains in their subsp. smithia (14 out of 19) were isolated from a
single host, the roach Rutilus rutilus. These findings provide evidence that the
isolation of these strains is often correlated with a specific host species. There is
no doubt that the taxonomy of atypical strains needs additional work, and DNA :
DNA hybridization studies are required to resolve their taxonomic uncertainty.
392
M. Hiney and G. Olivier
The recent increase in cases of atypical furunculosis reported from several
countries stresses the importance of this group of organisms for wild and
cultured stocks (Wichardt et al., 1989; Rintamki and Valtonen, 1991; Olivier,
1992; Bernoth, 1997a).
Genomic characterization is being increasingly used to determine the degree
of homology of bacterial isolates when serological analysis is not adequate.
Genomic analysis applied to A. salmonicida has clearly shown that A.
salmonicida isolates can be separated into subgroups. Both Belland and Trust
(1988) and McCormick et al. (1990) confirmed that typical isolates formed a
distinct group and that atypical isolates belonging to the subspp. achromogenes
and masoucida were genotypically diverse. Of particular interest is the fact that,
in addition to the recognized subspecies, there was evidence that additional
subgroups were likely to exist. Using 60 phenotypic tests, ribotyping and
plasmid profiles, Hnninen and Hirvel-Koski (1997) examined 53 atypical
strains originating from Finland, Denmark, Norway and Sweden. They found
that the division of strains into distinct phenotypic clusters (pigment +/, oxidase
+/) correlated well with ribotype and plasmid profile.
Recent developments in molecular biology have allowed scientists to
fingerprint various bacterial isolates in order to determine their degree of
homogeneity. One such technique is randomly amplified polymorphic DNA
analysis (RAPD). Using this technique, Miyata et al. (1995) and Inglis et al.
(1996) have shown that atypical strains can be differentiated from typical
isolates, although a limited number of atypical isolates were used in their study.
Kwon et al. (1997) have also used RAPD to analyse 29 Japanese atypical strains
isolated from six non-salmonids hosts. Their results indicate, as before, that
atypical isolates are genotypically different from typical ones. Even though a
high degree of homology was detected in their atypical isolates with four of their
RAPD primers, another primer (A31) gave an interesting result showing that all
isolates could be differentiated according to the host species the strains were
isolated from. Using RAPD and pulsed-field gel electrophoresis, OhIci et al.
(1998) analysed 39 atypical strains from Europe and North America isolated
from three salmonids and five non-salmonid hosts. They found the atypical
strains to be a genetically heterogeneous group with a similarity of less than
70%, but noted some correlation of groupings with different fish hosts and
locations of isolation.
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.
393 Furunculosis
In several cases, transmission of the disease seems to have been linked to the
transfer of infected fish (Wichardt et al., 1989; Hstein 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).
Diagnostic methods for atypical Aeromonas salmonicida
Diagnosis is usually based on culturing of the pathogen. The identification of
non-fastidious atypical isolates of the subsp. achromogenes is relatively
straightforward, because all these strains are reported to grow on conventional
media, such as TSA and BHIA. The strains can be chromogenic or not; however,
growth is often slower than that of typical isolates (Evelyn, 1971; Wichardt et
al., 1989; Rintamki and Valtonen, 1991; Olivier, 1992; Wiklund and Dalsgaard,
1995). Samples of ulcers or lesions, in addition to kidney tissues, should be
cultured to confirm the presence of atypical isolates, because some organisms
may not be found in internal organs. In Iceland, the recovery of A. salmonicida
subsp. achromogenes was superior when gills of subclinically or covertly
infected Atlantic salmon were cultured, rather than kidney tissues
(Benediktsdottir and Helgason, 1990).
Several problems are associated with the culture of fastidious strains
belonging to the subsp. nova. A culture medium containing blood is required for
the primary isolation of atypical strains of this subspecies, including isolates
from carp, goldfish and eel and some fastidious isolates recovered from
salmonids (Bootsma et al., 1977; Paterson et al., 1980; Bhm et al., 1986;
Ishiguro et al., 1986; Olivier, 1992). Ishiguro et al. (1986) investigated this
phenomenon and found that fastidious atypical strains isolated from goldfish
and salmon (including H. piscium) could grow well if conventional medium
(TSA) was supplemented with haemin (10 g ml
1
): this was confirmed by Nakai
et al. (1989) with atypical strains isolated from Japanese eel (A. japonica).
Isolation plates are often contaminated with what is believed to be
opportunistic pathogens, such as motile aeromonads and Pseudomonas spp.,
thus complicating the diagnosis. This phenomenon has been reported by several
investigators in both natural infections and during experimental challenges with
strains causing carp erythrodermatitis (Csaba et al., 1984; Evenberg et al.,
1986), goldfish ulcer disease (Whittington et al., 1987) and the causative agent
of the ulcerative disease of eels (Nakai et al., 1989). Often, the organism is only
isolated from skin lesions and the infection is not systemic (Elliot and Shotts,
1980; Csaba et al., 1984; Bhm et al., 1986; Noga and Berkhoff, 1990). In rare
instances, the diagnosis can be complicated by the presence of both typical and
atypical strains as reported by Noga and Berkhoff (1990) in American eels
(Anguilla rostrata).
394
M. Hiney and G. Olivier
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 37C. 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.
Serological identification of atypical Aeromonas salmonicida
Because it may be difficult to grow atypical A. salmonicida, other diagnostic
methods have been explored, including immunofluorescence (fluorescent
antibody test (FAT)), as described by Bhm et al. (1986). Using this method,
serological confirmation of atypical furunculosis in carp and goldfish was at
least 50% higher than by culture. Svnyi (1986) described a coagglutination
test for the diagnosis of carp erythrodermatitis and found that all experimentally
induced ulcerative lesions were positive by this test, with no cross reactions with
other fish pathogens being observed. The ELISA assay described by Adams and
Thompson (1990) was also able to detect atypical strains. Methods used to detect
typical isolates should be effective in detecting atypical isolates, because of their
serological similarities, and simple methods, such as FAT, could be used as an
initial screening for these pathogens. Modern techniques, such as PCR, will
undoubtedly become available to detect these isolates, although, as has been
discussed above, the routine diagnostic use of these methods cannot be
considered without extensive validation of the techniques.
Control and treatment
Chemotherapeutic treatment
Chemotherapy would appear to be the method of choice in dealing with
infections caused by atypical isolates (Csaba et al., 1984; Bhm et al., 1986;
395 Furunculosis
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. Hirvel-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
Svnyi 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
M. Hiney and G. Olivier
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398
M. Hiney and G. Olivier
Table 10.14. Biochemical characteristics distinguishing between typical and
atypical strains of Aeromonas salmonicida*.
Aeromonas salmonicida subspp.
Characteristic salmonicida masoucida acromogenes nova smithia
Pigment + V
Haemin requirement + .
Gas from glucose + + .
Arbutine + + V .
Sucrose + + V V
Salicin + V .
N-Acetylglucosamine + V .
Dextrin + + + .
Fructose + + + V .
Galactose + + V V
Glycerol + + +
Mannose + + + V .
Maltose + + + V
Trehalose + + + V
Indole + V +
Aesculin + +
Gelatinase + + V +
Lecithinase +
Elastase +
Protease + + V
Resistance to
Ampicillin (25 g) + + + +
Cephaloridine (15 g) + + V .
Polymyxin B (300 IU) V .
Salmonid source + + V V
*Differential characteristics of Aeromonas salmonicida subspecies based on
McCarthy (1978), Popoff (1984), Austin et al. (1989), Wichart et al. (1989) and
Olivier (1992, unpublished results).
+, , V indicate that >80%, <20% and 2179% of the strains gave positive
reactions, respectively. . indicates not tested.
Pathogenesis of atypical Aeromonas salmonicida
There is limited information on the pathogenicity of atypical strains of A.
salmonicida. They generally produce cutaneous ulceration, however, the
infection does not necessarily become systemic and the cause of death is not
always substantiated (reviewed by Trust, 1986). Work by Bucke (1980) provided
evidence that atypical isolates could be pathogenic not only for their original
host but for other species as well. In this particular study, Bucke (1980)
inoculated i.m. brown trout, perch, rudd, roach, carp and goldfish with three
strains of A. salmonicida, including a typical isolate, a cyprinid atypical isolate
and a goldfish isolate (unfortunately, the author did not provide the exact number
of bacteria injected). His results clearly indicated that there were differences in
the virulence of the isolates towards different hosts; for example, the typical
399 Furunculosis
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 LD
50
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 LD
50
values of 1 10
23
for Atlantic salmon (Olivier et al.,
1990), confirming the results of Gudmundsdttir 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 10
6
cfu (Evenberg et al., 1988). The LD
50
of an eel isolate
was approximately 1 10
3
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 LD
50
less than 1 10
4
cfu (Trust
et al., 1980b), while a wrasse isolate had a low LD
50
(5 10
2
) 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 LD
50
values
higher than 1 10
6
(Hstein 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 LD
50
values ranging from 1 10
2
to 1 10
3
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 10
6
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 10
4
A-layer-positive cells in goldfish killed all test animals (8/8), whereas
a similar injection of 10
8
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-
dttir 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 (Gudmundsdttir, 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-
401 Furunculosis
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-
mundsdttir 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 Gudmundsdttir 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.
REFERENCES
Adams, A. and Thompson, K. (1990) Development of an enzyme-linked immunosorbent
assay (ELISA) for the detection of Aeromonas salmonicida in fish tissue. Journal of
Aquatic Animal Health 20, 281288.
403 Furunculosis
Alderman, D.J., Rosenthal, H., Smith, P., Stewart, J. and Weston, D. (1994) Chemicals
Used in Mariculture. Technical Report of ICES Working Group, Environmental
Interactions of Mariculture, Report 202, International Council for the Exploration
of the Sea, Copenhagen.
Amlacher, E. (1961) Taschenbuch der Fischkrankheiten. Gustav Fisher Verlag, Jena,
(cited in Austin and Austin, 1993).
Anderson, D.P. (1969) Immunopathology. US Department of the Interior, Fish and
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Anderson, D.P., Rogerson, B.S. and Dixon, O.W. (1979) Induction of antibody-
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