10 furunculosis (aeromonas salmonicida)
TRANSCRIPT
341© CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
10Furunculosis (Aeromonas salmonicida)
M. Hiney1 and G. Olivier2
1Fish Disease Group, Department of Microbiology, National University ofIreland Galway, Galway City, Ireland; 2Department 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 100years. Emmerich and Weibel (1894) made the first authentic report of itsisolation during a disease outbreak at a Bavarian brown trout hatchery, themanifestations of the disease including furuncle-like swelling and, at a laterstage, ulcerative lesions on infected trout. Since that time a number ofsubspecies of A. salmonicida have been recognized, although the taxonomy ofthe species is far from settled. Aeromonas salmonicida is one of the most studiedfish pathogens, because of its widespread distribution, diverse host range andeconomically devastating impact on cultivated fish, particularly the salmonids(Austin and Austin, 1993). The continued importance of salmonids to rodfishermen, commercial fisheries and fish farmers and the extent of the impactthat A. salmonicida has on these various methods of exploitation have served tomaintain the status of A. salmonicida as an important fish pathogen. A number ofexcellent reviews of A. salmonicida have been published (McCarthy andRoberts, 1980; Austin and Austin, 1993; Bernoth et al., 1997). This chapterattempts to summarize the current information, both research and anecdotal,available on A. salmonicida and its associated pathologies in a manneraccessible 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 thisgroup. 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 iswide and that furunculosis is only one of several clinical diseases associatedwith A. salmonicida. Furthermore, infections of fish with A. salmonicida are notnecessarily associated with clinical manifestations and may remain covert(Hiney et al., 1997b). Typical A. salmonicida have been associated with clinicalor covert disease in a variety of salmonid and non-salmonid species in freshwater, brackish water and sea water. Reports of fish species that have beendocumented as suffering from diseases of typical A. salmonicida aetiology areoutlined in Tables 10.1–10.3. These tables do not represent all available reportsor all species but are intended to demonstrate the taxonomic breadth of fishspecies that are susceptible to infection with this organism. For a list of speciesfrom which atypical A. salmonicida have been isolated, readers are referred toTables 10.11 to 10.13.
Susceptibility to furunculosisMost 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 andHeartwell, 1986; Pérez et al., 1996), while brook trout, brown trout and manyother 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 differentgenetic lines (Dahle et al., 1996; Marsden et al., 1996) or with different historiesof exposure to A. salmonicida (St Jean, 1992). Because of the potentiallyinheritable nature of some disease resistance, directed breeding programmesaimed at raising stocks inherently resistant to furunculosis have beeninvestigated as a possible disease-control strategy in salmonids (Gjedrem et al.,1991; Lund et al., 1995; Gjedrem, 1997) and non-salmonids (Sövényi et al.,1988; Hjeltnes et al., 1995). However, the multifactorial nature of inheritablecharacteristics complicates selective breeding programmes and much workremains to be done in this area (Gjedrem, 1997). Species susceptibility toinfection 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 salmonidpopulations, furunculosis was mainly a disease of older fish (Plehn, 1911;Mettam, 1915; McCraw, 1952). Although this perception may, in part, have beendue to the easier observation of large carcasses in rivers, experimental evidencedid 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 essentiallyunknown but are probably non-specific (Krantz and Heist, 1970). Furthermore,not all workers agree that age plays a significant part in susceptibility tofurunculosis (McCarthy, 1977a; Inglis et al., 1993). McCarthy and Roberts(1980), referring to the disease in fingerlings, observed that fish of this sizecontract an acute form of the disease, which results in rapid death with littlemore than slight exophthalmos. Mortalities in infected fish in the 0+ age group
343Furunculosis
Tab
le 1
0.1
.S
alm
on
id f
ish
sp
ecie
s fr
om
wh
ich
typ
ical
Aer
om
on
as s
alm
on
icid
a h
as b
een
iso
late
d (
afte
r B
ern
oth
, 199
7a).
His
tory
of
Co
mm
on
nam
eS
cien
tifi
c n
ame
Hab
itat
iso
lati
on
Ref
eren
ce
Atl
anti
c sa
lmo
nS
alm
o s
alar
Fres
h w
ater
Co
vert
*M
cCar
thy
(197
7a)
Clin
ical
Ber
no
th (
1997
a)S
ea w
ater
Clin
ical
Sm
ith
(19
97)
Am
ago
sal
mo
nO
nco
rhyn
chu
s rh
od
uru
sS
ea w
ater
Clin
ical
†M
iyaz
aki a
nd
Ku
bo
ta (
1975
)B
roo
k tr
ou
tS
alve
linu
s fo
nti
nal
isFr
esh
wat
erC
ove
rtB
ullo
ck a
nd
Stu
ckey
(19
75)
Clin
ical
Jen
sen
(19
77)
Bro
wn
tro
ut
Sal
mo
tru
tta
m. l
acu
stri
sFr
esh
wat
erC
ove
rtJe
nse
n a
nd
Lar
sen
(19
80)
Clin
ical
Mac
kie
et a
l. (1
930)
Ch
ino
ok
salm
on
On
corh
ynch
us
tsh
awyt
sch
aN
ot
spec
ifie
dN
ot
spec
ifie
d†
Fulle
r et
al.
(197
7)C
hu
m s
alm
on
On
corh
ynch
us
keta
Fres
h w
ater
Co
vert
No
mu
ra e
t al
. (19
93)
Clin
ical
Sak
ai a
nd
Kim
ura
(19
85)
Sea
wat
erC
linic
alW
iklu
nd
et
al. (
1992
)C
oh
o s
alm
on
On
corh
ynch
us
kisu
tch
Fres
h w
ater
Clin
ical
Wik
lun
d e
t al
. (19
92)
Cu
tth
roat
tro
ut
Sal
mo
cla
rki
Fres
h w
ater
No
t sp
ecif
ied
McC
arth
y (1
975)
Do
lly V
ard
enS
alve
linu
s m
alm
aFr
esh
wat
erN
ot
spec
ifie
dM
cCar
thy
(197
5)Ja
pan
ese
char
Sal
velin
us
leu
com
aen
isN
ot
spec
ifie
dC
linic
alS
akai
an
d K
imu
ra (
1985
)La
ke t
rou
tS
alve
linu
s n
amay
cush
Fres
h w
ater
Co
vert
Dal
y an
d S
teve
nso
n (
1985
)C
linic
alM
cCar
thy
(197
5)M
asu
sal
mo
nO
nco
rhyn
chu
s m
aso
uFr
esh
wat
erC
ove
rtN
om
ura
et
al. (
1993
)P
ink
salm
on
On
corh
ynch
us
go
rbu
sch
aFr
esh
wat
erC
ove
rt*
No
mu
ra e
t al
. (19
93)
Clin
ical
Sak
ai a
nd
Kim
ura
(19
85)
Po
llan
Co
reg
on
us
po
llan
Fres
h w
ater
No
t sp
ecif
ied
†M
cCar
thy
(197
5)R
ain
bo
w t
rou
tO
nco
rhyn
chu
s m
ykis
sFr
esh
wat
erC
ove
rtB
ullo
ck a
nd
Stu
ckey
(19
75)
Clin
ical
Jen
sen
(19
77)
Sea
wat
erC
linic
alFe
rnán
dez
et
al. (
1995
)S
ea t
rou
tS
alm
o t
rutt
a m
. tru
tta
Fres
h w
ater
Co
vert
Hir
velä
-Ko
ski e
t al
. (19
88)
Clin
ical
Mac
kie
et a
l. (1
935)
So
ckey
e sa
lmo
nO
nco
rhyn
chu
s n
erka
No
t sp
ecif
ied
Clin
ical
Sak
ai a
nd
Kim
ura
(19
85)
*Ap
par
entl
y h
ealt
hy
fish
no
t sh
ow
ing
sig
ns
of
infe
ctio
n (
see
Hin
ey e
t al
., 19
97b
).†N
ot
spec
ifie
d w
het
her
typ
ical
or
atyp
ical
A. s
alm
on
icid
a b
ut
assu
med
to
be
typ
ical
.
Fish
sp
ecie
s
344 M. Hiney and G. Olivier
Tab
le 1
0.2
.Fr
esh
wat
er n
on
-sal
mo
nid
sp
ecie
s fr
om
wh
ich
typ
ical
Aer
om
on
as s
alm
on
icid
a h
ave
bee
n i
sola
ted
(af
ter
Ber
no
th,
1997
a).
His
tory
of
Co
mm
on
nam
eS
cien
tifi
c n
ame
iso
lati
on
Ref
eren
ce
Am
eric
an e
elA
ng
uill
a ro
stra
taC
linic
alN
og
a an
d B
erkh
off
(19
90)
Bra
ssy
min
no
wH
ybo
gn
ath
us
han
kin
son
iU
ncl
ear*
McF
add
en (
1970
)B
roo
k st
ickl
ebac
kC
ula
ea in
con
stan
sU
ncl
ear
McF
add
en (
1970
)C
arp
Cyp
rin
us
carp
ioC
linic
alM
acki
e et
al.
(193
0)U
ncl
ear
Ber
no
th (
1997
b)
Cat
fish
Silu
rus
gla
nis
Clin
ical
Mac
kie
et a
l. (1
930)
Ch
estn
ut
lam
pre
yIc
hth
yom
yzo
n c
asta
neu
mU
ncl
ear
Ber
no
th (
1997
a)C
om
mo
n s
hin
erN
otr
op
is c
orn
utu
sC
linic
alO
stla
nd
et
al. (
1987
)C
reek
ch
ub
Sem
oti
lus
atro
mac
ula
tus
Clin
ical
Ost
lan
d e
t al
. (19
87)
Un
clea
rM
cFad
den
(19
70)
Eu
rop
ean
eel
An
gu
illa
ang
uill
aIn
cid
enta
lS
lack
(19
37)
Fath
ead
min
no
wP
imep
hal
es p
rom
elas
Clin
ical
McF
add
en (
1970
)G
ob
yC
ott
us
go
bio
Clin
ical
Ber
no
th (
1997
b)
Go
lden
sh
iner
No
tem
igo
nu
s cr
yso
leu
cas
Clin
ical
Ost
lan
d e
t al
. (19
87)
Gro
per
Ro
ccu
s m
issi
ssip
pie
nsi
sU
ncl
ear
Her
man
(19
68)
Lam
pre
yN
ot
spec
ifie
dN
ot
spec
ifie
d†
McC
arth
y (1
975)
Min
no
wP
ho
xin
us
ph
oxi
nu
sU
ncl
ear
Ber
no
th (
1997
a)M
ott
led
scu
lpin
Co
ttu
s b
aird
iIn
cid
enta
lR
abb
an
d M
cDer
mo
tt (
1962
)N
on
-sal
mo
nid
sIn
cid
enta
lB
rag
g (
1991
)N
ort
her
n p
ike
Eso
x lu
ciu
sC
linic
alB
ern
oth
(19
97a)
Orn
amen
tal c
ypri
nid
sU
ncl
ear
Ber
no
th (
1997
a)P
add
lefi
shP
oly
od
on
sp
ath
ula
Clin
ical
Ford
et
al. (
1994
)R
edb
elly
dac
eC
hro
mo
mu
s eo
sC
linic
alM
cFad
den
(19
70)
Sm
allm
ou
th b
ass
Mic
rop
teru
s d
olo
mie
ui
Clin
ical
Le T
end
re e
t al
. (19
72)
Sti
ckle
bac
kG
aste
rost
eus
acu
leat
us
Un
clea
rB
arke
r an
d K
eho
e (1
995)
Fish
sp
ecie
s
345Furunculosis
Ten
chTi
nca
tin
caC
linic
alM
acki
e et
al.
(193
0)C
ove
rt†
Ber
no
th a
nd
Kö
rtin
g (
1992
)W
hit
e su
cker
Cas
tost
om
us
com
mer
son
iC
linic
alO
stla
nd
et
al. (
1987
)Ye
llow
bas
sM
oro
ne
mis
siss
ipp
ien
sis
Clin
ical
Bu
ckle
y (1
969)
Yello
w p
erch
Per
ca f
lave
scen
sU
ncl
ear
McF
add
en (
1970
)
*Un
clea
r fr
om
his
tory
wh
eth
er is
ola
tio
n w
as f
rom
a c
linic
al c
ase
or
was
an
inci
den
tal f
ind
ing
.†N
ot
spec
ifie
d w
het
her
typ
ical
or
atyp
ical
A. s
alm
on
icid
a b
ut
assu
med
to
be
typ
ical
.
346 M. Hiney and G. Olivier
can be high and have been reported to reach 93% to 40% during the egg to smoltstages (St Jean, 1992). In the experience of these authors, furunculosis can occurin Atlantic salmon alevin whose yolk sacs are still attached.
Seasonal variation in the incidence of infectionThere are predisposing factors, other than age and inherent susceptibility, thatare associated with the precipitation of clinical furunculosis in hatchery stocksthroughout the year. These include physical damage, poor water quality,presence of ectoparasites and other diseases, diet and physical and psychologicalstresses, such as grading, tagging, injection and netting (Olivier, 1997;Pickering, 1997). However, a marked seasonality in the incidence of bothclinical and covert furunculosis infections has been observed in hatchery stocksand wild populations. In hatchery stocks, both smolting and high watertemperatures have been implicated in this apparent seasonality. The period ofsmolting is associated with major physiological changes, including chroniccortisol elevation, which can bring about severe depression of the fish’s defencesystem and increased susceptibility to bacterial infections (Maule et al., 1986;Pickering, 1997). In addition, high water temperatures (12–15°C) in late spring–early summer increase the likelihood of furunculosis outbreaks in both freshwater and sea water (Klontz and Wood, 1972; Johansson, 1977; Novotny, 1978).In fact, Malnar et al. (1988) would contend that high temperature is the majorfactor influencing the development of furunculosis. High water temperature notonly influences the stress response of fish but may act at the level of thepathogen (Groberg et al., 1978), and it has been demonstrated that the growth of
Table 10.3. Marine non-salmonid species from which typical Aeromonassalmonicida have been isolated (after Bernoth, 1997a).
History ofCommon 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 anincidental finding.
Fish species
347Furunculosis
A. salmonicida in the blood of cherry salmon (Oncorhynchus masou) correlatedpositively with water temperatures in the range 5–20°C (Sako and Hara, 1981).Not surprisingly, the seasonal nature of clinical infection by A. salmonicida isnot confined to hatchery stocks. As early as 1926, Horne observed that theincidence of furunculosis in a riverine population of brown trout first appearedtowards the end of May and declined in October (Horne, 1928). Blake andClarke (1931) observed that spawning in salmon rendered them susceptible tofurunculosis. There is no reason to suspect that the temperature effects observedin hatchery reared stocks will not also apply to wild stocks. The influence ofspawning on increased susceptibility to furunculosis would appear to be similarin a wide range of salmonids (Nomura et al., 1993) and includes chronic cortisolelevation and associated lymphocytopaenia (Pickering, 1986), decline in anti-body production (Yamaguchi et al., 1980) and immunosupression associatedwith gonadal steroids (Slater and Schreck, 1993). The presence of wildspawning fish in the vicinity of freshwater hatcheries may also have an impacton the seasonality of furunculosis in stocks contained within those hatcheries.
The occurrence of covert A. salmonicida infections in hatchery populationshas also been observed to be seasonal (Jensen and Larsen, 1980; Scallan andSmith, 1984, 1993; Hiney, 1994). However, neither smolting, spawning nor highwater temperatures can explain other peaks in the incidence of clinical andcovert infections observed by these authors at a number of freshwater hatcheriesand supported by anecdotal evidence from the industry. Scallan (1983)suggested that these peaks may result from the stress induced in fish by both highwater temperatures and rapidly changing water temperature.
As a general rule, both clinical and covert furunculosis are more likely tooccur in smolting and spawning fish with the onset of higher water temperaturesin spring or during periods of rapid temperature change. However, it is importantto keep in mind that furunculosis outbreaks can also occur in very young fish(alevin and fry) and at temperatures as low as 2–4°C (Drinan, 1985).
Geographical distribution
At present, the geographical distribution of A. salmonicida subsp. salmonicida isalmost worldwide, including Japan (Miyazaki and Kubota, 1975) and themainland of Asia (Inglis et al., 1993), from where it was previously consideredto be absent (Fryer et al., 1988). The possible exceptions to this distribution areSouth America and New Zealand, from which reports of the isolation of A.salmonicida have yet to be made (Bernoth, 1997a). The introduction offurunculosis into Sweden in 1951 was reported by Wichardt et al. (1989) and itwas recognized in Norway in 1964 (Lunder and Håstein, 1990; Johnsen andJensen, 1994). This more recent identification of A. salmonicida in Scandinaviancountries has been tentatively traced to importation of live fish stocks, initiallyfrom other European countries (Egidius, 1987; Wichardt et al., 1989) and thenwithin Scandinavia (Rintamäki and Valtonen, 1991). To date, there have been noreports of ‘typical’ furunculosis in salmonids in Australia, despite many attemptsto 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 firstincident of infection of rainbow trout by an atypical A. salmonicida was noted byBoomker et al. (1984).
The widespread distribution of furunculosis is reflected by the fact that thedisease has never been listed by the Office Internationale des Epizooties (OIE)as one that merits special attention, being considered endemic in most countriesand capable of control (C. Michel, personal communication). Likewise, theEuropean 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 importationof stocks only with the approval of the Standing Veterinary Committee, whichmust 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 salmonicidaClassical furunculosis derives its name from the boil-like lesions observed byEmmerich and Weibel (1894) on the skin and in the musculature of infected fish.However, development of ‘furuncles’ on the dorsal body are the exception ratherthan the rule (Bernoth, 1997b) and, in the experience of this author, only occur inolder fish suffering from the chronic form of the disease. The clinicalmanifestations of furunculosis are often divided into peracute, acute andsubacute or chronic forms and will be discussed here under these headings(Table 10.4). It should be noted that clinical manifestations of more than oneform of the disease may be present in individual fish within a population(Bernoth, 1997b). Reviews on the macro- and microscopic features offurunculosis are given in Ferguson (1977), McCarthy and Roberts (1980),Frerichs and Roberts (1989), Armstrong (1992), Austin and Austin (1993) andBernoth (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 ofthe disease normally results in rapid death with little more that slightexophthalmus (McCarthy and Roberts, 1980; Frerichs and Roberts, 1989). Thegross pathology of peracute furunculosis is typical of a peracute septicaemia.Microcolonies can be observed histologically in a number of organs, with noinflammatory infiltration and only limited necrosis. McCarthy and Roberts(1980) considered that cardiac damage was the most likely cause of death inyoung fish.
ACUTE FURUNCULOSIS
In growing fish, furunculosis tends to occur in an acute form, which ismanifested clinically by a generalized bacterial septicaemia displaying the‘standard’ features (Table 10.4). As its name implies acute furunculosis is often
349Furunculosis
fatal in 2–3 days and, because of the short duration of the disease, furuncledevelopment is unusual. Fish with an acute infection show signs of ahaemorrhagic septicaemia, including bloody anal vents. Skin lesions may behaemorrhagic patches or blotches along the side or on the dorsal body surface,or, more typically, raised furuncles, which usually develop in the dermis ratherthan the hypodermis (Bernoth, 1997b).
SUBACUTE/CHRONIC FURUNCULOSIS
The chronic form of furunculosis is common in older fish and is probably theform first observed by Emmerich and Weibel (1894). In chronic cases, fish mayshow 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 observedduring the progress of a chronic infection and, where they do occur, more thanone lesion may be present. These furuncles may be large and, when ruptured, theviscous fluid may contain more necrotic material than furuncles found in acutecases (Bernoth, 1997b). For a description of the histopathology of furuncles, seethe reviews of McCarthy and Roberts (1980) and Frerichs and Roberts (1989).
INTESTINAL FURUNCULOSIS
A fourth form of furunculosis associated with low mortality, intestinalfurunculosis, has been described by Amlacher (1961) (cited in Austin andAustin, 1993). The only clinical sign of this form of the disease was prolapseof the anus, although examination revealed haemorrhage and intestinalinflammation.
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). Theepizootiological importance of fish with covert furunculosis in the maintenanceand spread of the disease within and between susceptible fish populations wasunderstood by early workers. In 1935 the Scottish Furunculosis Committeeconcluded that clinically unapparent infections by A. salmonicida could persistin fish populations, that these infections could be latent and that clinicalfurunculosis could be precipitated in covertly infected fish populations by stress.In addition, fish with covert infections were capable of acting as carriers andcould shed sufficient bacteria to transmit the infection to other fish (Mackie etal., 1930, 1933, 1935). However, despite the large body of work that exists oncovert furunculosis, a number of important questions on the nature, persistence,location, lack of clinical signs, host defence mechanisms and transmission ofcovert 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 havebeen performed have employed a variety of methods, of differing efficiency, to
350 M. Hiney and G. Olivier
Tab
le 1
0.4
.D
iag
no
sis
of
clin
ical
infe
ctio
ns
of
typ
ical
A. s
alm
on
icid
a ae
tio
log
y (d
ocu
men
ted
sig
ns)
.
Typ
e o
f cl
inic
al in
fect
ion
Per
acu
teA
cute
Su
bac
ute
/Ch
ron
ic
Ag
e o
f fi
shV
ery
you
ng
fis
hG
row
ing
fis
hU
sual
ly o
lder
fis
hC
linic
al s
ign
sD
arke
nin
g o
f sk
in; t
ach
ybra
nch
iaD
arke
nin
g o
f sk
in; i
nap
pet
ence
;S
ligh
t d
arke
nin
g o
f sk
in; i
nap
pet
ence
;(r
apid
bre
ath
ing
); s
ligh
tle
thar
gy
(slu
gg
ish
nes
s);
leth
arg
y; c
on
ges
ted
blo
od
ves
sels
at
exo
ph
thal
mu
s (p
op
-eye
d)
tach
ybra
nch
ia; s
mal
lb
ase
of
fin
s; s
ligh
t ex
op
hth
alm
us;
hae
mo
rrh
ages
at
bas
e o
f fi
ns
exp
ress
ion
of
sero
san
gu
ineo
us
flu
idfr
om
nar
es a
nd
ven
t; m
ay h
ave
pal
e o
r co
ng
este
d g
ills;
may
hav
efu
run
cles
on
fla
nk
or
do
rsal
su
rfac
eG
ross
pat
ho
log
yD
ilate
d b
loo
d-v
esse
ls; p
un
ctat
eH
yper
aem
ia o
f se
rosa
lG
ener
al v
isce
ral c
on
ges
tio
n a
nd
hae
mo
rrh
ages
in p
arie
tal a
nd
surf
aces
; pu
nct
ate
per
ito
nit
is; m
ult
iple
hae
mo
rrh
ages
invi
scer
al p
erit
on
eum
an
d o
ver
hae
mo
rrh
ages
sca
tter
ed o
ver
mu
scle
an
d li
ver;
sp
len
om
egal
y;m
yoca
rdiu
m; m
ay h
ave
foca
lab
do
min
al w
alls
, vis
cera
an
dn
ecro
tic
kid
ney
; ad
hes
ion
s b
etw
een
hae
mo
rrh
ages
in g
ills
hea
rt; s
oft
an
d f
riab
le o
rvi
scer
a an
d b
etw
een
vis
cus
and
liqu
efie
d k
idn
ey; e
nla
rged
,ab
do
min
al c
avit
y; in
test
inal
cher
ry-r
ed s
ple
en w
ith
ro
un
din
flam
mat
ion
; sep
tic,
nec
roti
c an
ded
ges
; pal
e liv
er w
ith
hae
mo
rrh
agic
mu
scle
lesi
on
ssu
bca
psu
lar
hae
mo
rrh
ages
or
(fu
run
cles
)m
ott
led
ap
pea
ran
ce; s
tom
ach
and
inte
stin
e vo
id, m
ay c
on
tain
slo
ug
hed
ep
ith
eliu
m, m
ucu
san
d b
loo
d; s
wim
-bla
dd
er w
all
clo
ud
ed a
nd
hyp
erae
mic
;h
aem
orr
hag
ic p
atch
es a
lon
gb
od
y si
de
or
rais
ed f
uru
ncl
esin
der
mis
351Furunculosis
His
top
ath
olo
gy
Sm
all b
acte
rial
co
lon
ies
inP
rin
cip
al b
acte
rial
fo
cus
may
Hea
rt a
nd
sp
leen
mo
st in
fect
edb
ran
chia
l mes
ench
yme,
be
in a
ny
sin
gle
org
an o
r b
eo
rgan
s; m
icro
colo
nie
s in
vas
cula
rm
yoca
rdiu
m, a
nte
rio
r ki
dn
eym
ult
iply
loca
ted
; to
xic
end
oth
elia
; may
be
mas
sive
and
sp
leen
; lim
ited
loca
lized
hae
mat
op
oie
tic
nec
rosi
s;d
estr
uct
ion
of
sple
en e
llip
soid
s,n
ecro
sis;
car
dia
c d
amag
e as
myo
card
ial a
nd
ren
al t
ub
ula
rre
sult
ing
in v
ascu
lar
colla
pse
;p
oss
ible
cau
se o
f d
eath
deg
ener
atio
n; f
oca
l hep
atic
dam
age
to s
ple
en e
llip
soid
s m
ay b
en
ecro
sis;
init
ial l
esio
ns
inac
com
pan
ied
by
reti
cula
r ce
llg
ills
may
cau
se la
mel
lar
pro
lifer
atio
n a
nd
lym
ph
ocy
teth
rom
bo
sis
accu
mu
lati
on
; deg
ener
atio
n o
fca
rdia
c ve
ntr
al e
pic
ard
ium
fib
rin
oid
and
co
llag
en; m
arke
d in
flam
mat
ion
of
epic
ard
ium
; ret
icu
lin a
nd
co
llag
end
amag
e in
sp
leen
an
d h
eart
; to
xic
card
iac
nec
rosi
s, e
spec
ially
of
atri
allin
ing
Ou
tco
me
ifM
ay d
ie r
apid
lyM
ay d
ie w
ith
in 2
–3 d
ays
Low
mo
rtal
ity,
hea
led
fu
run
cles
may
infe
ctio
nle
ave
scar
tis
sue
352 M. Hiney and G. Olivier
detect covert infections. In addition, the variety of names that have been appliedto these infections makes it extremely difficult to present comparisons of thesestudies (Hiney et al., 1997b). In an effort to clear up some of the confusion thatsurrounds both the nomenclature and the exact nature of what is being studied,Hiney et al. (1997b) proposed a number of alternative definitions of clinicallyunapparent infections, strictly related to the diagnostic methods that have beenused. Using this approach, three categories could be identified and all studies oncovert infections were reassigned to one of these three categories:
1. Covert infection: demonstration of A. salmonicida, its antigens or itsdeoxyribonucleic acid (DNA) in, or on, a fish that manifests no clinical signs offurunculosis.2. (Covert) carrier infection: demonstration of the shedding of A. salmonicidaor its antigens or DNA into the environment by a fish that manifests no clinicalsigns of furunculosis; demonstration of the ability of fish that manifest noclinical signs of disease to transmit, in cohabitation experiments, furunculosis tofish free of this disease.3. (Covert) stress-inducible infection (stress-inducible furunculosis (SIF)):demonstration of clinical disease following the stressing of a fish that manifestsno 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 ourunderstanding about the mechanisms of these types of infections. Whenconsidering covert infections, these clinically unapparent infections of fishmay, in fact, represent a number of infection types, which are mediated byfundamentally different processes (Hiney et al., 1997b). However, the under-lying processes of covert infection will remain unknown until we developmethods 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 ofA. salmonicida subsp. salmonicida in covertly infected fish. Convincingevidence 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 administrationof chloramine T and have isolated typical A. salmonicida from the mucusof apparently healthy fish (Cipriano et al., 1992, 1994, 1996a,c). Thepathogenicity of mucus-isolated A. salmonicida has been demonstrated byHiney et al. (1994), who could induce clinical furunculosis in disease-freebrown trout by injection with a mixture of mucus and gill scrapings collectedfrom Atlantic salmon with SIF.
An internal primary location of A. salmonicida in covertly infected fish hasbeen proposed by a number of authors (cited by Hiney et al., 1997b). However,the experimental protocols employed in many of these studies make meaningfulinterpretation of the results difficult. In particular, experiments that involved thetransport of fish would have been stressful and, when examined in the
353Furunculosis
laboratory, fish that originally had a commensal infection might be experiencingthe early phase of a clinical infection. Preliminary work carried out in Galway,Ireland, on the salmon intestinal mucosa suggests that bacteria resident in theintestine may breach this barrier following a brief transport stress. Thus,arguments for an internal location of A. salmonicida in fish must be viewed withcaution in the absence of detailed descriptions of the protocols employed forhandling and transport. Nomura and his coworkers in Japan have, however,presented evidence suggesting an internal location for A. salmonicida in covertlyinfected 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 isrelatively simple to understand if the location of A. salmonicida is external to thefish. The clinical signs of furunculosis are those of a systemic bacteraemia andare unlikely to be present where the bacterium is confined to the gills, mucus orintestine. If, however, the location is internal, then the absence of diseasebecomes much more difficult to explain. A number of studies have demonstratedthat the median lethal dose (LD50) of virulent A. salmonicida subsp. salmonicidastrains for salmonids is between 102 and >10 colony-forming units (cfu), wheninjected 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 Körting, 1992). However,Scallan (1983) could isolate over 106 cfu of A. salmonicida from the kidneys ofsalmonid mortalities within 4 days of their being stressed. Knowledge of thepossible mechanisms whereby A. salmonicida might avoid the fish’s internaldefence systems during a covert infection would facilitate an understanding ofthe processes underlying these infections. In practically the only attempt to offerany explanation for the temporary reduction of virulence that would appear to benecessary 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 providea favourable niche for L-forms, because of the high electrolyte concentration inthis 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 occurringA. salmonicida L-forms failed to produce disease, even after stressingexperimentally infected fish (McIntosh and Austin, 1990).
PERSISTENCE OF COVERT INFECTIONS
Although persistence of covert infections has frequently been suggested, there isno definitive evidence that covert infections of individual fish can persist forextended periods. Early furunculosis research demonstrated the existence ofpersistent covert infections (Plehn, 1911), with no doubt that the duration ofthese infections had major epizootiological significance (Mackie et al., 1935). Astudy by Scallan (1983), involving the regular sampling of a population, wouldsuggest that high frequencies of stress-inducible infections may persist in suchpopulations 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, thepossibility of continual reinfections by A. salmonicida derived from the watercannot 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 natureand that there is, in theory, more than one form of covert infection, whoseunderlying 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 apopulation without obvious harm to the fish. Scallan (1983) also demonstrated,using a quantitative stress test, that the level of covert infection in a populationvaried throughout the year. Cipriano (1997) also reported that the numbers of A.salmonicida that could be isolated from the mucus of salmonids were variableover the growing season. Covertly infected fish may act as carriers of disease orbe latently infected. Both internal and external locations have been proposed forcovert infections, but, to date, the locations of A. salmonicida in fish sufferingfrom commensal covert infections have not been resolved.
The microorganism
Taxonomic classification of Aeromonas salmonicida subsp. salmonicidaAeromonas salmonicida is a member of the genus Aeromonas, which alsoincludes the mesophilic aeromonads (Popoff, 1984). The species has beendescribed by a number of different names since its original isolation, beingvariously called Bacillus der Forellenseuche, or bacillus of contagious troutdisease (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 coworkersrecommended the reclassification of B. salmonicida to the newly created genusAeromonas, as A. salmonicida, a taxonomic position the species has held to thisday. Since Aeromonas species share common properties with members of theEnterobacteriaceae, the Vibrionaceae and the Pseudomonadaceae, there hasbeen some controversy over the classification of these species within the familyVibrionaceae. Molecular genetic studies have led to proposals that the genusAeromonas be placed in a new family, Aeromonadaceae (Colwell et al., 1986).
There is agreement that A. salmonicida is the only psychrophilic member ofthe genus Aeromonas. Within this group, a number of subspecies have beenproposed, which have been traditionally referred to as ‘typical’ and ‘atypical’ A.salmonicida. There has been much confusion about what actually constitutes atypical or atypical A. salmonicida strain. McCarthy (1978) proposed a functionalsplit of A. salmonicida subspecies based on the host from which they wereisolated and the associated pathology. Other workers, reviewed in a later section,have favoured separating A. salmonicida subspecies by phenotypic/genotypic
355Furunculosis
characteristics. Guidelines for subspecies separation of the psychrophilicaeromonads are presented in Table 10.5. Within these guidelines, A. salmonicidasubsp. salmonicida, associated with classical furunculosis, is termed ‘typical’,while all isolates that do not fit this description are termed ‘atypical’. Thetaxonomy of typical A. salmonicida will be discussed in this section, while alater section presents a discussion of the taxonomy of ‘atypical’ strains.
The most striking taxonomic feature of A. salmonicida subsp. salmonicidastrains is their homology. Taxonomic studies conclude that this subspecies is acompact phenotypic and genotypic group, with a relationship to the mesophilicaeromonads at the genetic level, unlike ‘atypical’ A. salmonicida, which are ahighly 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; O’hIci et al., 1998). The homogeneity of typical A.salmonicida strains creates problems for substrain identification, which couldprove useful for improving our understanding of both the ecology of typical A.salmonicida and the epizootiology of classical furunculosis. Techniques basedon phenotypic or immunological expression, which have found application inepidemiological studies of other organisms, have not proved useful fordifferentiating between strains of A. salmonicida subsp. salmonicida (Table10.6). A number of newer techniques, based on the heterogeneity of the geneticmaterial contained within bacterial cells, have also been applied to thedifferentiation 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 subtypingtechniques can provide valuable information about the origin of an epizootic offurunculosis in a region, the impact of fish movements between freshwater andsea-water sites and the relative success of disease control strategies employed.Because so much is still unknown about the epizootiology of furunculosis andthe ecology of typical A. salmonicida, the search will continue for othersubtyping 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 andLallier, 1984) and produces cytochrome oxidase, although isolation of anoxidase-negative ‘typical’ A. salmonicida has been reported from coho salmon(Teska et al., 1992). Staining has a tendency to be bipolar, and the organismmeasures approximately 1.0 µm × 2.0 µm, varying morphologically from analmost coccoid form in freshly isolated cultures to distinct rods in culturesmaintained on artificial media, the latter often proving avirulent. Aeromonas
356 M. Hiney and G. Olivier
Tab
le 1
0.5
.G
ener
al g
uid
elin
es f
or
sub
spec
ies
sep
arat
ion
of
the
psy
chro
ph
ilic
aero
mo
nad
s.
Pro
per
ties
Do
min
ant
Gen
etic
Bio
chem
ical
Des
ign
atio
nS
ub
spec
ies
Dis
trib
uti
on
ho
stP
ath
olo
gy
pro
file
*p
rofi
le*
Typ
ical
salm
on
icid
aA
lmo
st w
orl
dw
ide
Sal
mo
nid
sC
lass
ical
fu
run
culo
sis
Ho
mo
gen
eou
sH
om
og
eneo
us
Aty
pic
alm
aso
uci
da
Jap
anS
alm
on
ids
Oth
er p
ath
olo
gie
s–
–ac
hro
mo
gen
esE
xten
sive
No
n-s
alm
on
ids/
salm
on
ids
Oth
er p
ath
olo
gie
sH
eter
og
eneo
us
Het
ero
gen
eou
sn
ova
Ext
ensi
veN
on
-sal
mo
nid
s/sa
lmo
nid
sO
ther
pat
ho
log
ies
Het
ero
gen
eou
sH
eter
og
eneo
us
smit
hia
En
gla
nd
(m
ost
ly)
No
n-s
alm
on
ids
Oth
er p
ath
olo
gie
s–
–
*In
suff
icie
nt
dat
a av
aila
ble
on
so
me
sub
spec
ies
to s
uff
icie
ntl
y ev
alu
ate
pro
file
.
357Furunculosis
Tab
le 1
0.6
.S
ub
stra
in id
enti
fica
tio
n t
ech
niq
ues
ap
plie
d t
o t
he
dif
fere
nti
atio
n o
f A
ero
mo
nas
sal
mo
nic
ida
sub
sp. s
alm
on
icid
a.
Exp
ress
ion
Tech
niq
ue
Bas
is o
f te
chn
iqu
eR
esu
ltR
efer
ence
Ph
eno
typ
icA
nti
bio
gra
m t
ypin
gA
nti
bio
tic
resi
stan
ceR
esu
lts
inco
nsi
sten
t. T
oo
Dal
sgaa
rd e
t al
. (19
94);
Bar
ker
pro
file
s ex
pre
ssed
as
dep
end
ent
on
his
tory
of
and
Keh
oe
(199
5)ab
ility
to
gro
w o
n s
olid
anti
bio
tic
exp
osu
rem
edia
aro
un
d a
nan
tib
ioti
c co
nta
inin
g d
isc
Bio
typ
ing
Sco
rin
g o
f a
wid
e ra
ng
e o
fN
ot
suff
icie
ntl
yS
mit
h (
1963
); S
chu
ber
tb
ioch
emic
al a
nd
cu
ltu
ral
dis
crim
inat
ory
(197
4); D
alsg
aard
et
al.
reac
tio
ns
(199
4); H
änn
inen
et
al.
(199
5)M
ult
ilocu
s en
zym
eS
cree
nin
g o
f ra
nd
om
lyFo
un
d o
nly
on
eB
oyd
et
al. (
1994
)el
ectr
op
ho
resi
sse
lect
ed c
yto
pla
smic
elec
tro
ph
ore
tic
typ
een
zym
es f
or
vari
atio
nco
rres
po
nd
ing
to
typ
ical
refl
ecti
ng
str
uct
ura
l gen
eA
. sal
mo
nic
ida
vari
atio
nP
hag
e ty
pin
gS
urf
ace
rece
pto
rs a
llow
Use
ful f
or
dif
fere
nti
atio
nP
op
off
(19
71);
Ro
dg
ers
et a
l.b
ind
ing
by
spec
ific
vir
use
so
f g
eog
rap
hic
ally
dis
tin
ct(1
981)
; Oliv
ier
(199
2)an
d s
elec
tive
infe
ctio
n a
nd
stra
ins
lysi
s o
f ce
llsP
seu
do
mo
nas
Dif
fere
nti
al in
hib
itio
n o
fR
esu
lts
dif
ficu
lt t
oR
ud
den
an
d S
mit
h (
1995
)in
hib
ito
ry a
ssay
A. s
alm
on
icid
a o
n s
olid
inte
rpre
t. N
ot
med
ium
by
a p
anel
of
dis
crim
inat
ory
en
ou
gh
Pse
ud
om
on
as s
pp
.
Co
nti
nu
ed o
ver
358 M. Hiney and G. Olivier
Imm
un
olo
gic
alLP
S a
nti
bo
dy
bin
din
gA
nti
gen
ic v
aria
tio
n in
No
t su
ffic
ien
t LP
SE
zura
et
al. (
1980
); C
har
t et
al.
A. s
alm
on
icid
a LP
San
tig
enic
het
ero
gen
eity
(198
4); R
ock
ey e
t al
. (19
91)
det
ecte
d b
y b
ind
ing
of
in A
. sal
mo
nic
ida
to b
esp
ecif
ic m
on
ocl
on
alu
sefu
lan
tib
od
ies
Ser
oty
pin
gM
icro
sco
pic
slid
eFe
w s
ero
log
ical
Pat
erso
n e
t al
. (19
80);
Hah
nel
agg
luti
nat
ion
an
dd
iffe
ren
ces
fou
nd
et a
l. (1
983)
; Ber
no
th a
nd
cro
ss-a
bso
rpti
on
of
Bö
hm
(19
88);
Dal
sgaa
rdA
. sal
mo
nic
ida
wit
het
al.
(199
4)an
tise
rum
rai
sed
ag
ain
std
iffe
ren
t st
rain
sG
enet
icP
lasm
id p
rofi
ling
Gen
erat
ion
of
pro
file
by
Pla
smid
pro
file
s o
fTo
ran
zo e
t al
. (19
83);
Bas
t et
al.
pla
smid
ext
ract
ion
an
dA
. sal
mo
nic
ida
too
(198
8); N
iels
en e
t al
. (19
93);
sizi
ng
of
pla
smid
s b
y g
elu
nif
orm
to
be
Hän
nin
en e
t al
. (19
95)
elec
tro
ph
ore
sis
dis
crim
inat
ory
RA
PD
an
alys
isP
CR
am
plif
icat
ion
of
Res
ult
s d
iffi
cult
to
Hän
nin
en e
t al
. (19
95);
Miy
ata
po
lym
orp
hic
DN
A w
ith
ain
terp
ret.
May
hav
eet
al.
(199
5)p
anel
of
ran
do
m p
rim
ers,
pro
mis
e b
ut
very
sizi
ng
of
pro
du
cts
by
gel
un
rep
rod
uci
ble
at
pre
sen
tel
ectr
op
ho
resi
sR
EF
anal
ysis
En
zym
e re
stri
ctio
n o
f to
tal
Use
fuln
ess
limit
ed b
yM
cCo
rmic
k et
al.
(199
0)ce
llula
r D
NA
siz
ing
of
gen
etic
ho
mo
gen
eity
of
frag
men
ts b
y g
elA
. sal
mo
nic
ida
elec
tro
ph
ore
sis
Tab
le 1
0.6
.C
on
tin
ued
Exp
ress
ion
Tech
niq
ue
Bas
is o
f te
chn
iqu
eR
esu
ltR
efer
ence
359Furunculosis
RFL
P/D
NA
pro
be
anal
ysis
En
zym
e re
stri
ctio
n o
fC
ou
ld d
etec
t n
oH
enn
igan
et
al. (
1989
)p
oly
mo
rph
ic D
NA
an
dp
oly
mo
rph
ism
sh
ybri
diz
atio
n w
ith
a p
anel
of
pro
bes
tar
get
ed a
gai
nst
A. s
alm
on
icid
aR
ibo
typ
ing
En
zym
e re
stri
ctio
n o
fLo
w d
iscr
imin
ato
ry p
ow
erN
iels
en e
t al
. (19
94);
Hän
nin
enrR
NA
gen
es a
nd
for
A. s
alm
on
icid
aan
d H
irve
lä-K
osk
i (19
95);
hyb
rid
izat
ion
wit
h a
Hän
nin
en e
t al
. (19
95)
suit
able
pro
be
LPS
, lip
op
oly
sacc
har
ide;
RA
PD
, ran
do
mly
am
plif
ied
po
lym
orp
hic
DN
A; r
RN
A, r
ibo
som
al r
ibo
nu
clei
c ac
id; R
FLP,
res
tric
tio
nfr
agm
ent
len
gth
po
lym
orp
his
m.
360 M. Hiney and G. Olivier
salmonicida is normally isolated from the kidney of infected fish, although it canalso 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 diameterafter 72 h incubation (Drinan, 1985). Temperatures of 18–22°C are optimal forgrowth. A. salmonicida grows poorly at 4°C and does not grow at 37°C(Snieszko, 1957), although isolation of an atypical strain capable of growth at37°C has been reported (Austin, 1993).
On agar media containing tryptone, typical A. salmonicida normally, thoughnot always (Wiklund et al., 1993), produces a brown, melanin-like, water-soluble pigment. This pigment and its production pathway have been describedin detail by Donlon et al. (1983). Virulent colonies are small and friable and, oninitial 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 optimumtemperatures, produces non-aggregating variants with altered cell morphology(Ishiguro et al., 1981). The hydrophobic nature of A. salmonicida is due to thepossession of an additional surface-protein layer (A-layer), first described byUdey 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 itsrole in resistance to host defence mechanisms (Sakai and Kimura, 1985;Secombes and Olivier, 1997). However, the detection of an intact A-layer inlaboratory culture cannot be used, in isolation, as a predictor of the virulence ofan isolate (Ellis et al., 1988; Olivier, 1990).
Diagnosis in clinically infected fish
The boil-like lesions observed by Emmerich and Weibel (1894), from whichclassical furunculosis derives its name, are the exception in clinical cases, ratherthan the rule (Bernoth, 1997b) and are normally only observed during chronicinfections in older fish. Table 10.4 summarizes the main clinical signs, grosspathology and histopathological features that have been described for thedifferent forms of typical furunculosis (peracute, acute, subacute/chronic). Thegross pathological signs of peracute and acute furunculosis in young fish areoften indistinguishable from other bacterial septicaemias on preliminaryexamination. The inexperienced diagnostician may also have difficulty indifferentiating the morphology and Gram-staining behaviour of A. salmonicidain fish tissue, as seen under a microscope, from other Gram-negative bacteriaoccurring in fish (Bernoth, 1997b). Thus, a firm diagnosis of clinical furun-culosis requires isolation of the dominant infecting organism on agar media andidentification by morphology, combined with either biochemical or serologicaltests, as A. salmonicida.
Detection of typical Aeromonas salmonicida during clinical infectionsUnder ordinary circumstances, typical A. salmonicida can be recovered fromclinically diseased fish, especially from the kidney and surface lesions, wherepresent (Austin and Austin, 1993). However, culturing samples from several
361Furunculosis
organs, rather than kidney alone, has been demonstrated to increase the detectionrate in fish populations where the incidence of infection is low (Bernoth, 1997b).The recommended diagnosis of the presence of A. salmonicida in clinicallydiseased fish is based on isolation of the organism from the kidney on eithertryptone soya agar (TSA) or brain–heart infusion agar (BHIA) (Department ofFisheries and Oceans, 1984; Shotts, 1984). The brown water-soluble pigmentproduced by typical A. salmonicida on TSA after 2–4 days’ incubation at20–25°C is used as a presumptive identification. However, caution must beexercised when pigmentation on TSA is used as a presumptive identification ofA. salmonicida. Other bacteria have also been found to produce a browndiffusible pigment on TSA, such as mesophilic aeromonads and Pseudomonasfluorescens (McCarthy, 1975; Frerichs and Holliman, 1991). Neither TSA norBHIA is selective for A. salmonicida, allowing the growth of competingorganisms, which may inhibit pigmentation or the growth of A. salmonicida(Austin and Austin, 1993). Inhibition of growth may result from the ability offaster-growing organisms to sequester the available nutrients in the medium(particularly iron) or from the production of inhibitory substances by thesecompeting 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 theseand other authors to aid in the preliminary differentiation of A. salmonicida fromcompeting bacteria (Cipriano and Bertolini, 1988; Markwardt et al., 1989;Cipriano et al., 1992). On this medium, A-layer-positive A. salmonicida coloniesstain deep blue to navy and can be easily distinguished, the intensity of stainingbeing dependent on the source of the dye and the batch of TSA. However, CBBAcannot be totally relied upon, because bacteria other than A. salmonicida canproduce 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 bacteriathat need to be screened to ensure definitive identification.
Occasional failure to isolate typical A. salmonicida from diseased fish withmacroscopic and histological signs of furunculosis has been noted. There are anumber 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 cellspresent in the original sample may be below the lower detection limit of culturalisolation (Bernoth, 1997b). Attempts to overcome this limitation, by incorpor-ating a pre-enrichment step, carried out in liquid media, prior to plating on solidmedia, were reported by Daly and Stevenson (1985). Pre-enrichment of kidneysamples in tryptone soya broth (TSB) for 48 h more than doubled the A.salmonicida detection rate in a fish population undergoing a furunculosisepizootic. A second reason for lack of growth might be the unsuitability oflaboratory media, such as TSA and BHIA, to support the growth of A.salmonicida. Little is known about the specific nutrient requirements of typicalA. salmonicida, other than that it requires methionine and arginine (Nerland etal., 1993). Most artificial media have been formulated for the isolation ofmedically important bacteria and do not, therefore, present an ideal environmentfor terrestrial and aquatic organisms. Another potential problem with the use ofTSA 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 thispossibility, the Galway laboratory now routinely checks the ability of each TSAbatch to support the growth of a positive control A. salmonicida.
Biochemical identification of typical Aeromonas salmonicidaPresumptive identification of typical A. salmonicida colonies by pigmentationon TSA or dark blue to navy staining on CBBA after 2–4 days’ incubation andgrowth at 20–25°C but not 37°C is not sufficient to make a firm diagnosis offurunculosis, 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, incombination with a limited number of biochemical tests, should be sufficient toconfirm the identity of an isolate as typical A. salmonicida and to exclude othermembers of the Vibrionaceae, Enterobacteriaceae and Pseudomonadaceae. Adiagnostic protocol to confirm the identity of typical A. salmonicida is presentedin Table 10.7. More extensive tables of biochemical tests for A. salmonicidacharacterization are presented elsewhere (Austin and Austin, 1993; Munro andHastings, 1993), but performance of these tests is usually unnecessary, unless itis suspected that the isolate is an atypical A. salmonicida. A number ofcommercial rapid test kits for biochemical identification, such as API-BioMerieux and Biolog, have become available. These test systems weredeveloped for bacteria of medical importance and function best at incubationtemperatures of 30–37°C. Their application to the biochemical characterizationof A. salmonicida has been reported to generate inconsistent results followingincubation at lower temperatures (Bernoth, 1997b). However, incubation of A.salmonicida at 30°C, although above its optimal growth temperature (Snieszko,1957), will generate consistent results.
Serological identification of typical Aeromonas salmonicidaAeromonas salmonicida subsp. salmonicida requires at least 48 h incubation toproduce colonies suitable for morphological and biochemical identification and,as a result, diagnosis of furunculosis may take up to 1 week. This oftenrepresents an unacceptable time-lag for the fish farmer or veterinarian, whoneeds to make rapid decisions on the treatment and fate of infected fish. Rapididentification methods that could be applied directly to colonies after 48 h havethe potential to overcome the current delays in identifying presumptive A.salmonicida. Bernoth and Artz (1989) suggested that serological tests may bemore sensitive than cultural isolation for the detection of A. salmonicida in fishtissue. A comprehensive list of studies on the development of serologicalidentification tests for typical A. salmonicida has been presented by Bernoth(1997b). She also provides a discussion of the technical difficulties that may beencountered, 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 ininfected tissue, they can facilitate more rapid confirmation of presumptive A.salmonicida subsp. salmonicida colonies on agar plates (Bernoth, 1997b).
363Furunculosis
Diagnosis of covertly infected fish
Diagnosis of covert A. salmonicida subsp. salmonicida infections poses anumber of important problems for the diagnostician, veterinarian, fish healthworker or regulator. As discussed by Bernoth (1997b), disease diagnosis in fishmust be understood as diagnosis in a population, rather than in an individual fish,and therefore the method of sampling becomes an important diagnosticconsideration when covert furunculosis is suspected. The first samplingconsideration must be the number of fish that should be sampled in order toreflect the health status of the population as a whole. Ossiander and Wedemeyer(1973) have published sampling tables which specify statistically relevantnumbers of fish that should be sampled from populations of a given size. Thesetables have been incorporated into regulations laying down the sampling plansand diagnostic methods for the detection and confirmation of fish diseases by theEuropean Commission (Commission Decision 92/532/EEC), the USA(Department of the Interior, 1993; Thoesen, 1994) and Canada (Department ofFisheries and Oceans, 1984). However, Bernoth (1997b) was sceptical about thereliability of such sampling tables in situations where the prevalence of covertinfection is low, but acknowledged that, in the absence of any detailedepizootiological statistics, such recommendations will remain, for the moment,our ‘best guess’. Even if statistically representative numbers of fish from a fishpopulation are sampled, detection of covert furunculosis remains problematic. Anumber of approaches for the detection of covert infections have been presentedover the history of the disease. Hiney et al. (1997b) grouped these methodsaccording to what they demonstrated about the infection, i.e. the ability ofinfected 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 fishor the precipitation of clinical furunculosis following the application of stress.
Demonstration of shedding and transmission of typicalAeromonas salmonicida
The shedding of A. salmonicida by covertly infected fish can be demonstratedindirectly, by cohabitation studies, or directly, by detection of the organism inthe environment of such fish. Cohabitation studies have been used by manyauthors to demonstrate shedding and transmission of furunculosis. A number ofdifferent transmission scenarios have been reported, including transmission ofclinical furunculosis from clinically infected fish to healthy fish, transmission ofclinical furunculosis from covertly infected fish to healthy fish, transmission ofcovert furunculosis from clinically infected fish to healthy fish and transmissionof 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 diagnosticstrategy for detection of covert infections.
Direct shedding of A. salmonicida into the environment of subclinically orclinically infected fish has been demonstrated by culture methods (Scallan,1983; Ford, 1994; Cipriano et al., 1996a), using immunological assays (Engerand Thorsen, 1992; Gilroy and Smith, 1995) and polymerase chain reaction
364 M. Hiney and G. Olivier
Tab
le 1
0.7
.C
on
firm
ato
ry id
enti
fica
tio
n o
f ty
pic
al A
ero
mo
nas
sal
mo
nic
ida.
Test
Ch
arac
teri
stic
/res
ult
Cav
eats
/co
mm
ents
Mo
rph
olo
gic
al t
ests
Gro
wth
on
TS
A o
r B
HIA
Bro
wn
dif
fusi
ble
pig
men
t; s
mal
l, fr
iab
leP
igm
ent
may
be
inh
ibit
ed b
y o
ther
bac
teri
a;co
lon
ies
afte
r 2–
4 d
ays’
incu
bat
ion
at
18–2
2°C
colo
ny
size
var
ies
fro
m 0
.5 t
o 3
mm
aft
er 7
2 h
incu
bat
ion
Gro
wth
on
CB
BA
Dar
k b
lue
to n
avy
stai
nin
g; s
mal
l, fr
iab
leO
ther
bac
teri
a m
ay a
lso
sta
in d
ark
blu
e; A
-lay
er-
colo
nie
s af
ter
2–4
day
s’ in
cub
atio
n a
t 18
–22°
Cn
egat
ive
iso
late
s m
ay n
ot
stai
n
Gro
wth
tem
per
atu
reG
row
th a
t 18
–25°
C; n
o g
row
th a
t 37
°CS
edim
enta
tio
n t
est
Cel
ls a
uto
agg
luti
nat
e in
0.8
5% P
BS
Au
toag
glu
tin
atio
n a
lso
occ
urs
in b
roth
Gra
m s
tain
Sh
ort
Gra
m-n
egat
ive
rod
s; s
tain
ing
ten
ds
to b
eb
ipo
lar
Mic
rosc
op
yN
on
-mo
tile
; alm
ost
co
cco
id r
od
s; t
end
ing
to
agg
reg
ate
Han
gin
g d
rop
tes
tN
on
-mo
tile
Bio
chem
ical
tes
tsC
yto
chro
me
oxi
das
eO
xid
ase
po
siti
veO
xid
ase
neg
ativ
e is
ola
tes
hav
e b
een
des
crib
edp
rod
uct
ion
(Ch
apm
an e
t al
., 19
91);
tes
t co
lon
ies
fro
m T
SA
on
lyC
atal
ase
pro
du
ctio
nC
atal
ase-
po
siti
veTe
st c
olo
nie
s fr
om
TS
A o
nly
Oxi
dat
ion
/fer
men
tati
on
Ferm
enta
tive
Incu
bat
e at
25°
C; c
hec
k af
ter
48 h
an
d 5
day
ste
st (
Hu
gh
/Lei
fso
n)
0/12
9 se
nsi
tivi
tyR
esis
tan
t o
n b
loo
d a
gar
Incu
bat
e at
25°
C; c
hec
k af
ter
48 h
an
d 5
day
s
365Furunculosis
Ser
olo
gic
al t
ests
Late
x ag
glu
tin
atio
nC
lum
pin
g (
sou
rin
g)
of
anti
bo
dy
coat
ed la
tex
Alw
ays
incl
ud
e p
osi
tive
an
d n
egat
ive
con
tro
lin
so
luti
on
wit
h A
. sal
mo
nic
ida
susp
ensi
on
stra
ins
bec
ause
of
po
ssib
le a
uto
agg
luti
nat
ion
of
A. s
alm
on
icid
aA
. sal
mo
nic
ida-
targ
eted
Co
lou
r re
spo
nse
fro
m b
ind
ing
of
con
jug
ated
Alw
ays
incl
ud
e p
osi
tive
an
d n
egat
ive
con
tro
lE
LIS
Aan
tib
od
y to
A. s
alm
on
icid
a an
tig
en(s
)st
rain
s
ELI
SA
, en
zym
e-lin
ked
imm
un
oso
rben
t as
say.
366 M. Hiney and G. Olivier
(PCR)/DNA probe assays (Gustafson et al., 1992; O’Brien et al., 1994). Culture-based detection of A. salmonicida in the environment has traditionally been seenas problematic (Cornick et al., 1969). However, the success of Ford (1994) andCipriano et al. (1996a), using dilution filtration and CBBA, in isolating typicalA. salmonicida from hatchery water suggests that these methods are promisingin routine monitoring of hatchery water-supplies (Cipriano, 1998). None theless, the use of culture-based techniques are complicated, as A. salmonicida mayenter a non-culturable-but-viable state once it has been shed into theenvironment (Roszak and Colwell, 1987; Enger, 1997). Therefore, techniquesthat do not rely on culture, such as PCR and enzyme-linked immunosorbentassay (ELISA), have received considerable attention and may have the potentialto overcome the problems encountered by culture-based techniques. However,these non-culture-based detection techniques present significant problems ofinterpretation 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 orits components
The difficulty of isolating A. salmonicida or its components (antigens, DNA)from covertly infected fish, in terms of not only the lack of selectivebacteriological methods (Cornick et al., 1969; Gustafson et al., 1992) but alsothe reliability of current diagnostic techniques (Inglis et al., 1993; Crane andBernoth, 1996; Hiney and Smith, 1998) and the time-consuming nature of someof these techniques, has been discussed (Austin and Austin, 1993). The problemof deciding which organs of the fish should be examined is central to all methodsthat fall into this group. However, as discussed, the location of A. salmonicida incovertly infected fish remains uncertain.
Attempts to culture A. salmonicida from the internal organs of fishsuspected of covert infection constitute the method used by many authors.However, there has been some disagreement about precisely which organ ororgans 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 ofcovert infections (Mackie et al., 1935; Mackie and Menzies, 1938; McCarthy,1977a), but others have demonstrated that examination of a number of organs ismore 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 thekidney.
The application of non-culture-based techniques to the detection ofcomponents of A. salmonicida in tissues of covertly infected fish has notresolved the issue of location in this infection type. In two studies which appliedan A. salmonicida-targeted ELISA to examination of the kidneys of similarsalmonid populations with SIF, A. salmonicida antigens were detected in onestudy (Rose et al., 1989) but not in the other (Hiney et al., 1994). Similarly, usingPCR amplification and A. salmonicida-targeted DNA probes, positive responseshave been detected in a variety of salmonid organs, including spleen and kidney(Gustafson et al., 1992; Høie et al., 1997) and blood (Mooney et al., 1995; Høie
367Furunculosis
et al., 1997). However, in none of these studies was covert infectiondemonstrated, and few successfully isolated the organism in primary culturefrom the samples tested, making it difficult to interpret the meaning of thesedata.
The use of external surfaces, including the intestine, as sampling sites whenattempting to detect typical A. salmonicida or its components has also beenreported. Although the intestine has been demonstrated as a site of colonizationof 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. Themain reason for this is rapid overgrowth by concomitant flora, whichcomplicates isolation of A. salmonicida (Bernoth, 1997b). None the less, theintestine is currently recommended as a sampling site for covertly infected fishin the UK (Munro and Hastings, 1993) and USA (Shotts, 1984). In the opinion ofthe present authors, this recommendation is misplaced. The difficultiesencountered with culture from the intestine make it unlikely that a covertinfection will be detected by this means. The use of non-culture-based detectiontechniques for A. salmonicida may have the potential to overcome thedifficulties of bacteriological culture from the intestine. Intestinal contents havebeen reported to yield positive results using both an A. salmonicida-targetedELISA 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 havebeen adequately validated for routine analysis they will remain research tools.
Perhaps the most extensive work on detection from external locations hasbeen carried out by Cipriano and coworkers. Using CBBA as a differentialmedium (Cipriano and Bertolini, 1988; Markwardt et al., 1989), theydemonstrated that A. salmonicida could be isolated from the external mucus ofhatchery-reared salmon (Cipriano et al., 1996a), hatchery trout (Cipriano et al.,1992, 1994) and feral salmon returning to spawn (Cipriano et al., 1996c) morefrequently 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 inrestoration programmes and when assessing valuable brood stock (Cipriano,1997).
Detection of covert furunculosis by the application of stressBacteriological culture from one or more organs may detect preclinicalinfections or covert infections present at high levels in a fish population, butculturing 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 18°C for 14 days, activated latentinfections in covertly infected fish and facilitated their detection. The stress testproposed by Bullock and Stuckey (1975) was demonstrated to be more efficientat 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 ofBullock 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 amethod for the bath administration of the corticosteroid prednisolone acetate tofish of 5 g or less, which might not survive injection administration. She alsodemonstrated that the amount of corticosteroid injected into fish was not acritical factor in precipitating overt disease, with concentrations over a fourfoldrange being effective. In a recent comparison of the stress test and culture-basedassays, Cipriano et al. (1997) found that the probability of detecting A.salmonicida in apparently healthy salmon and trout, where the prevalence ofcovert infection was assumed to be low, was 17 times greater using stress testingthan 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 ofdetecting A. salmonicida was 10 and 27 times greater for stress testing, ascompared with kidney and mucus culture, respectively (Cipriano et al., 1997). Itshould be noted that infections detected by the stress test of Bullock and Stuckey(1975) or its later modifications should properly be termed SIF, and therelationship between SIF and covert infections detected by other methods shouldbe considered unknown (Hiney et al., 1994).
Limitation on the use of the stress-inducible furunculosis test fordetection of covert furunculosis infections
In Ireland, reliance on stress testing is widespread at the level of individualcompanies and has been successful, when used, in limiting the spread offurunculosis to marine farms (Smith, 1992; Scallan and Smith, 1993). Stresstesting of salmon smolts prior to their transport to sea is a regulatory requirementin New Brunswick, Canada, and has also been found to be a successful measurethere (Olivier, 1992). However, although stress testing is valuable in the field, itis not without its problems. In the experience of the present authors, stresstesting tends to result in the isolation of mixed cultures from the kidney, makingidentification of A. salmonicida from this tissue difficult (Cornick et al., 1969).Pigmentation of A. salmonicida strains, often used as a preliminary confirmationof their presence, is inhibited in the presence of other organisms, requiringcareful examination and repeated subculture of kidney streaks to confirmdiagnosis (McCarthy, 1977a). Even so, the presence of A. salmonicida may bemissed (Scallan, 1983). No selective medium exists for the isolation of A.salmonicida from mixed cultures, although CBBA has been found by the presentauthors to simplify identification of putative A. salmonicida colonies isolatedfrom the kidneys of stress tested fish. Furthermore, the stress test is by definitionlethal. It requires 14–16 days for a definitive diagnosis and uses large numbers(150–200) of fish to ensure detection of the infection where prevalence is low.However, Bullock and Cipriano (1997) found, following sampling of the mucusand gills of stress-tested fish, that, from day 5 of the test onwards, A. salmonicidacould be detected in these samples, thus eliminating the need to continue the testto 14 days. This method may, therefore, represent a more rapid alternative to thecurrent stress test.
Prior vaccination of SIF-tested fish may complicate interpretation of theresults obtained from a stress test. Hiney (1995) demonstrated that, followingstress testing by the method of McCarthy (1997a), A. salmonicida could be
369Furunculosis
isolated from the kidney of Atlantic salmon and brown trout that had beenvaccinated against furunculosis. Attempts to culture A. salmonicida from thekidneys of parallel, unstressed, groups of salmon and trout were unsuccessful,confirming that the infection was of a stress-inducible nature. The results ofHiney (1995) suggested that vaccinated fish could become or remain covertlyinfected following vaccination and that the extent of protection provided byvaccination was not sufficient to prevent SIF. Hiney et al. (1997b) and Smith(1997) have suggested that covertly infected fish with increased systemicimmunity (i.e. vaccinated) might act as ‘immune carriers’. If such fish exist, thenthey will present a number of important questions for managers of fish farms andwild fisheries. For example, should potential ‘immune carriers’ be treated withantimicrobial agents immediately prior to transfer to a sea site or restocking intofresh water in order to eliminate any carried A. salmonicida? In theory, ‘immunecarriers’, while remaining disease-free, could act as a source of infection forunvaccinated fish. However, at present not enough is known about theinteractions 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 hasbeen the move to detect microorganisms directly in clinical samples or in theenvironment of the host, without the need to culture. In line with this generaltrend, non-culture-based microbial detection techniques are being increasinglydeveloped for the identification of typical A. salmonicida. Since 1990, inparticular, a number of genetic-based assays for A. salmonicida have beendescribed, and these are presented in Table 10.8, along with immunologicalassays for the organism developed during the same period. For a moreexhaustive list of immunological assays developed prior to 1990 readers arereferred to Bernoth (1997b). Although many of the techniques presented inTable 10.8 are intended for clinical diagnosis of furunculosis their potential fordetecting A. salmonicida in covertly infected fish and their environment hasalso been investigated. In theory, non-culture-based techniques can bedesigned to specifically detect low numbers of a target organism in a mixedmicrobial flora. In addition, because culturing of the target organism is not arequisite for its detection, they circumvent the numerous problems of loop-and-plate microbiology (Bernoth, 1997b; Pace, 1997). Non-culture-basedtechniques developed for the detection of A. salmonicida fall into two broadcategories, those based on immunological principles and those based ongenetic principles.
Immunological detection techniquesImmunological detection techniques offer many advantages over culture-basedtechniques, particularly when attempting to detect organisms, such as A.salmonicida, whose growth on laboratory media can be inhibited by the presenceof other bacteria. These techniques have been revolutionized in recent years by
370 M. Hiney and G. Olivier
Tab
le 1
0.8
.N
on
-cu
ltu
re-b
ased
tec
hn
iqu
es d
evel
op
ed f
or,
or
app
lied
to
, th
e d
etec
tio
n o
f ty
pic
al A
ero
mo
nas
sal
mo
nic
ida
sin
ce19
90.
Targ
et s
amp
le
Det
ecti
on
pri
nci
ple
Ass
ay t
ype
Clin
ical
En
viro
nm
enta
lR
efer
ence
Imm
un
olo
gic
alE
LIS
AK
idn
eyA
dam
s an
d T
ho
mp
son
(19
90)
ELI
SA
Kid
ney
Ber
no
th (
1990
b)
IFA
T, E
LIS
AK
idn
ey, l
iver
Lalli
er e
t al
. (19
90)
Imm
un
ofl
uo
resc
ence
Wat
er, s
edim
ent
En
ger
an
d T
ho
rsen
(19
92)
ELI
SA
Blo
od
Yosh
imiz
u e
t al
. (19
92)
ELI
SA
Kid
ney
, in
test
ine
Hin
ey e
t al
. (19
94)
Imm
un
od
ot-
blo
tV
ario
us
tiss
ues
Ban
nek
e an
d B
ern
oth
(19
94)
ELI
SA
Kid
ney
Sed
imen
tG
ilro
y an
d S
mit
h (
1995
)E
LIS
AK
idn
ey, i
nte
stin
eS
edim
ent
Hin
ey e
t al
. (19
97a)
ELI
SA
Kid
ney
, mu
cus,
gill
s, s
ple
enB
ullo
ck e
t al
. (19
98)
Gen
etic
16S
rD
NA
-PC
RP
ure
cu
ltu
reB
arry
et
al. (
1990
)D
NA
-PC
RS
ple
en, k
idn
eyFi
sh f
aece
s, t
ank
effl
uen
tG
ust
afso
n e
t al
. (19
92)
DN
A-P
CR
Pu
re c
ult
ure
Hin
ey e
t al
. (19
92)
DN
A-P
CR
Fres
hw
ater
mic
roco
smM
org
an e
t al
. (19
93)
DN
A-P
CR
Mar
ine
sed
imen
t, w
ater
Hin
ey (
1994
)D
NA
-PC
RH
atch
ery
effl
uen
tO
’Bri
en e
t al
. (19
94)
DN
A-P
CR
Blo
od
Mo
on
ey e
t al
. (19
95)
DN
A-P
CR
Fres
hw
ater
mic
roco
smP
icku
p e
t al
. (19
96)
16S
rD
NA
-PC
RK
idn
ey, s
ple
enH
øie
et
al. (
1996
)D
NA
-PC
RK
idn
eyM
iyat
a et
al .
(199
6)D
NA
-PC
RK
idn
ey, i
nte
stin
eFr
esh
wat
er s
edim
ent
Hin
ey e
t al
. (19
97a)
DN
A-P
CR
Kid
ney
, gill
sw
abs
Hø
ie e
t al
. (19
97)
DN
A-P
CR
Kid
ney
Jen
çiç
and
Pia
no
(19
97)
DN
A-P
CR
Kid
ney
, in
test
ine
Fres
hw
ater
sed
imen
tP
adle
y et
al.
(199
7)D
NA
-PC
RK
idn
eyS
øru
m e
t al
. (19
98)
IFA
T, in
dir
ect
flu
ore
scen
t an
tib
od
y te
st; r
DN
A, r
ibo
som
al D
NA
.
371Furunculosis
the introduction of monoclonal antibodies and ELISAs, which allow for morespecific assays that can be semiautomated. As a result, a number of ELISAs havebeen developed for screening of clinical samples for signs of A. salmonicida(Bernoth, 1997b; Table 10.8). In a comparison of ELISA and an indirectfluorescent antibody test (IFAT), similar to the fluorescent antibody microscopy(FAM) technique, Lallier et al. (1990) found that ELISA was more sensitive thanIFAT when tested on pure cultures of A. salmonicida and both methods werefound to be more sensitive than bacteriological culture. However, it has beenargued that the use of IFAT coupled with experience overcomes the problems oflesser specificity of this technique, making it comparable to ELISA (E.-M.Bernoth, CSIRO, 1995, personal communication). Perhaps the most usefulapplication of immunological assays would be in the detection of covert A.salmonicida infections, and a number of ELISA tests have been applied for thispurpose. 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 weresupported by Hiney et al. (1994), who found that ELISA examination of thekidney, mucus and intestine of covertly infected fish detected A. salmonicidaantigens in 45% of fish as compared with culture of the organism from thekidney 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 wasmore sensitive than ELISA for identification of A. salmonicida in fish that hadbeen stress-tested. Enger and Thorsen (1992) have reported on an IFAT whichwas applied to detection of A. salmonicida antigens in the environment of a fishfarm. None of these tests have, however, been applied successfully under truefield conditions, nor are they recommended with any conviction in diagnosticmanuals (Crane and Bernoth, 1996).
There are a number of problems to resolve when attempting to detectbacterial antigens in situ in tissue or environmental samples. Many of theimmunological assays developed do not appear to offer any greater sensitivity orreliability than conventional bacteriological methods (Inglis et al., 1993). Thelower limit of detection has been found to be 103 cells ml–1 or greater in bothclinical and environmental samples (Sakai et al., 1986; Adams and Thompson,1990; Bernoth, 1997b). A major problem with cross-reactivity of antisera orantibodies to epitopes expressed by ubiquitous motile Aeromonas species orother aquatic microorganisms has been reported, which requires thatconfirmatory bacteriological isolation of the pathogen be performed (Bernoth,1990b). Importantly, immunological assays do not differentiate between live anddead cells (Rose et al., 1989) and A. salmonicida-targeted ELISA has beenshown to generate positive results from the spleen and intestine of fish whichwere immunized with a killed whole-cell furunculosis vaccine (Gilroy andSmith, 1997). Another problem with immunological assays is that the antiserumraised against bacteria grown in vitro must detect the organism as it occurs in situin a clinical or environmental sample. For A. salmonicida, at least, cells grown invivo have been found to express novel antigens, including an antigenically newform of lipopolysaccharide (LPS), which were not induced under in vitro growthconditions (Garduño 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 techniquesA second family of non-culture-based detection techniques that have beeninvestigated for the detection of components of A. salmonicida is based ongenetic principles. Typical A. salmonicida is homogeneous at the genetic level(Vaughan, 1997) and presents, in theory, an ideal candidate for a genetic-baseddetection technique. Since 1990, a number of assays targeted against 16Sribosomal ribonucleic acid (rRNA) or DNA sequences of A. salmonicida havebeen developed for this organism (Table 10.8). Most of these assays also utilizethe ability of PCR to amplify the number of copies of the target sequence in asample between several thousand- and 1 millionfold, making detection ofinitially minute quantities of that sequence possible. However, it is difficult toestimate the true lower detection limits of PCR-based assays for A. salmonicidaDNA sequences, because the kinetics of PCR amplification are essentially non-linear and amplification is prone to variable inhibition by facets of both clinicaland environmental samples (Wilson, 1997).
Similar to immunological assays, the ultimate goal of genetic techniques isto specifically detect DNA sequences of A. salmonicida in the tissues orenvironment of both clinically and covertly infected fish. Clearly, then, the keyparameter of this type of technique is its specificity for the target organism.Because of the relative newness of genetic detection techniques in comparisonwith immunological techniques, there have been few reports to date about cross-reactivity of these techniques. None the less, Høie (1995) found that his PCR-based assay for an A. salmonicida DNA sequence cross-reacted with anuncharacterized aquatic organism, and Hiney and Smith (1998) have expressedconcern about the relevance of laboratory studies of specificity for fieldapplications. In addition, PCR-based techniques are prone to false-positiveresults through sample contamination, especially if a double cycle of PCRamplification is employed, so-called ‘nesting’ (Byers et al., 1997). As withimmunological assays, many PCR-based assays do not differentiate betweenlive and dead cells, and they have been demonstrated to detect DNA from killedwhole cells in the spleen and head kidney of fish vaccinated against furunculosis(Høie et al., 1996), suggesting that this type of assay is unsuitable for applicationto vaccinated stocks.
Validation of non-culture-based detection techniquesThe biggest drawback to the use of non-culture-based detection techniques for A.salmonicida is the difficulty of interpreting the results generated by thesetechniques in field samples. Genetic techniques ‘see’ a small segment of themicrobial genome, while immunological techniques ‘see’ one or more epitopeson the microbial surface. Therefore, the key characteristic of these techniques isthat they are proxy measurements of microbial presence, that is, indirect indicesthat are presumed to signal the presence of the target organism in a sample(Wildavsky, 1995). Where the target organism is a pathogen, the target sequenceor epitope(s) may also be used as a proxy measurement of disease potential. In
373Furunculosis
fish health studies, it is probable that this will, in fact, be the meaning attributedto any data generated by a non-culture-based detection technique. The use ofproxy measurements does not preclude the generation of meaningful data butdoes present difficulties about the interpretation of results (Wildavsky, 1995).Hiney (1997) and Hiney and Smith (1998) have argued strongly that thesedifficulties can only be overcome by validation, that is, demonstration that atechnique does what it is supposed to do (Thrusfield, 1986). Although importantfor all techniques, validation is essential for techniques that involve proxymeasurements.
Validation is not the property of a technique but, rather, is a property of itsapplication. It establishes that a technique can be correctly and properly used fora particular purpose. A formal structure for the validation of non-culture-baseddetection techniques in laboratory studies was presented by Hiney and Smith(1998). However, no amount of laboratory studies can validate the application ofsuch techniques under field conditions. Comparative and predictive validationrepresent the only two available strategies for the validation of suchapplications. In comparative methods, the technique under test can be comparedeither with a technique that has been previously validated or with one which is,itself, also unvalidated. This second approach, mutual covalidation, is the morefrequently encountered but is, however, limited in power.
Predictive validation requires that the application intended for the techniquemust be clearly defined in terms which are empirically meaningful. For example,the intended application might be the prediction of the disease incidence, forhatchery smolts covertly infected with A. salmonicida following transfer to a seasite. An empirically meaningful measure of ‘disease’ could, in this case, be thefrequency of the isolation of A. salmonicida from the kidney of moribund fish,following transfer. The process of predictive validation would then involvemeasuring 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 anumber of years and in a variety of environmental contexts. When, and onlywhen, it has been established that a satisfactory correlation exists can thetechnique be used alone and the data it generates be interpreted as a proxymeasurement of disease. Simply stated, techniques can only be used to predict anevent if they have been shown to predict it.
Despite the importance of validation, few papers that have reported on thedevelopment of non-culture-based assays for A. salmonicida have paid adequateattention to demonstrating that these techniques have field validity. Hiney et al.(1997a) illustrated the danger of assuming that a positive response, generated bya non-culture-based assay for A. salmonicida in a field sample, was indicative ofthe 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 healthstatus of fish at that hatchery over a 2-year period, as assessed by routinebacteriological analysis and stress testing. These results suggested thatinterpretation of the results generated by either of these techniques as indicatorsof 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-basedtechniques for the detection of A. salmonicida have the potential to improve ourunderstanding of, and limit the impact of, diseases caused by this pathogen.However, as discussed, adequate validation of the development and utilizationof these techniques is critical to their correct interpretation, especially if thatinterpretation will be used to impose regulatory limitations on aquacultureactivities.
CONTROL AND TREATMENT
Transmission of the disease
The identification of the reservoir of the pathogen, the vector system it employsto move from this reservoir to a new host and the mode of entry into the new hostare key questions in developing an understanding of the epizootiology of anydisease. Unfortunately, for A. salmonicida and its associated diseasefurunculosis, none of these questions can be answered with certainty.
Standard textbooks of bacterial taxonomy refer to A. salmonicida as anobligate fish pathogen (Bergey, 1984), suggesting that the bacterium is incapableof sustained growth outside its piscine hosts. A number of laboratory microcosmstudies have suggested short survival times for the bacterium in aquaticenvironments. It should, however, be noted that such experimental systems arenotoriously difficult to work with and the data they generate should only beextrapolated to the real world with great caution (Enger, 1997). It is also true thatthere are few reports of the isolation of the bacterium from the non-piscineenvironment. However, colony formation by A. salmonicida can be inhibited bya 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 afunction of the inadequacy of our methods, rather than the absence of theorganism. A second factor that may have contributed to our failure is the extremehydrophobicity of the bacterium. This surface property would suggest that, innature, A. salmonicida is more likely to be isolated from particles (Sakai, 1986),making traditional culture techniques unsuitable for the organism from theenvironment. Michel and Dubois-Darnaudpeys (1980) demonstrated that A.salmonicida can survive in a virulent form for many months in a sterile watermicrocosm 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 bothELISA and a PCR/DNA probe assay, Hiney et al. (1997a) found no correlationbetween positive detection of the signs of A. salmonicida in hatchery sedimentand the disease status of hatchery stocks over a 2-year period, as assessed bykidney culture and stress testing. These non-culture-based assays are, however,capable of detecting non-culturable-but-viable cells and dead cells, and theresults generated by them cannot be used, therefore, as an indication ofvirulence.
375Furunculosis
Treatment and protection
Treatment of overt clinical epizooticsThere are important differences between the treatment of large land-basedanimals with antimicrobial agents and the treatment of populations of fish tocontrol furunculosis (Smith et al., 1994). In the former case, the primary aimof 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 overtfurunculosis have seriously reduced appetite, and orally administered agentsare unlikely to achieve therapeutic levels in such fish. The primary aim oftreatments of fish populations is, therefore, to prevent the initiation of newinfections within the population. There are two major practical consequencesthat flow from this prophylactic nature of fish treatment. The first is that allinfections initiated prior to the start of therapy can be expected to run theircourse. Thus, significant mortalities will be experienced in a population forsome days after the commencement of therapy. The second is that the speedwith which therapy is initiated is critical. Laboratory confirmation of the natureof an infection and the antimicrobial susceptibility of the isolate may take 4–5days. Such a delay in the initiation of therapy will result in a significantincrease in mortalities. To achieve maximum reduction in mortalities, it isessential that treatment is initiated at the first sign of mortalities which areconsistent with a septicaemia. With respect to Atlantic salmon smolts, theoccurrence, within any population, of mortalities that present evidence ofhaemorrhage at the anus or the base of the fins (Bernoth, 1997b) should besufficient reason for initiating treatment. If such a rapid response is to beachieved, the choice of agent to be employed will have to made on historicaldata. Experience has shown that the sensitivity patterns of strains isolated at aparticular farm tend, in the absence of importation of ’foreign’ populations, toremain reasonably consistent.
CHOICE OF ANTIMICROBIAL CHEMOTHERAPEUTANTS
In the laboratory, typical A. salmonicida can be shown to be sensitive to a widerange 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 widespreadresistance to ampicillin that is a property of Aeromonas hydrophila and othermesophilic members of this genus (Sakazaki and Balows, 1981). The range ofagents available for the control of epizootics of furunculosis is limited by threemain factors. If the mode of administration is to be oral, then the agents must becapable of achieving therapeutic levels when administered in this manner.Secondly, the agents must be palatable. There have, for example, beensignificant palatability problems associated with some preparations ofpotentiated sulphonamides (Mitchell, 1992). It has been reported thatpopulations of fish may accept the first presentations of feed medicated withthese agents but that they will refuse subsequent presentations. The third factorlimiting 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 limitsignificantly 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 ofisolates is essential in every epizootic. There are considerable data that strainsmanifesting more than one sensitivity pattern can be isolated from a singleepizootic 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 thatA. salmonicida can acquire resistance during therapy (Brazil et al., 1986),suggesting that sensitivity testing should, particularly if mortalities continue, berepeated during a treatment period. Even following a successful treatment, it iscommon for a population to experience a second, normally less severe,recurrence of the epizootic a few weeks later. Sensitivity testing of isolates fromsuch 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, atpresent, no standard methods for measuring such sensitivities and therefore noaccepted means of interpreting the clinical significance of any sensitivity dataobtained (Smith et al., 1994). Serious errors can be made by employingrelationships between sensitivity and clinical resistance that have beendeveloped for humans or other land-based animals. These problems are mostacute with respect to the sensitivity to the quinolone group. Quinolone resistanceis mediated by reduced membrane permeability, is frequently low-level andincreases in a stepwise manner (Tsoumas et al., 1989). There are data to suggestthat strains with a minimum inhibitory concentration (MIC) as low as0.75 µg ml–1 and which give an inhibition zone of up to 18 mm when tested witha disc containing 2 mg oxolinic acid are refractory to treatment (O’Grady et al.,1987). In practice, it is probably safer to treat an isolate showing any reducedsensitivity 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 frequentlysupplied by the manufacturers. Table 10.9 presents a summary of these data. Asa general rule, it can be assumed that sea water interferes with oral uptake andtherefore higher doses should be employed if treatment is of fish in the marineenvironment.
Treatments of covert infectionsHiney et al. (1997b) have presented a detailed review of the chemotherapeutictreatment of covert furunculosis infection. Fish with covert infections can, withgood husbandry, be reared without any experience of overt disease (Scallan,1983). Further, the presence of covert infections in a population are probably areflection of the presence of A. salmonicida in their water-supply. Therefore,therapy of such covert infections is frequently neither justified nor liable, in the
377Furunculosis
long run, to be effective. Under certain conditions, however, prudent healthmanagement might suggest implementation of therapies to control suchinfections.
Covert infections are latent and therefore, if it is known in advance thatpopulations 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 fishpopulations is of course a major and predictable stressful event but othermanagement procedures, such as grading or tagging, can also provide sufficientstress to activate latent covert infections. In designing therapeutic interventionsto deal with these situations, it is necessary to decide whether the aim of theintervention is prophylactic or therapeutic. In prophylactic treatments, the aim isto provide adequate concentrations of the agent in the fish during the period ofthe 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 incontrolling overt clinical epizootics of furunculosis (Table 10.9) would alsoprovide some degree of prophylactic cover against overt infections arising fromthe activation of covert infections. There are, however, data that suggest that oraladministration of the quinolones oxolinic acid or flumequine would be thetreatment of choice. Standard practice would be to initiate a 10-day oraltreatment designed to end immediately prior to the predicted stressful event andprobably 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 mannerand actually eliminate A. salmonicida from a population of covertly infected fish.They are both significantly intrusive and stressful and therefore should only beemployed when the epizootiological situation demands them. When fish transportis 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 DosageEnvironment agent (% active) (mg kg–1) Regime*
Fresh water Oxytetracycline 50–100 80 Orally for 10 daysOxolinic acid 50–100 10 Orally for 10 daysRomet 100 50 Orally for 5 daysFlorfenicol 100 20 Orally for 10 daysAmoxycillin 100 40–80 Orally for 10–12 days
Sea water Oxytetracycline 50–100 120 Orally for 10–12 daysOxolinic acid 50–100 30 Orally for 10 daysMethasul 40 75 Orally for 10 daysSulfatrim 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 fishare being moved. Thus, in this situation, the attempt is to control both the carrierand the latent dimensions of covert infections, and therefore the extra effort andrisks associated with therapeutic treatments may be justified.
Scallan and Smith (1985) reported the successful therapeutic treatment ofcovert infections with a protocol that involved a standard oral administration offlumequine followed, 2 days before transport, by the i.p. injection of all fish with30 µg kg–1 flumequine. Protocols involving the bath administration of flume-quine have also been reported (O’Grady et al., 1988; O’Grady and Smith, 1992;Cazabon et al., 1994; Hiney et al., 1995). The uptake by fish of flumequine andoxolinic acid from their water can be extremely efficient and rapid. Almostuniquely in treatments of fish with antimicrobial agents, bath administration ofthese agents runs a significant risk of administering a lethal overdose (Coyne etal., 1994). The rate of uptake of these agents is strongly dependent on waterquality and is favoured by soft acid conditions (O’Grady et al., 1988). In hardalkaline waters, uptake is greatly reduced and in sea water it is practicallynegligible. Rates of uptake are also dependent on the condition of the fish andare frequently greater during commercial-scale treatments than in small-scalelaboratory trials (Cazabon et al., 1994). It is recommended that, before any newtreatment of fish is performed, trials of the safety of the procedure are madebefore a full treatment of a commercial population is initiated. When bathadministration of quinolones is contemplated, this precautionary requirementbecomes absolutely essential. The aim of bath treatments is to produce serumlevels of 30 µg ml–1 and this should be monitored by appropriate analyticalprocedures. Serum concentration in excess of approximately 60 µg ml–1, whichcan easily be achieved by bath administration, is lethal for salmonids (Cazabonet al., 1994).
In any treatment aimed at protecting fish during a movement from freshwater to sea water, the influence of the change in the environment of the fish onthe internal concentrations of antimicrobial agents must be taken into account.Movement to the sea has been shown to result in a very rapid excretion offlumequine from fish (Hiney et al., 1995). It is probable that this is associatedwith the rapid excretion of Mg2+ ions that fish perform on entering the sea. If thisexplanation is correct, then it is probable that any antimicrobial agents that formcomplexes with Mg2+ (oxytetracycline and the quinolones) will also be rapidlyexcreted under these conditions. As a consequence, it is probably unwise toassume that any agent introduced into fish in fresh water will persist inmeaningful concentrations after their entry into the sea.
VaccinationVaccination strategies designed to control furunculosis were reported as early asthe 1940s (Duff, 1941). Midtlyng (1997) has reviewed the attempts that weremade, over the next 50 years, to produce vaccines and vaccine administrationmethods that would provide adequate control of this disease. Succinctly, andpossibly rather unfairly, these attempts can be summarized, at least from theperspective of commercial salmon farmers, as failures. Oral, immersion andinjection administrations of a variety of bacterins were developed and a few
379Furunculosis
were commercially produced and marketed. Some, particularly thoseadministered by injection, were cost-effective in that the value of the fishprotected probably exceeded the cost of the vaccination. None, however, weresufficiently effective to have a significant influence on the epizootiology of thedisease. This situation changed dramatically at the beginning of the 1990s. Atthis 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 hastransformed the significance of furunculosis in commercial salmon farming. Inthe 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 ofthese vaccines is now almost universal throughout the European industry andthis has resulted in the perceived disappearance of furunculosis as a cause ofsalmon mortality. Munro and Gauld (1996), for example, reported a reduction inmortality of salmon in sea cages from 35% to approximately 10% that wascoincident with the introduction of these vaccines. Similarly Markestad et al.(1995) related the 75% reduction in the use of antimicrobials in Norway thatoccurred in 1994 due to the increased use of oil-adjuvant vaccines. Thedevelopment of these vaccines has been accompanied by the development ofmachinery that allows the injection of a large number of fish in a short time. Notonly have these machines substantially minimized the logistical problemsassociated with the administration of vaccines by injection, but they have alsosignificantly reduced the health risks to farm workers. Accidental self-injectionof furunculosis vaccines, particularly those that contain oil adjuvants, can resultin significant damage to workers. In any large-scale vaccination, significantthought must be given to worker health issues, and clearly the use of machines toperform the injection will significantly reduce the potential risks. Midtlyng(1997) has also discussed the animal-welfare aspects of the use of oil-adjuvantvaccines. While he accepts that there is evidence that such vaccines do result insuffering for fish, he argues that any such suffering must be balanced against thatwhich would result from a devastating epizootic of furunculosis.
While the importance of oil-adjuvant vaccines for the salmon farmingindustry is well attested (Markestad et al., 1995), the application of thistechnology to salmonid farms whose primary function is restocking presentsproblems. These problems are primarily associated with the persistence of theadjuvant within the vaccinated fish. In restocking operations, there is inherentlyless 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 thepurpose of competition fishing, considerations of the health risks that may beassociated with the consumption of highly immunogenic adjuvants may placeconsiderable constraints on the use of oil-adjuvant vaccines. Hiney (1995) hasdemonstrated that covert infections can persist in oil-adjuvant-vaccinated fish.Thus, there is a clear possibility that such fish can act as ‘immune carriers’. Themovement of such fish between water bodies may therefore be capable of actingas 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 ofvaccinated fish.
380 M. Hiney and G. Olivier
The success of oil-adjuvant vaccines for the control of furunculosis incommercial fish farming has had a number of side-effects, which should also beconsidered. Stress is the major precipitating factor associated with furunculosis.In many situations, the fear of furunculosis has been a significant factor in theintroduction of husbandry practices that were aimed at reducing stress. Inparticular, furunculosis has exerted a major constraint on stocking densities. Theeffective removal, by vaccination, of the fear of furunculosis may result in therelaxation of some farm practices, particularly when these place economicconstraints on the operation of a farm. It is axiomatic that any increase in stresslevels will ultimately result in reduction in fish health. The effective control offurunculosis may, therefore, become the predisposing factor for another disease.A second side-effect of the efficiency of modern vaccination is that funding forresearch into furunculosis has been drastically reduced. Whether this will haveany 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 thepathogenicity of A. salmonicida and the advances that have been made in thisarea, the mechanisms whereby this organism produces disease are only partlyunderstood. Virulence mechanisms fall broadly into two categories, these beingcell-surface structures and extracellular products (ECPs) excreted by the cell.
Cell-surface structuresIn common with many pathogenic bacteria A. salmonicida can manifest anadditional surface protein microcapsule (Kay and Trust, 1997). These crystallinesurface protein arrays are generally referred to as S-layers, but, for historicalreasons, in A. salmonicida the term A-layer is more commonly used (Udey andFryer, 1978). SDS-polyacylamide gel electrophoresis (PAGE) analysis and X-ray diffraction studies have shown the A-layer of A. salmonicida to be a proteinof approximately 50 kDa, with a tetragonal structural arrangement (Trust et al.,1980a; Kay et al., 1981; Evenberg et al., 1982; Garduño and Kay, 1992a; Kayand Trust, 1997). A-layers isolated from a wide variety of A. salmonicida strainswere shown to be immunologically conserved (Kay et al., 1984). The evidenceimplicating the A-layer of A. salmonicida as a primary virulence factor is verystrong. Authors have demonstrated that typical strains possessing the A-layer areboth 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; Ciprianoand Blanch, 1989; Noonan and Trust, 1995). Kay and Trust (1997) havesuggested that the ability of the A-layer to bind immunoglobulins and otherextracellular proteins may result in the masking of bacterial immunogenicreceptors, thus allowing A. salmonicida to evade the host’s immune response.The A-layer has been reported to promote bacterial penetration and adhesion and
381Furunculosis
to inhibit complement-mediated lysis in host serum (Trust et al., 1983; Sakai andKimura, 1985; Garduño and Kay, 1992b; Garduño et al., 1995). It was alsoreported that the net negative charge of A-layer-containing A. salmonicida cellsplays a crucial role in their long-term survival in a freshwater microcosm (Sakai,1986) and that the production of exopolysaccharides under low nutrientconditions, 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 dyeCBB into common growth media could provide a differentiation between strainscontaining an intact A-layer (A+), which grew as blue colonies, and those lackingit (A–), which grew as white colonies. Using CBB-containing media, Ciprianoand Bertolini (1988) demonstrated a correlation between the A+ phenotype onthis medium and virulence for brook trout, although Bernoth (1990a) reportedthat colony colour on CBB-containing media did not always correlate withvirulence of A. salmonicida. Anomalies have been reported which mightchallenge the importance of the A-layer as a virulence factor. Specifically, oneA– 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 didnot always correlate with virulence. It must be remembered, however, that A.salmonicida strains lacking an A-layer are laboratory artefacts. To ourknowledge, no strain lacking an intact A-layer has ever been isolated from anatural furunculosis infection.
The other major component of the cell surface of A. salmonicida, incommon with all Gram-negative cells, is LPS. In A. salmonicida, LPS isnormally 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 ofLPS in the structure of the A-layer and the virulence of A. salmonicida has beenelusive. Observations of O-polysaccharide-deficient mutants have indicated thatthey 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 thatonly A. salmonicida strains containing both an intact A-layer and LPS werevirulent for brook trout.
It must be remembered, however, that virtually all of the studies cited abovewere carried out on A. salmonicida strains grown in vitro on artificial laboratorymedia. It is unlikely that these conditions will reflect the behaviour of A.salmonicida occurring naturally either in a fish host or in the environment.Garduño et al. (1993) found that cells grown in vivo in diffusion chambers whichhad been implanted in rainbow trout peritoneal cavities displayed enhancedresistance to host-mediated serum and oxidative killing, and expressed apolysaccharide capsular layer, which they suggested might act as a mechanismof phagocytosis resistance or evasion of the host immune system. Thornton et al.(1993) have also reported that A. salmonicida cells grown in vivo expressednovel 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 surfacecomponents in the pathogenicity of A. salmonicida.
Extracellular productsIn common with other pathogenic organisms, A. salmonicida has been found toproduce a number of ECPs many of which have enzyme activity. Injection ofcrude extracellular material of A. salmonicida has been clearly demonstrated tokill susceptible fish (Munro et al., 1980; Ellis et al., 1981; Ellis, 1991) and aconsiderable amount of work has been performed to analyse the constituents ofECPs and to understand their role in virulence and pathogenesis. Ellis (1997a)has provided an excellent review of the known ECPs of A. salmonicida, whichtogether make for a long list. These are grouped by Ellis (1997a) into three types,namely proteases, membrane-damaging toxins and other toxins, including H-lysin, which have not yet been fully investigated.
Tajima et al. (1983) were the first to report a lethal 70 kDa protease in A.salmonicida ECP, which was found to have an LD50 of 2.4 µg g–1 when injectedinto young salmon and to produce haemorrhaging and muscle liquefaction (Leeand 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 ahaemolytic factor in the ECP, suggesting an interaction between thesecomponents (Lee and Ellis, 1991b). It is believed that the protease toxin isproduced by A. salmonicida to digest host proteins as a nutrient source, althoughit has been found to have only limited specificity, mainly towards proteins with arelatively open structure (Price et al., 1990). The 70 kDa protease has also beenshown to reduce the clotting time of trout blood, which may account for thepresence of microthrombi in fish tissue in cases of clinical furunculosis andfollowing 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 lackof correlation between the amount of protease in ECP and the killing ability ofthat ECP has been reported (Drinan et al., 1989), and Sakai (1985) reported thata protease-deficient mutant of A. salmonicida was avirulent, while other workershave identified virulent isolates which do not produce any protease understandard 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). Asecond protease, whose preferred substrates are gelatine and collagen, has beenidentified (Sheeran and Smith, 1981), but its physiochemical properties have notyet been determined in detail.
A number of membrane-damaging activities of ECP have been described.Present evidence suggests that, of these membrane-damaging toxins,glycerophospholipid–cholesterol acyltransferase (GCAT) complexed with LPS,so called GCAT/LPS, is the most important factor in the lethal toxicity andpathology of the ECP (Ellis, 1997a). The LD50 of purified GCAT/LPS forAtlantic 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 thishaemolysis was incomplete in the absence of the 70 kDa protease (Lee and Ellis,
383Furunculosis
1990), which has been suggested by Ellis (1997b) to be necessary for activationof GCAT activity. However, there is no evidence for in vivo haemolysis inclinical furunculosis (Lee and Ellis, 1991a) and Ellis (1997a) has suggested thatin vivo GCAT/LPS may function in destabilization of host red cell membranesrather than active haemolysis. The histopathological effects of GCAT/LPS arenot extensive and cannot account for the death, within 20 h, of fish injected withpurified product, and it has been suggested that in vivo toxicity may be due tometabolic effects, although this has yet to be confirmed (Ellis, 1997a). AlthoughGCAT/LPS is clearly an important ECP, it has recently been demonstrated that aGCAT-deficient A. salmonicida mutant could produce the manifestations ofclassical furunculosis (A.E. Ellis, Aberdeen, 1997, personal communication).Therefore, it has become clear, with respect to the pathogenesis of furunculosisand the lethal toxicity of the exotoxins, that, instead of one toxin being criticalfor 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 againstfurunculosis have led to a much improved understanding of the salmonidimmune system, and a number of comprehensive reviews have been published(Warr and Cohen, 1991; Secombes, 1994a; Secombes and Olivier, 1997). Ingeneral, most components of the fish immune system are analogous to those ofhigher vertebrates. These include physical barriers and chemical barriers toprevent infection, inducible but non-specific humoral factors, phagocytes andnon-specific cytotoxic cells, which mediate an inflammatory response, and,finally, specific immunity, effected by lymphocytes. It is this last type that isresponsible for ‘immunological memory’, ensuring that responses to a secondexposure are faster and stronger than the initial response, thus conferringimmunity (Secombes and Olivier, 1997). Specific immune responses includeboth humoral immunity, based on the production of antibodies by B cells, andcellular immunity, based on the production of activated macrophages withenhanced bacterial activity, following release of cytokines from T cells(Secombes and Olivier, 1997).
Non-specific humoral factorsSalmonids present physical barriers to infection by A. salmonicida in thestructure of their skin and intestinal mucosa and there is some evidence foractive immune cells on both of these surfaces (Ellis, 1985; Cipriano, 1986). Fishalso have a variety of non-specific humoral factors with which to counterinfection by A. salmonicida (Lund et al., 1991). These include enzyme systemsto lyse bacteria, such as lysozyme and complement, antiproteases to neutralizebacterial proteases, proinflammatory molecules, which can attract leucocytesand neutrophils to an infection site and increase local capillary permeability,substances to sequester essential nutrients and thus limit bacterial growth, andmolecules which bind to the bacterial cell and trigger the complement systemand 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 humoralresponse serve to neutralize toxins excreted by A. salmonicida, for example, α2-macroglobulin, which has antiprotease activity (Salte et al., 1992). There is alsoevidence that the complement system may have a role in neutralizing A.salmonicida ECP (Sakai, 1984).
Non-specific cellular factorsShould A. salmonicida successfully breach the non-specific immune defences offish, it will encounter specialized cells of the host immune system, namely thephagocytic cells. These include monocytes, or macrophages, and granulocytes(neutrophils, eosinophils and basophils). The macrophages of fish are similar tothose of higher animals in both morphology and function and they act as antigen-presenting cells, cytokine-secreting cells and effector cells, with the ability toactively phagocytose bacterial cells (Secombes and Fletcher, 1992; Secombesand Olivier, 1997). Less is known about the function of neutrophils in theimmune reaction of salmonids. Lamas and Ellis (1994a) demonstrated thatneutrophils isolated from Atlantic salmon migrated in fish serum in response tothe presence of A. salmonicida, although, strangely, A-layer-deficient strainsacted as stronger chemoattractants. Neutrophils were also found to bephagocytic, but phagocytosis was low compared with macrophages (Lamas andEllis, 1994b). Eosinophils may play an important role in the host responseagainst infection by A. salmonicida, as they are widely distributed in connectivetissues, especially in the intestine and gills (Ellis, 1985). In the intestine,eosinophilic granular cells (EGC) form a layer called the stratum granulosumbetween the muscle layer and the stratum compactum. A number of studies havesuggested 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 intestinalEGC, coincident with the appearance of histamine in the blood (Ellis, 1985;Vallejo and Ellis, 1989).
Specific cellular responsesThe specific immune response of salmonids includes three strategies, that is,lymphocyte proliferation, antibody production and cytokine response. In highervertebrates, lymphocyte responses are characterized by their ability toproliferate following contact with bacterial antigens, generating a clone ofantigen-specific cells. Some of these cells give rise to the primary response,while others remain dormant as specific memory cells, able to proliferate inresponse to subsequent contact with the same antigen (Secombes and Olivier,1997). There are relatively few studies on lymphocyte proliferation in salmonidsin response to A. salmonicida infection. However, those that have been carriedout suggest that lymphocyte proliferation does occur in response to bothformalin-killed and live A. salmonicida cells (Reitan and Thuvander, 1991;Vaughan et al., 1993), although this response is dependent on the dose and typeof antigen presented (Erdal and Reitan, 1992; Secombes and Olivier, 1997).
Antibody production to A. salmonicida, on the other hand, has beenextensively studied in salmonids, particularly following immunization with
385Furunculosis
killed whole cells or ECP. High serum antibody titres can be routinely elicited insalmonids by both i.p and i.m. injection of A. salmonicida (Secombes andOlivier, 1997). At permissible temperatures (10–15°C) antibody titres risewithin 2–3 weeks and peak within 8–12 weeks, although water temperature iscritical to this response and at lower temperatures responses may be slower orabsent (Ellis et al., 1992; Eggset et al., 1997). The sites of antibody production insalmonids would primarily seem to be spleen and head kidney (Reitan andThuvander, 1991; Davidson et al., 1993). Antibody-secreting cells have alsobeen found in mucosal sites, such as the gut, but these can take up to 7 weekspost-vaccination to appear (Davidson et al., 1993). Antibody responses havealso been elicited following administration of A. salmonicida by subcutaneousinjection (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, invaccines administered by a parental route has been shown to enhance theantibody titre and may act as a depot of antigen, allowing vaccination at lowtemperatures (Cipriano and Pyle, 1985; Anderson et al., 1997; Ellis, 1997b;Midtlyng, 1997; Secombes and Olivier, 1997). It should be noted, however, thathigh antibody titres do not necessarily correlate with protection and it is thespecificity of the antibodies that appears to be important (Hirst and Ellis, 1994;Ellis, 1997b). In older fish, proliferation studies suggest that specific B-cellmemory can be established to A. salmonicida and that a second exposure to theorganism results in a faster and stronger antibody response (Secombes andOlivier, 1997). However, in younger fish, it is possible that a primary exposureto A. salmonicida may induce tolerance rather than specific immunologicalmemory (Manning et al., 1982).
Unlike higher vertebrates, antibody response to A. salmonicida in salmonidsis essentially independent of T cells. In fish the regulatory role of T cells in theimmune response is thought to be mediated by released cytokines, analogous tointerleukin-2, chemokines and macrophage-activating factor (MAF), followingexposure to specific antigens (Secombes, 1994b). Although little is generallyknown about cytokine release in response to A. salmonicida, release of MAF hasbeen demonstrated in vitro from cultured cells, removed from fishimmunologically primed with killed whole A. salmonicida or ECP, 2–3 weekspost-exposure, peaking 4–5 weeks post-exposure (Marsden et al., 1994). In vitrostudies have also shown that MAF-treated macrophages acquired the ability tokill A. salmonicida (Graham et al., 1988). In fish, MAF production has also beendemonstrated to correlate with both lymphocyte proliferation and antibodyproduction following vaccination with whole cells (Secombes and Olivier,1997). Therefore, unlike antibody production, any epitope on A. salmonicida canpotentially induce a cell-mediated response, such as MAF release. In commonwith antibody production, however, cytokine release is temperature dependentand has been shown to be absent in fish cells kept at 7°C or less (Hardie et al.,1994).
386 M. Hiney and G. Olivier
‘ATYPICAL’ AEROMONAS SALMONICIDA
Introduction
Aeromonas salmonicida subsp. salmonicida reviewed in the previous sections isan important salmonid pathogen that has probably been studied more than anyother fish pathogen. However, the species A. salmonicida comprises a largenumber of strains, routinely referred to as ‘atypical’ strains, that is, strains thatdo not conform to the general guidelines presented in Table 10.5 for typical A.salmonicida. These atypical strains are responsible for several diseaseconditions in both salmonids and non-salmonid species. Our knowledge of‘atypical’ strains is growing rapidly and their importance is being increasinglyrecognized. In the last decade, atypical isolates of A. salmonicida have become agrowing concern in several countries, because of their frequent association withmortalities of both salmonids and non-salmonid species. Recent studies fromCanada and northern Europe indicate that cases of ‘furunculosis’ in salmonidscaused by atypical isolates are increasing, probably because of better awarenessof these subspecies. This confirms the belief that atypical isolates are morewidely distributed than previously suspected (Wichardt et al., 1989; Rintamäkiand Valtonen, 1991; Olivier, 1992; Pedersen et al., 1994; Wiklund et al., 1994).
The literature on ‘atypical’ strains of A. salmonicida is expanding rapidlybut remains confusing, largely because of the problems associated with theirtaxonomy and the variety of disease conditions they cause in different species offish. In previous reviews of A. salmonicida, atypical strains have always beenincluded with typical isolates (McCarthy and Roberts, 1980; Trust, 1986; Austinand Austin, 1993). Although there are numerous similarities, we believe thatatypical strains are distinct subspecies and should be considered independently.
Diseases and disease agents
Species susceptibility to atypical Aeromonas salmonicidaThe number of hosts from which atypical A. salmonicida isolates have beencultured 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 farmedpopulations of salmonids in northern Europe (Wichardt et al., 1989; Håstein andLindstad, 1991; Rintamäki and Valtonen, 1991; Hirvelä-Koski et al., 1994;Pedersen et al., 1994; Gudmundsdóttir et al., 1995). The most common clinicalsign in these infections is skin ulceration (Wichardt et al., 1989), but a number ofother clinical signs have been recognized some of which are similar to those seenin cases of typical furunculosis (Rintamäki and Valtonen, 1991).
Mortality of up to 60% has been reported from cultured sea trout in Sweden(Wichardt et al., 1989) and 15–20% in Finland and Iceland (Gudmundsdóttiret al., 1995: Rintamäki and Valtonen, 1991). In North America, an ulcer diseaseof trout, originally described by Sniezko et al. (1950), was associated with afastidious organism (Haemophilus piscium), which was subsequently
387Furunculosis
Tab
le 1
0.1
0.
Sal
mo
nid
fis
h s
pec
ies
fro
m w
hic
h a
typ
ical
Aer
om
on
as s
alm
on
icid
a h
ave
bee
n is
ola
ted
(af
ter
Ber
no
th, 1
997a
).
His
tory
of
Co
mm
on
nam
eS
cien
tifi
c n
ame
Hab
itat
iso
lati
on
Ref
eren
ce
Atl
anti
c sa
lmo
nS
alm
o s
alar
Fres
h w
ater
Clin
ical
Gro
man
et
al. (
1992
)In
cid
enta
lB
ened
ikts
dó
ttir
an
d H
elg
aso
n (
1990
)B
rack
ish
wat
erC
linic
alH
arm
on
et
al. (
1991
)S
ea w
ater
Clin
ical
Oliv
ier
(199
2)In
cid
enta
lB
ened
ikts
dó
ttir
an
d H
elg
aso
n (
1990
)A
rcti
c ch
arS
alve
linu
s al
pin
us
Fres
h w
ater
Clin
ical
Wic
har
dt
et a
l. (1
989)
Sea
wat
erC
linic
alO
livie
r (1
992)
Bro
ok
tro
ut
Sal
velin
us
fon
tin
alis
Fres
h w
ater
Clin
ical
Lju
ng
ber
g a
nd
Jo
han
sso
n (
1977
)B
row
n t
rou
tS
alm
o t
rutt
a m
. lac
ust
ris
Fres
h w
ater
Clin
ical
Rin
tam
äki a
nd
Val
ton
en (
1991
)C
hu
m s
alm
on
On
corh
ynch
us
keta
Sea
wat
erC
linic
alE
vely
n (
1971
)C
oh
o s
alm
on
On
corh
ynch
us
kisu
tch
Fres
h w
ater
Clin
ical
Ch
apm
an e
t al
. (19
91)
Gra
ylin
gT
hym
allu
s th
ymal
lus
Fres
h w
ater
Clin
ical
Rin
tam
äki a
nd
Val
ton
en (
1991
)La
ke t
rou
tS
alve
linu
s n
amay
cush
Fres
h w
ater
Clii
nic
alLj
un
gb
erg
an
d J
oh
anss
on
(19
77)
Mas
u s
alm
on
On
corh
ynch
us
mas
ou
Fres
h w
ater
Clin
ical
Kim
ura
(19
69)
Pin
k sa
lmo
nO
nco
rhyn
chu
s g
orb
usc
ha
Fres
h w
ater
Clin
ical
Kim
ura
(19
69)
Rai
nb
ow
tro
ut
On
corh
ynch
us
myk
iss
Fres
h w
ater
Clin
ical
Wic
har
dt
et a
l . (1
989)
Sea
tro
ut
Sal
mo
tru
tta
m. t
rutt
aS
ea w
ater
Clin
ical
Kro
vace
k et
al .
(198
7)In
cid
enta
lR
inta
mäk
i an
d V
alto
nen
(19
91)
So
ckey
e sa
lmo
nO
nco
rhyn
chu
s n
erka
Sea
wat
erC
linic
alE
vely
n (
1971
)
Fish
sp
ecie
s
388 M. Hiney and G. Olivier
reclassified as an atypical strain of A. salmonicida (Paterson et al., 1980; Trustet al., 1980b; Adams and Thompson, 1990). On the Atlantic coast of Canada,fastidious atypical strains have been isolated from Atlantic salmon (Paterson etal., 1980; Groman et al., 1992; Olivier, 1992) with mortality of up to 50% beingrecorded in some instances (Groman et al., 1992). Thus, disease of atypical A.salmonicida aetiology can have a significant economic impact, depending on thefish 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. (Shottset al., 1980) and the ‘head ulcer disease’ of cultured Japanese eels, Anguillajaponica (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 sablefish, Anopoploma fimbria. A similar strain was isolated from ling cod, Ophiodon
Table 10.11. Freshwater non-salmonid species from which atypicalAeromonas salmonicida have been isolated (after Bernoth, 1997a).
History ofCommon 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)
specifiedGoldfish Carassius auratus Clinical Elliot and Shotts (1980)Japanese eel Anguilla japonica Clinical Kitao et al. (1984)Minnow Phoxinus phoxinus Clinical Håstein et al. (1978)Northern pike Esox lucius Clinical Wiklund (1990)
Unclear Wichardt et al. (1989)Ornamental Unclear Bernoth (1997a)cyprinids
Perch Perca fluviatilis Clinical Bernoth (1997a)Unclear Wichardt et al. (1989)
River bleak Alburnus alburnus Clinical Bernoth (1997a)Roach Rutilus rutilus Clinical Austin (1993)
Unclear* Wichardt et al. (1989)Rudd Scardinius Unclear Barker and Kehoe (1995)
erythrophthalmusSilver 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 anincidental finding.
Fish species
389Furunculosis
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 aquaculturepurposes, and, according to some authors, disease conditions of atypical A.salmonicida aetiology may become a restricting factor for the mass culture ofthese species (Pedersen et al., 1994). In several cases, the disease has beendiagnosed in wild fish transferred and maintained in aquarium facilities,reinforcing the hypothesis that there could be strains of A. salmonicida of marineorigin (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 Aeromonassalmonicida have been isolated (after Bernoth, 1997a)
History ofCommon 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)
aeglefinusJapanese 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 andlanceolatus Paulsen (1986)
Shotted halibut Eopsetta grigorjewi Clinical Nakatsugawa (1994)Tom cod Gadus microgadus Clinical Olivier (1992)Turbot Scophthalmus Clinical Pedersen et al. (1994)
maximusWrasse 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 andthe disease condition will be expressed when animals are stressed.
The microorganisms
Biological characteristics of atypical Aeromonas salmonicidaThe description and characterization of several atypical isolates over the last fewyears confirm the close relationship between typical and atypical strains.Although their growth characteristics and their biochemical profiles are quitedistinctive, almost all atypical isolates investigated so far possess phenotypicproperties similar to those of typical strains. They are all stronglyautoaggregating, forming small convex raised colonies on agar, which arecompact, are strongly adherent and slide on the surface of agar media whenpushed with a loop (friable). Their cell-surface structure is similar to typicalstrains. Most strains investigated have an A-layer and LPS similar to those of thetypical 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-proteinappears to be conserved in both typical and atypical isolates, as demonstrated byChu et al. (1991), using Southern blot analysis: a 2.5 kb portion of the gene wasdetected in typical and atypical strains alike, but not in A. hydrophila isolatestested. Atypical isolates grown above 30°C can give rise to the phenotypicvariants A–LPS+, A–LPS- and A+LPS–, which are similar to the recognizedphenotypes of typical strains (Evenberg et al., 1985; Griffiths and Lynch, 1990;Rockey et al., 1991). Using a preparation of A-protein obtained from a typicalisolate, Griffiths and Lynch (1990) demonstrated that the A layer could bereconstituted on the surface of A–LPS+ phenotypes of both typical and atypicalstrains.
Typical and atypical isolates are serologically cross-reactive, using mono-clonal or polyclonal antibodies (Paterson et al., 1980; Sövényi et al., 1984;Evenberg et al., 1985; Kitao et al., 1985; Austin et al., 1986; Böhm et al., 1986;Adams and Thompson, 1990). Rockey et al. (1991), using monoclonalantibodies, have demonstrated different epitopes on the LPS of typical andatypical isolates. In addition, using restriction endonuclease fingerprinting andplasmid profiles, strong homology between typical and atypical strains has alsobeen demonstrated (Bast et al., 1988; McCormick et al., 1990).
Taxonomy of atypical Aeromonas salmonicidaThe classification of psychrophilic Aeromonas is not clear-cut. Table 10.5attempted to provide guidelines for separating typical from atypical strains,based on associated pathology and its correlation with genetic and phenotypiccharacteristics. However, the atypical group still presents major difficulty whenattempting to assign isolates to a particular subspecies. In most cases, one mustresort to referring to the isolate as simply ‘atypical’ A. salmonicida. Austin andAustin (1993) have extensively reviewed the history of the taxonomy ofatypical A. salmonicida as they are currently classified in Bergey’s Manual of
391Furunculosis
Determinative Bacteriology (Holt et al., 1994), in which atypical isolates areseparated into three subspecies, masoucida, achromogenes and smithia. Strainsbelonging to the subsp. masoucida were described by Kimura (1969). Becausesubsp. masoucida is clearly restricted to Japan, its biochemical profile is quitedifferent 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 beplaced in only two recognized subspecies, achromogenes or smithia. Thissituation epitomizes the basic problem facing diagnosticians when atypicalisolates are recovered from diseased fish. Often these strains are biochemicallydifferent 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 29atypical isolates, he found that atypical strains could be separated into twophenons (B1 and B2), distinguished by their biochemical and enzymaticproperties. He proposed to combine the two subspecies achromogenes andmasoucida (corresponding to his phenon B1) into a single subspecies,achromogenes. Interestingly, all 18 strains of this phenon had been isolated fromsalmonid hosts. His second suggestion was to create a new subsp., nova, toaccommodate the 11 isolates of phenon B2, ten of which had been isolated fromnon-salmonid hosts. A subsequent DNA : DNA hybridization study of a series oftypical and atypical isolates carried out by Belland and Trust (1988) supportedthis new classification, but these authors cautioned that there could be additionalsubspecies present, because some strains could not be properly assigned to eitherof the two proposed subspecies. The later study on A. salmonicida taxonomy, byAustin et al. (1989), disagreed with the previous findings and these authors wereof the opinion that the subspp. achromogenes and masoucida should be retainedand that a new subspecies, smithia, should be created. It would incorporate acluster 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 : DNAhybridization, their results tend to agree with those of McCarthy (1977b, 1978)and Belland and Trust (1988). Two major groups were identified according totheir biochemical and enzymatic profiles; these were similar to the subspp.achromogenes and nova described by McCarthy (1978). In addition to thisprimary separation into subspecies, different biotypes could be identified withineach of these two groups. Analysis of these results strongly suggests that there isa definite correlation between biotypes and host species and also betweenbiotypes 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 asingle host, the roach Rutilus rutilus. These findings provide evidence that theisolation of these strains is often correlated with a specific host species. There isno 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 severalcountries stresses the importance of this group of organisms for wild andcultured stocks (Wichardt et al., 1989; Rintamäki and Valtonen, 1991; Olivier,1992; Bernoth, 1997a).
Genomic characterization is being increasingly used to determine the degreeof 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 adistinct group and that atypical isolates belonging to the subspp. achromogenesand masoucida were genotypically diverse. Of particular interest is the fact that,in addition to the recognized subspecies, there was evidence that additionalsubgroups were likely to exist. Using 60 phenotypic tests, ribotyping andplasmid profiles, Hänninen and Hirvelä-Koski (1997) examined 53 atypicalstrains originating from Finland, Denmark, Norway and Sweden. They foundthat 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 tofingerprint various bacterial isolates in order to determine their degree ofhomogeneity. One such technique is randomly amplified polymorphic DNAanalysis (RAPD). Using this technique, Miyata et al. (1995) and Inglis et al.(1996) have shown that atypical strains can be differentiated from typicalisolates, 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 strainsisolated from six non-salmonids hosts. Their results indicate, as before, thatatypical isolates are genotypically different from typical ones. Even though ahigh degree of homology was detected in their atypical isolates with four of theirRAPD primers, another primer (A31) gave an interesting result showing that allisolates could be differentiated according to the host species the strains wereisolated from. Using RAPD and pulsed-field gel electrophoresis, O’hIci et al.(1998) analysed 39 atypical strains from Europe and North America isolatedfrom three salmonids and five non-salmonid hosts. They found the atypicalstrains to be a genetically heterogeneous group with a similarity of less than70%, but noted some correlation of groupings with different fish hosts andlocations of isolation.
Disease transmissionThe transmission of atypical A. salmonicida has not been thoroughlyinvestigated, 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 survivedbetter in salt water compared with fresh water. In another study, Wiklund(1995a) investigated the survival of atypical strains isolated from flounders inmicrocosms. Best survival was observed in the presence of sediments and athigh water temperature and the highest survival was observed in brackish watercompared with fresh and salt water. Additional studies on the ecology of thesestrains is required if the epizootiology of these isolates is to be better understood.
393Furunculosis
In several cases, transmission of the disease seems to have been linked to thetransfer of infected fish (Wichardt et al., 1989; Håstein and Lindstad, 1991). Thebest evidence has been provided by the example of the goldfish ulcer diseaseagent, which was introduced into Australia through the importation of liveinfected or subclinically infected goldfish in 1974. Following this introduction,the agent has been recovered from wild goldfish and the disease is now thoughtto be endemic in several areas of Australia (Whittington et al., 1987; Humphreyand Ashburner, 1993).
Diagnostic methods for atypical Aeromonas salmonicida
Diagnosis is usually based on culturing of the pathogen. The identification ofnon-fastidious atypical isolates of the subsp. achromogenes is relativelystraightforward, because all these strains are reported to grow on conventionalmedia, 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 etal., 1989; Rintamäki and Valtonen, 1991; Olivier, 1992; Wiklund and Dalsgaard,1995). Samples of ulcers or lesions, in addition to kidney tissues, should becultured to confirm the presence of atypical isolates, because some organismsmay not be found in internal organs. In Iceland, the recovery of A. salmonicidasubsp. achromogenes was superior when gills of subclinically or covertlyinfected Atlantic salmon were cultured, rather than kidney tissues(Benediktsdottir and Helgason, 1990).
Several problems are associated with the culture of fastidious strainsbelonging to the subsp. nova. A culture medium containing blood is required forthe primary isolation of atypical strains of this subspecies, including isolatesfrom carp, goldfish and eel and some fastidious isolates recovered fromsalmonids (Bootsma et al., 1977; Paterson et al., 1980; Böhm et al., 1986;Ishiguro et al., 1986; Olivier, 1992). Ishiguro et al. (1986) investigated thisphenomenon and found that fastidious atypical strains isolated from goldfishand salmon (including H. piscium) could grow well if conventional medium(TSA) was supplemented with haemin (10 µg ml–1): this was confirmed by Nakaiet al. (1989) with atypical strains isolated from Japanese eel (A. japonica).
Isolation plates are often contaminated with what is believed to beopportunistic pathogens, such as motile aeromonads and Pseudomonas spp.,thus complicating the diagnosis. This phenomenon has been reported by severalinvestigators in both natural infections and during experimental challenges withstrains causing carp erythrodermatitis (Csaba et al., 1984; Evenberg et al.,1986), goldfish ulcer disease (Whittington et al., 1987) and the causative agentof the ulcerative disease of eels (Nakai et al., 1989). Often, the organism is onlyisolated from skin lesions and the infection is not systemic (Elliot and Shotts,1980; Csaba et al., 1984; Böhm et al., 1986; Noga and Berkhoff, 1990). In rareinstances, the diagnosis can be complicated by the presence of both typical andatypical strains as reported by Noga and Berkhoff (1990) in American eels(Anguilla rostrata).
394 M. Hiney and G. Olivier
Biochemical identificationSimilar to typical isolates, the presumptive identification of atypical isolates isbased on a few characteristics, i.e. the isolates are Gram-negative coccobacilli,oxidase-positive and fermentative and do not grow at 37°C. It is, however,important to note that some atypical strains are oxidase-negative, includingisolates recovered from flounder (Wiklund and Bylund, 1991), herring (one outof four isolates) (Traxler and Bell, 1988), tom cod (Olivier, 1992) and turbot(Scophthalmus maximus) (Pedersen et al., 1994). These results strongly suggestthat care is needed to properly identify some of these atypical isolates, and thateven discrepancies in basic characteristics, such as the oxidase reaction, shouldnot necessarily cause a tentative identification to be rejected. Additional tests areneeded to ensure that atypical strains are not implicated in the condition underinvestigation. A schematic representation of the various steps that should betaken for the culture and identification of atypical strains of A. salmonicida ispresented in Table 10.13.
Differentiation between typical and atypical isolates is achieved based onthe following properties; atypical isolates are generally achromogenic, lack thecapacity to produce gas from glucose, utilize sucrose, produce indole and aregelatinase-negative (Popoff, 1984). All of these characteristics are useful for thedifferentiation of atypical isolates, although it must be borne in mind thatexceptions to the above have been reported. A more comprehensive list ofbiochemical characteristics is presented in Table 10.14, although this level ofinvestigation may not be necessary for routine diagnosis.
Serological identification of atypical Aeromonas salmonicidaBecause it may be difficult to grow atypical A. salmonicida, other diagnosticmethods have been explored, including immunofluorescence (fluorescentantibody test (FAT)), as described by Böhm et al. (1986). Using this method,serological confirmation of atypical furunculosis in carp and goldfish was atleast 50% higher than by culture. Sövényi (1986) described a coagglutinationtest for the diagnosis of carp erythrodermatitis and found that all experimentallyinduced ulcerative lesions were positive by this test, with no cross reactions withother fish pathogens being observed. The ELISA assay described by Adams andThompson (1990) was also able to detect atypical strains. Methods used to detecttypical isolates should be effective in detecting atypical isolates, because of theirserological similarities, and simple methods, such as FAT, could be used as aninitial screening for these pathogens. Modern techniques, such as PCR, willundoubtedly become available to detect these isolates, although, as has beendiscussed above, the routine diagnostic use of these methods cannot beconsidered without extensive validation of the techniques.
Control and treatment
Chemotherapeutic treatmentChemotherapy would appear to be the method of choice in dealing withinfections caused by atypical isolates (Csaba et al., 1984; Böhm et al., 1986;
395Furunculosis
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 beingevaluated; for example, Heo and Seo (1996) have found that ciprofloxacin canbe effective in protecting carp against infection with an atypical carp isolate. Aswith typical strains, development of antibiotic resistance has been recognized inatypical A. salmonicida. Hirvelä-Koski et al. (1994) reported resistance tosulphonamides, and strains isolated from turbot (Pedersen et al., 1994) werefound to be resistant to trimethoprim. In addition, Sandaa and Enger (1996)found that a plasmid encoding for multiple antibiotic resistance could betransferred from atypical strains to several marine bacteria.
VaccinationThere is a paucity of studies on vaccine development against diseases of atypicalA. salmonicida aetiology. Protection of carp against carp erythrodermatitis hasbeen investigated by Evenberg et al. (1986, 1988). These authors developed areproducible experimental challenge in carp, using the subcutaneous route. Theyfound that the i.m. route of immunization was superior to the i.p. route and thatwhole-cell vaccines or purified antigens conferred only marginal protection. Thebest 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 inthese fish was thought to be related to humoral immunity (antitoxoid antibodies)and not cellular immunity. A subsequent study (Daly et al., 1994) demonstratedthat carp could be successfully challenged by bath administration, and animalsexhibited classical signs of carp erythrodermatitis. During these studies, carpthat received sublethal infections were able to withstand subsequent lethalinfections 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 onthe mechanism of protection, but their results suggest that carp vaccinated with alive vaccine (sublethal infection with virulent bacteria) can be protected,indicating that cellular immunity might be important in providing protection inthis fish species.
Host resistanceAs an alternative to chemotherapy and vaccination, disease resistant fish havebeen investigated. The susceptibility to carp erythrodermatitis of a strain ofHungarian carp and F1 crosses with Japanese coloured carp was investigated bySövényi et al. (1988), who found that the morbidity, calculated by lesion size, ofthe hybrid fish was only half that of the homozygous group. A subsequent studyby Houghton et al. (1991), on the resistance of carp to erythrodermatitis,confirmed that some strains of carp were more resistant to this disease thanothers, the Hungarian line was more resistant than the Polish line and there weredifferences within each strain. These results indicate that breeding carp fordisease resistance could improve the health of fish stocks.
396 M. Hiney and G. Olivier
Tab
le 1
0.1
3.
Dia
gn
ost
ic p
roce
du
re t
o c
ult
ure
an
d id
enti
fy a
typ
ical
Aer
om
on
as s
alm
on
icid
a .
Test
Ch
arac
teri
stic
/res
ult
Cav
eats
/co
mm
ents
Sam
plin
g p
roce
du
reE
xter
nal
sam
ple
sS
amp
le f
rom
lesi
on
mat
eria
lS
amp
ling
pre
fera
ble
du
rin
g e
arly
or
acu
te p
has
e;te
st s
ever
al f
ish
Inte
rnal
sam
ple
sS
amp
les
fro
m k
idn
ey, s
ple
en, h
eart
Test
sev
eral
fis
hM
orp
ho
log
ical
tes
tsG
row
th o
n T
SA
or
BH
IAO
nly
no
n-f
asti
dio
us
stra
ins
will
gro
w o
n t
hes
eM
any
stra
ins
do
no
t p
rod
uce
pig
men
t; in
cub
ate
for
agar
s; d
elay
ed p
igm
ent
pro
du
ctio
n a
fter
at le
ast
7 d
ays
exte
nd
ed in
cub
atio
n a
t 18
–22°
C
Gro
wth
on
blo
od
ag
arFa
stid
iou
s st
ain
s re
qu
ire
this
med
ium
; sm
all,
Incu
bat
e fo
r at
leas
t 7
day
scr
eam
y co
lon
ies
stro
ng
ly a
dh
erin
g t
o t
he
med
ium
Gro
wth
tem
per
atu
reG
row
th a
t 18
–22°
C; n
o g
row
th a
t 37
°CS
trai
ns
wh
ich
gro
w a
t 37
°C h
ave
bee
n r
epo
rted
(Au
stin
, 199
3)S
edim
enta
tio
n t
est
Cel
ls a
uto
agg
luti
nat
e in
0.8
5% P
BS
Gra
m s
tain
Sh
ort
Gra
m-n
egat
ive
rod
sH
ang
ing
dro
p t
est
No
n-m
oti
leS
ero
log
ical
tes
tsS
lide
agg
luti
nat
ion
Clu
mp
ing
(so
uri
ng
) o
f an
tib
od
y-co
ated
late
x in
Alw
ays
incl
ud
e p
osi
tive
an
d n
egat
ive
con
tro
l str
ain
s(p
resu
mp
tive
solu
tio
n w
ith
cel
l su
spen
sio
nb
ecau
se o
f p
oss
ible
au
toag
glu
tin
atio
n o
f A
.id
enti
fica
tio
n o
nly
)sa
lmo
nic
ida
Bio
chem
ical
tes
tsC
yto
chro
me
oxi
das
eO
xid
ase-
po
siti
veO
xid
ase-
neg
ativ
e is
ola
tes
hav
e b
een
des
crib
edp
rod
uct
ion
(Tra
xler
& B
ell,
1988
; Oliv
ier,
199
2; P
eder
son
et
al.,
1994
; Wik
lun
d e
t al
., 19
94)
397Furunculosis
Cat
alas
e p
rod
uct
ion
Cat
alas
e-p
osi
tive
Cat
alas
e-n
egat
ive
iso
late
s h
ave
bee
n d
escr
ibed
(Csa
ba
et a
l., 1
984;
Bö
hm
et
al.,
1986
)In
do
le p
rod
uct
ion
Ind
ole
-po
siti
veIn
do
le-n
egat
ive
iso
late
s h
ave
bee
n d
escr
ibed
(Au
stin
et
al.,
1989
; Wik
lun
d, 1
990;
Oliv
ier,
199
2)O
xid
atio
n/f
erm
enta
tio
nFe
rmen
tati
veIn
cub
ate
at 2
5°C
; ch
eck
afte
r 5
and
7 d
ays
test
(H
ug
h/L
eifs
on
)S
ub
stra
te u
tiliz
atio
nS
ucr
ose
-po
siti
ve; d
o n
ot
pro
du
ce g
as f
rom
No
t al
l str
ain
s w
ill b
e p
osi
tive
fo
r th
ese
test
sg
luco
seR
esis
tan
ce t
oR
esis
tan
t to
am
pic
illin
(25
mg
) an
dIn
cub
ate
at 2
5°C
; ch
eck
afte
r 5
and
7 d
ays;
no
tan
tim
icro
bia
l ag
ents
cep
hal
ori
din
(15
mg
)al
l str
ain
s w
ill b
e p
osi
tive
fo
r th
ese
test
s
398 M. Hiney and G. Olivier
Table 10.14. Biochemical characteristics distinguishing between typical andatypical strains of Aeromonas salmonicida*.
Aeromonas salmonicida subspp.
Characteristic salmonicida masoucida acromogenes nova smithia
Pigment + – V – –Haemin requirement – – – + .Gas from glucose + + – – .Arbutine + + V – .Sucrose – + + V VSalicin + – 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 + – + – VResistance toAmpicillin (25 µg) – + + + +Cephaloridine (15 µg) – + + V .Polymyxin B (300 IU) – – – V .Salmonid source + + V V –
*Differential characteristics of Aeromonas salmonicida subspecies based onMcCarthy (1978), Popoff (1984), Austin et al. (1989), Wichart et al. (1989) andOlivier (1992, unpublished results).+, –, V indicate that > 80%, < 20% and 21–79% of the strains gave positivereactions, 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, theinfection does not necessarily become systemic and the cause of death is notalways substantiated (reviewed by Trust, 1986). Work by Bucke (1980) providedevidence that atypical isolates could be pathogenic not only for their originalhost but for other species as well. In this particular study, Bucke (1980)inoculated i.m. brown trout, perch, rudd, roach, carp and goldfish with threestrains of A. salmonicida, including a typical isolate, a cyprinid atypical isolateand a goldfish isolate (unfortunately, the author did not provide the exact numberof bacteria injected). His results clearly indicated that there were differences inthe virulence of the isolates towards different hosts; for example, the typical
399Furunculosis
isolate was virulent for trout, roach and rudd, while the cyprinid isolate was onlyvirulent for brown trout and the goldfish isolate was virulent for all the speciestested.
In some cases, atypical strains isolated from salmonids can be almost asvirulent as typical isolates. The atypical strain isolated from Atlantic salmon inNewfoundland, Canada, has an LD50 of less than 50 bacteria for juvenile Atlanticsalmon, following an i.p. challenge (Olivier et al., 1990; Olivier, 1992). Othersalmonid isolates from Sweden, Norway and Iceland were also virulent forsalmonids, with LD50 values of 1 × 102–3 for Atlantic salmon (Olivier et al.,1990), confirming the results of Gudmundsdóttir et al. (1997) with similarisolates 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 betweendorsal scales with 1 × 106 cfu (Evenberg et al., 1988). The LD50 of an eel isolatewas approximately 1 × 103 cfu by i.m. injection into healthy eels (Ohtsuka et al.,1984); similarly, herring isolates were highly virulent for their host, killing 50%of fish injected with fewer than 100 cfu (Traxler and Bell, 1988). A goldfishisolate is highly virulent for goldfish, with LD50 less than 1 × 104 cfu (Trustet al., 1980b), while a wrasse isolate had a low LD50 (5 × 102) when determinedin wrasse by i.p. injection. Other isolates were not found to be as virulent fortheir host: isolates from cod, flounder, turbot and minnow had LD50 valueshigher than 1 × 106 (Håstein et al., 1978; Cornick et al., 1984; Wiklund, 1995b).
As salmonid culture represents an important asset in several countries,several non-salmonid isolates have been tested for their possible virulence insalmonids. There is no doubt that one goldfish isolate can be highly virulent forsalmonids after i.p. injection, with LD50 values ranging from 1 × 102 to 1 × 103
injected cells. This has been confirmed by other studies, in which goldfishisolates were found to be virulent for Atlantic salmon, brook trout and rainbowtrout under different challenge conditions, including i.p. injection and bathchallenge, with or without prior skin abrasion, and by cohabitation (Carson andHandlinger, 1988; Whittington and Cullis, 1988). In addition, a sand eel isolatekilled 50% of rainbow trout injected i.p. with 8 × 106 cfu (Dalsgaard andPaulsen, 1986). Isolates from other species, including flounder, turbot, wrasseand 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 tobe virulent for salmonids under laboratory challenge conditions, there are fewreports of atypical strains of non-salmonid origin causing losses in salmonids inthe field. However, in one instance a sable fish isolate was found to infectcultured Pacific salmon, but other virulent non-salmonid isolates, such as thegoldfish strain, have not been reported from salmonids to date. Furthermore,there is little evidence of crossover of atypical strains from one non-salmonidspecies to another, although one goldfish isolate has been found to infect silverperch (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, unpublishedresults).
400 M. Hiney and G. Olivier
Virulence factorsThe virulence factors of atypical A. salmonicida strains are not as wellcharacterized as those of typical strains (Trust, 1986). Atypical strains are able tosequester iron under conditions of iron limitation, but their ability is less thanthat reported for typical strains and some atypical strains are able to utilizesiderophores produced by typical isolates (Chart and Trust, 1983). According toHirst et al., (1991, 1994), the iron-uptake mechanism of atypical strains issiderophore-independent, in contrast to typical strains. These results wererecently confirmed when Hirst and Ellis (1996) demonstrated that typical andatypical 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 extracellularserine protease. Atypical strains utilize transferrin by a siderophore-independentmechanism, probably involving the proteolytic degradation of transferrin by theextracellular metalloprotease.
As stated previously, most atypical strains examined so far possess surfacestructures (A-layer and LPS) similar to those of typical isolates. Smalldifferences have been noted in the electrophoretic mobility of both the A-layerand LPS of atypical strains (Kay et al., 1984; Evenberg et al., 1985; Griffiths andLynch, 1990). In the only report where a correlation between surface structureand virulence was tested, Trust et al. (1980a) demonstrated that an i.m. injectionof 1 × 104 A-layer-positive cells in goldfish killed all test animals (8/8), whereasa similar injection of 108 cfu of an A-layer-negative isogenic strain killed onlyone goldfish. Except for these results, the role of surface structure in thepathogenicity of atypical strains has not been fully assessed, but it would not besurprising if the important role of the A-layer and additional surface structures asvirulence factors were confirmed in atypical isolates.
Extracellular virulence factors produced by atypical strains are poorlyunderstood. Pol et al. (1981) have reported that the ECP of one atypical strainwas 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 growthconditions. Hastings and Ellis (1985) noted differences in the production ofhaemolysin and protease between typical and atypical strains, and Gudmunds-dóttir et al. (1990) have purified and characterized a new toxic protease from anatypical 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 intypical isolates and thus may be specific for atypical strains. A recent study of 25atypical strains isolated from a variety of hosts indicates that the proteasesproduced by atypical strains are variable, and the author was able to differentiatefive groups, based on protease properties (Gudmundsdóttir, 1996). Comparedwith typical strains which were homogeneous in producing protease andgelatinase, atypical strains were heterogeneous.
The possibility that atypical strains can cause immunosuppression in carpwas investigated by Evenberg et al. (1986). Infected carp given a sublethal i.m.injection showed a progressive decrease in total serum protein and immuno-
401Furunculosis
globulin (Ig) levels but differential blood counts did not differ. In carp immunizedwith sheep erythrocytes, a sublethal infection produced a reduction of plaque-forming cells and serum antibodies against sheep erythrocytes, but the cellularresponse, tested by skin allograft rejection, was enhanced as the disease progressed(Pourreau et al.,1986). Further studies by Pourreau et al. (1987) indicated thatcrude supernatants of a virulent atypical strain modulated the carp immuneresponse, when tested by mitogen stimulation of carp leucocytes. Stimulation ofleucocytes was enhanced by the supernatant from a young culture (20 h) of thevirulent strain but was severely inhibited by supernatants from older cultures (96h). This inhibitory activity was lost after heat treatment, suggesting it was due to aproteinaceous structure, but the substance was not characterized further.
With respect to atypical strains causing ‘furunculosis’ in salmonids, Gud-mundsdóttir et al. (1995) have provided evidence that bacterins prepared froman atypical strain isolated from salmonids in Iceland can provide protectionagainst the disease. Further work by Gudmundsdóttir et al. (1997) showed thatAtlantic salmon vaccinated with detoxified ECP provided better protection thanformalin-killed cells or a mixture of both bacterins. Since passive immunizationwith rainbow trout or rabbit antiprotease (AsaP1) was demonstrated to beefficacious and since the same antisera, but containing anti-A-layer antibodies,were not protective, the authors conclude that the protection of Atlantic salmonagainst atypical A. salmonicida is probably due to humoral immunity, withantibodies directed against bacterial toxins contained in the ECP.
FUTURE RESEARCH
There is still much to be learned about A. salmonicida and its associatedpathologies. However, as long as there is continued availability of oil-basedvaccines, furunculosis will no longer be perceived as a major problem forcommercial marine salmonid farmers. Therefore, future research must focus onthe appropriate and sustainable management of freshwater fisheries, inparticular restoration programmes and native fisheries. Many of the diseasecontrol strategies available to commercial farmers such as vaccination andchemotherapy may not be appropriate in these situations. For example, theadvisability of vaccinating fish with oil-based preparations prior to their releaseinto river systems, from which they may be removed for consumption by anglersmust be questioned. Oil based adjuvants are highly immunogenic, not only forfish but for consumers of those fish. Likewise, the presence of chemotherapeuticresidues in the flesh of fish which are available for consumption is undesirable.Therefore, the control strategies that can be adopted by fresh water fisheriesmanagers will be affected by different concerns than the more controlledcommercial sector.
A number of important questions can be identified for fresh water fisheriesin relation to furunculosis. These may be summarized as follows:
• do fresh water hatcheries act as amplifiers of disease for the water body onwhich 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 haveon their survival and on resident fish populations, and how can this impactbe minimized or eliminated
• does vaccination of covertly infected hatchery stocks prior to releaseconstitute the creation of ‘immune carriers’, what impact are these ‘immunecarriers’ likely to have on non-vaccinated fish and how can this impact beminimized or eliminated
• how can hatchery reared stocks be protected from the impact of anadromousfish in the water body on which the hatchery is situated.
Many of these questions have yet to be answered. It is hoped, therefore, thatfuture research into furunculosis will address the fundamental concerns outlinedhere. Only in this way will we limit both the commercial and biological impactof furunculosis among valuable and endangered fish populations. Non-culture-based methods may have a valuable contribution to make to the study offurunculosis epizootiology but much work on the validation of these techniquesneeds to be done before they can be applied to field studies. The presence ofatypical strains in wild marine fish with ulcerations is also of concern and theirimpact on wild fish populations is still unknown. The importance of theseisolates originating from wild marine fish could represent a threat to the futureculture of these species in aquaculture conditions.
A second important problem associated with atypical A. salmonicidaisolates is that they are highly variable in their biochemical profile and cannot beeasily placed in known or accepted subspecies. Further work on their taxonomywill be necessary to unravel this basic problem. Results so far indicate thatatypical strains are for the most part highly restricted to precise geographicareas, however, some strains including goldfish and carp isolates have a widedistribution probably due to the international trade of these species. The limitedwork carried out to date would indicate that atypical strains are restricted to theirhost of origin, this is especially true for atypicals isolated from non-salmonidfish. Most of the strains isolated from these various hosts have so far beenbiochemically different. More work on this issue will, however, be necessary togain a fuller epizootiological picture of atypical A. salmonicida and itsassociated 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 helpfulcomments and editorial advice.
REFERENCES
Adams, A. and Thompson, K. (1990) Development of an enzyme-linked immunosorbentassay (ELISA) for the detection of Aeromonas salmonicida in fish tissue. Journal ofAquatic Animal Health 20, 281–288.
403Furunculosis
Alderman, D.J., Rosenthal, H., Smith, P., Stewart, J. and Weston, D. (1994) ChemicalsUsed in Mariculture. Technical Report of ICES Working Group, EnvironmentalInteractions of Mariculture, Report 202, International Council for the Explorationof 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 andWildlife Service Resource Publication 88, 65–67.
Anderson, D.P., Rogerson, B.S. and Dixon, O.W. (1979) Induction of antibody-producing cells in rainbow trout, Salmo gairdneri Richardson, by flush exposure.Journal of Fish Biology 15, 317–322.
Anderson, D.P., Jeney, G., Rumsey, G.L. and Siwicki, A.K. (1997) Adjuvants andimmunostimulants for potentiating protection against furunculosis in fish. In:Bernoth, E.-M., Ellis, A.E., Midtlyng, P.J., Olivier, G. and Smith, P. (eds)Furunculosis: Multidisciplinary Fish Disease Research. Academic Press, London,pp. 345–365.
Andrews, C. (1981) Latent infections with Aeromonas salmonicida. Bulletin of theEuropean Association of Fish Pathologists 1, 25.
Arkwright, J.A. (1912) An epidemic disease affecting salmon and trout in Englandduring the summer of 1911. Journal of Hygiene 12, 391–413.
Armstrong, R. (1992) Histopathological lesions associated with salmonid furunculosis.Bulletin of the Aquaculture Association of Canada 92–1, 31–35.
Austin, B. (1993) Recovery of “atypical” isolates of Aeromonas salmonicida, whichgrow at 37°C, from ulcerated non-salmonids in England. Journal of Fish Diseases16, 165–168.
Austin, B. and Austin, D.A. (1993) Bacterial Fish Pathogens: Diseases in Farmed andWild Fish, 2nd edn. Ellis Horwood, London.
Austin, B. and McIntosh, D. (1988) Natural antibacterial compounds on the surface onrainbow trout, Salmo gairdneri Richardson. Journal of Fish Diseases 11, 275–277.
Austin, B., Bishop, I., Gray, C., Watt, B. and Dawes, J. (1986) Monoclonal antibodybased enzyme linked immunosorbent assays for the rapid diagnosis of clinical casesof enteric redmouth and furunculosis in fish farms. Journal of Fish Diseases 9,469–474.
Austin, D.A., McIntosh, D. and Austin, B. (1989) Taxonomy of fish associatedAeromonas spp., with the description of Aeromonas salmonicida subsp. smithiasubsp. nov.. Systematic and Applied Microbiology 11, 277–290.
Banneke, S. and Bernoth, E.-M. (1994) Immuno-dotblots vor Forellengeweben:Reduzierung der Hintergrundfärbung durch Bleichung mit Kaliumpermanganat undWasserstoffperoxide. In: Proceedings of Conference of the German Branch of theEuropean Association of Fish Pathologists, Wolfegg, Germany. (Cited in Bernoth,1997a).
Barker, G.A. and Kehoe, E. (1995) Assessment of disc diffusion methods forsusceptibility testing of Aeromonas salmonicida. Aquaculture 134, 1–8.
Barry, T., Powell, R. and Gannon, F. (1990) A general method to generate DNA probesfor microorganisms. Biotechnology 8, 233–236.
Bast, L., Daly, J.G., DeGrandis, S.A. and Stevenson, R.M.W. (1988) Evaluation ofprofiles of Aeromonas salmonicida as epidemiological markers of furunculosisinfections. Journal of Fish Diseases 11, 133–145.
Bell, G.R., Hoffman, R.W. and Brown, L.L. (1990) Pathology of experimental infectionsof the sablefish (Anoplopoma fimbria) with Renibacterium salmoninarum, the agentof bacterial kidney disease in salmonids. Journal of Fish Diseases 13, 355–367.
404 M. Hiney and G. Olivier
Belland, R.J. and Trust, T.J. (1985) Synthesis, export, and assembly of Aeromonassalmonicida A-layer analyzed by transposon mutagenesis. Journal of Bacteriology163, 877–881.
Belland, R.J. and Trust, T.J. (1988) DNA:DNA reassociation analysis of Aeromonassalmonicida. Journal of General Microbiology 134, 307–315.
Benediktsdóttir, E. and Helgason, S. (1990) The isolation of Aeromonas salmonicidasubsp. achromogenes from the gills of salmonid fish. Bulletin of the EuropeanAssociation of Fish Pathologists 10, 104–105.
Bergey, D.H. (ed.) (1984). Bergey’s Manual of Systematic Bacteriology. Williams andWilkins, Baltimore.
Bernoth, E.-M. (1990a) Autoagglutination, growth on tryptone-soy-coomassie-agar,outer membrane protein patterns and virulence of Aeromonas salmonicida strains.Journal of Fish Diseases 13, 145–155.
Bernoth, E.-M. (1990b) Screening for the fish disease agent Aeromonas salmonicidawith an enzyme-linked immunosorbent assay (ELISA). Journal of Aquatic AnimalHealth 2, 99–103.
Bernoth, E.-M. (1997a) Furunculosis: the history of the disease and of disease research.In: Bernoth, E.-M., Ellis, A.E., Midtlyng, P.J., Olivier, G. and Smith, P. (eds)Furunculosis: Multidisciplinary Fish Disease Research. Academic Press, London,pp. 1–20.
Bernoth, E.-M. (1997b) Diagnosis of furunculosis: the tools. In: Bernoth, E.-M., Ellis,A.E., Midtlyng, P.J., Olivier, G. and Smith, P. (eds) Furunculosis: MultidisciplinaryFish Disease Research. Academic Press, London, pp. 98–1158.
Bernoth, E.-M. and Artz, G. (1989) Presence of Aeromonas salmonicida in fish tissuemay be overlooked by sole reliance on furunculosis-agar. Bulletin of the EuropeanAssociation of Fish Pathologists 9, 5.
Bernoth, E.-M. and Böhm, K.M. (1988) Serological comparison between isolated strainsof Aeromonas salmonicida by agglutination and enzyme-linked immunosorbentassay (ELISA). Journal of Veterinary Medicine 35, 637–647.
Bernoth, E.-M. and Körting, W. (1992) Identification of a cyprinid fish, the tench Tincatinca L., as a carrier of the bacterium Aeromonas salmonicida, causative agent offurunculosis in salmonids. Journal of Veterinary Medicine 39, 585–594.
Bernoth, E.-M., Ellis, A.E., Midtlyng, P.J., Olivier, G. and Smith, P. (eds) (1997)Furunculosis: Multidisciplinary Fish Disease Research. Academic Press, London,529 pp.
Blake, I. and Clark, J.C. (1931) Observations on experimental infection on trout by B.salmonicida: with particular reference to ‘carriers’ of furunculosis, and to certainfactors influencing susceptibility. Fisheries Board of Scotland, Salmon Fisheries 7,1–13.
Böhm, K.H., Furhmann, H., Schlotfeldt, H.J. and Körting, W. (1986) Aeromonassalmonicida from salmonids and cyprinids, serological and cultural identification.Journal of Veterinary Medicine B33, 777–783.
Bonet, R., Simon-Pujol, M.D. and Congregado, F. (1993) Effects of nutrients onexopolysaccharide production and surface properties of Aeromonas salmonicida.Applied and Environmental Microbiology 59, 2437–2441.
Boomker, J., Henton, M.M., Naudè, T.W. and Hunchzermeyer, F.W. (1984) Furunculosisin rainbow trout (Salmo gairdneri) raised in sea water. Onderstepoort Journal ofVeterinary Research 51, 91–94.
Bootsma, R., Fijan, N. and Blommaert, J. (1977) Isolation and preliminary identificationof the causative agent of carp erythrodermatitis. Veterinarski Arhiv 47, 291–302.
Boyd, E.F., Hiney, M.P., Peden, J.F. and Smith, P.R. (1994) Assessment of genetic
405Furunculosis
diversity among Aeromonas salmonicida isolates by multilocus enzyme electro-phoresis. Journal of Fish Diseases 17, 97–98.
Bragg, R.R. (1991) Health status of salmonids in river systems in Natal. III. Isolation andidentification of bacteria. Onderstepoort Journal of Veterinary Research 58, 67–70.
Brazil, G., Curley, D., Gannon, F. and Smith, P. (1986) Persistence and acquisition ofantibiotic resistance plasmids in Aeromonas salmonicida. In: Antibiotic ResistanceGenes: Ecology, Transfer, and Expression. Branbury Report 24, Cold Spring HarborLaboratory, New York, pp. 107–114.
Bricknell, I.R. and Ellis, A.E. (1993) The development of an effective vaccine againstfurunculosis. Fish Farmer 16, 16–19.
Bucke, D. (1980) Experimental and naturally occurring furunculosis in various fishspecies: a comparative study. In: Ahne, W. (ed.) Fish Diseases, Springer-Verlag,Springer-Verlag, Berlin, pp. 82–86.
Buckley, R.V. (1969) A furunculosis epizootic in clear lake yellow bass. Bulletin of theWildlife Diseases Association 5, 322–327.
Bullock, G.L. and Cipriano, R.C. (1997) Detection of Aeromonas salmonicida by cultureand serodiagnostic assays following stress induced furunculosis tests. Abstract. In:Proceedings of 22nd Annual Eastern Fish Health Workshop, Atlantic Beach, NorthCarolina.
Bullock, G.L. and Stuckey, H.M. (1975) Aeromonas salmonicida: detection ofasymptomatically infected trout. Progressive Fish Culturist 37, 237–239.
Bullock, G.L., Cipriano, R.C. and Schill, W.B. (1997). Culture and serodiagnosticdetection of Aeromonas salmonicida from covertly infected rainbow trout given thestress induced furunculosis test. Biomedical Letters 55, 169–177.
Byers, H., Gudkovs, N. and Crane, M. (1997) Identification of Aeromonas salmonicida –validation of PCR tests. Abstract. In: Proceedings of Diseases of Fish and Shellfish.8th International Conference of the European Association of Fish Pathologists,Edinburgh, Scotland.
Carson, J. and Handlinger, J. (1988) Virulence of the aetiological agent of goldfish ulcerdisease in Atlantic salmon, Salmo salar L. Journal of Fish Diseases 11, 471–479.
Cazabon, D., O’Grady, P., Moloney, M. and Smith, P. (1994) Factors affecting theefficiency of bath administration of flumequine. Bulletin of the EuropeanAssociation of Fish Pathologists 14, 197–199.
Chapman, P.F., Cipriano, R.C. and Teska, J.D. (1991) Isolation and phenotypiccharacterization of an oxidase-negative Aeromonas salmonicida causingfurunculosis in Coho salmon (Oncorhynchus kisutch). Journal of Wildlife Diseases27, 61–67.
Chart, H., and Trust, T.J. (1983) Acquisition of iron by Aeromonas salmonicida. Journalof Bacteriology 156, 758–764.
Chart, H., Shaw, D.H., Ishiguro, E.E. and Trust, T.J. (1984) Structural andimmunochemical homogeneity of Aeromonas salmonicida lipopolysaccharide.Journal of Bacteriology 158, 16–22.
Chu, S., Cavaignac, S., Feutrier, J., Phipps, B.M., Kostrzynska, M., Kay, W.W. and Trust,T.J. (1991) Structure of the tetragonal surface virulence array protein and gene ofAeromonas salmonicida. Journal of Biological Chemistry 23, 15258–15265.
Cipriano, R.C. (1986) Immunodetection of antibodies in the mucus of rainbow troutinfected with or immunized against the furunculosis agent Aeromonas salmonicida.Microbios Letters 32, 105–112.
Cipriano, R.C. (1997) Strategies for management of furunculosis in Atlantic salmoneffected by non-lethal detection of Aeromonas salmonicida. Bulletin of theEuropean Association of Fish Pathologists 17, 215–219.
406 M. Hiney and G. Olivier
Cipriano, R.C. and Bertolini, J. (1988) Selection for virulence in the fish pathogenAeromonas salmonicida using Coomassie brilliant blue agar. Journal of WildlifeDiseases 24, 672–678.
Cipriano, R.C. and Blanch, A.R. (1989) Different structural characteristics in the cellenvelope of the fish pathogen Aeromonas salmonicida. Microbios Letters 40, 87–95.
Cipriano, R.C. and Heartwell, C.M. (1986) Susceptibility of salmonids to furunculosis:differences between serum and mucus responses against Aeromonas salmonicida.Transactions of the American Fisheries Society 115, 83–88.
Cipriano, R.C. and Pyle, S.W. (1985) Adjuvant-dependent immunity and the agglutininresponse of fishes against Aeromonas salmonicida, cause of furunculosis. CanadianJournal of Fisheries and Aquatic Sciences 38, 1322–1326.
Cipriano, R.C. and Starliper, C.E. (1982) Immersion and injection vaccination ofsalmonids against furunculosis with an avirulent strain of Aeromonas salmonicida.Progressive Fish Culturist 44, 167–169.
Cipriano, R.C., Griffin, B.R. and Lidgerding, B.C. (1981) Aeromonas salmonicida:Relationship between extracellular growth products and isolate virulence.Canadian Journal of Fisheries and Aquatic Sciences 38, 1322–1326.
Cipriano, R.C., Ford, L.A., Teska, J.D. and Hale, L.E. (1992) Detection of Aeromonassalmonicida in the mucus of salmonid fishes. Journal of Aquatic Animal Health 4,114–118.
Cipriano, R.C., Ford, L.A., Schachte, J.H. and Petrie, C. (1994) Evaluation of mucus asa valid site to isolate Aeromonas salmonicida among asymptomatic populations oflake trout (Salvelinus namaycush). Biomedical Letters 49, 229–233.
Cipriano, R.C., Ford, L.A., Nelson, J.T. and Jenson, B.G. (1996a) Monitoring for earlydetection of Aeromonas salmonicida to enhance antibiotic therapy and controlfurunculosis in Atlantic salmon (Salmo salar). Progressive Fish Culturist 58, 203–208.
Cipriano, R.C., Ford, L.A., Starliper, C.E. and Teska, J.D. (1996b) Control of externalAeromonas salmonicida: topical disinfection of salmonids with chloramine-T.Journal of Aquatic Animal Health 8, 52–57.
Cipriano, R.C., Ford, L.A., Teska, J.D., Schachte, J.H., Petrie, C., Movak, B.M. andFlint, D.E. (1996c) Use of non-lethal procedures to detect and monitor Aeromonassalmonicida in potentially endangered or threatened populations of migrating andpost-spawning salmon. Diseases of Aquatic Organisms 27, 233–236.
Cipriano, R.C., Ford, L.A., Smith, D.R., Schachte, J.H. and Petrie, C.J. (1997)Differences in detection of Aeromonas salmonicida in covertly infected salmonidfishes by the stress-inducible furunculosis test and culture-based assays. Journal ofAquatic Animal Health 9, 108–113.
Colwell, R.R., McDonnell, M.T. and Deley, J. (1986) Proposal to recognise the familyAeromonadaceae fam. nov. International Journal of Systematic Bacteriology 36,473–477.
Cornick, J.W., Chudyk, R.V. and McDermott, L.A. (1969) Habitat and viability studieson Aeromonas salmonicida, causative agent of furunculosis. Progressive FishCulturist 31, 90–93.
Cornick, J.W., Morrison, C.M., Zwicker, B. and Shum, G. (1984) Atypical Aeromonassalmonicida infection in Atlantic cod, Gadus morhua L. Journal of Fish Diseases 7,495–499.
Coyne, R., Hiney, M. and Smith, P. (1994) Evidence associating overfeeding on asalmon farm with a prolonged half-life of oxytetracycline in undercage sediments.Bulletin of the European Association of Fish Pathologists 14, 207–210.
Crane, M., St. J. and Bernoth, E.-M. (1996) Molecular biology and fish disease
407Furunculosis
diagnosis: Current status and future trends. In: Asche, V. (ed.) Recent Advances inMicrobiology. Australian Society for Microbiology, Melbourne, pp. 41–82.
Csaba, G. and Szakolczai, J. (1991) Erythrodermatitis in silver carp and big head.Abstract. In: Proceedings of Diseases of Fish and Shellfish, 5th InternationalConference of the European Association of Fish Pathologists, Budapest, Hungary.
Csaba, G., Kormendy, B. and Békési, L. (1984) Observations on the causative agent ofcarp erythrodermatitis in Hungary. Symposia Biologica Hungarica 23, 63–74.
Dahle, G., Hjeltnes, B. and Jørstad, K.E. (1996) Infection of Atlantic salmon siblinggroups with infectious salmon anaemia (ISA) and furunculosis. Bulletin of theEuropean Association of Fish Pathologists 16, 192–195.
Dalsgaard, I. and Paulsen, H. (1986) Atypical Aeromonas salmonicida isolated fromdiseased sand-eels, Ammodytes lancea (Cuvier) and Hyperoplus lanceolatus(Lesauvege). Journal of Fish Diseases 9, 361–364.
Dalsgaard, I., Nielsen, B. and Larsen, J.L. (1994) Characterization of Aeromonassalmonicida subsp. salmonicida: a comparative study of strains of differentgeographic origin. Journal of Applied Bacteriology 77, 21–30.
Daly, J.G. and Stevenson, R.M.W. (1985) Importance of culturing several organs todetect Aeromonas salmonicida in salmonid fish. Transactions of the AmericanFisheries Society 114, 909–910.
Daly, J.G., Wiegertjes, G.F. and Van Muiswinkel, W.B. (1994) Protection against carperythrodermatitis following bath or subcutaneous exposure to sublethal numbers ofvirulent Aeromonas salmonicida subsp. nova. Journal of Fish Diseases 17, 67–75.
Davidson, G.A., Ellis, A.E. and Secombes, C.J. (1993) Route of immunizationinfluences the generation of antibody secreting cells in the gut of rainbow trout(Oncorhynchus mykiss). Developmental and Comparative Immunology 17, 373–376.
Department of Fisheries and Oceans, Canada (1984) Fish Health ProtectionRegulations: Manual of Compliance, Revised. Miscellaneous Special Publications31, Ottawa, Canada.
Department of the Interior (1993) Fish and Wildlife Service. 50 CFR Part 16. SalmonidImportation Regulations. Federal Register 58(213), 976–981.
Donlon, J., McGettigan, S., O’Byrne, P. and O’Carra, P. (1983) Re-appraisal of thenature of the pigment produced by Aeromonas salmonicida. FEMS MicrobiologyLetters 19, 285–290.
Drinan, E. (1985) Studies on the pathogenesis of furunculosis in salmonids. PhD thesis,National University of Ireland.
Drinan, E.M. and Smith, P.R. (1985) Histopathology of a mutant Aeromonassalmonicida infection in Atlantic salmon (Salmo salar). In: Ellis, A.E. (ed.) Fishand Shellfish Pathology. Academic Press, London, pp. 79–83.
Drinan, E.M., Donlon, J., Molloy, S. and Smith, P.R. (1989) Observations on the role ofthe caseinase enzyme in the pathogenesis of furunculosis. Journal of Fish Diseases12, 45–50.
Duff, D.C.B. (1941) The oral immunization of trout against Bacterium salmonicida.Journal of Immunology 44, 87–94.
Eggset, G., Mikkelsen, H. and Killie, J.-E.A. (1997) Immunocompetence and duration ofimmunity against Vibrio salmonicida and Aeromonas salmonicida after vaccinationof Atlantic salmon (Salmo salar L.) at low and high temperatures. Fish and ShellfishImmunology 7, 247–260.
Egidius, E. (1987) Import of Furunculosis to Norway with Atlantic Salmon Smolts fromScotland. International Council for the Exploration of the Sea Report C.M 1987/F:8.
408 M. Hiney and G. Olivier
Elliot, D.G. and Shotts, E.B. (1980) Aetiology of an ulcerative disease in goldfish,Carassius auratus (L): microbiological examination of diseased fish from sevenlocations. Journal of Fish Diseases 3, 13–143.
Ellis, A.E. (1982) Histamine, mast cells and anaphylactic responses in fish.Developmental and Comparative Immunology Suppl. 2, 147–155.
Ellis, A.E. (1985) Eosinophilic granular cells (EGC) and histamine response in fish.Developmental and Comparative Immunology 9, 251–260.
Ellis, A.E. (1991) An appraisal of the extracellular toxins of Aeromonas salmonicida ssp.salmonicida. Journal of Fish Diseases 14, 265–277.
Ellis, A.E. (1997b) Furunculosis: protective antigens and mechanisms. In: Bernoth,E.-M., Ellis, A.E., Midtlyng, P.J., Olivier, G. and Smith, P. (eds) Furunculosis:Multidisciplinary Fish Disease Research. Academic Press, London, pp. 366–381.
Ellis, A.E. (1997a) The extracellular toxins of Aeromonas salmonicida subsp.salmonicida. In: Bernoth, E.-M., Ellis, A.E., Midtlyng, P.J., Olivier, G. and Smith, P.(eds) Furunculosis: Multidisciplinary Fish Disease Research. Academic Press,London, pp. 248–268.
Ellis, A.E., Hastings, T.S. and Munro, A.L.S. (1981) The role of Aeromonas salmonicidaextracellular products in the pathology of furunculosis. Journal of Fish Diseases 4,41–51.
Ellis, A.E., Burrows, A.S. and Stapleton, K.J. (1988) Lack of relationship betweenvirulence of Aeromonas salmonicida and the putative virulence factors: A-layer,extracellular proteases, and extracellular haemolysins. Journal of Fish Diseases 11,309–323.
Ellis, A.E., Bricknell, I.R. and Munro, A.L.S. (1992) Current status of a Scottishfurunculosis vaccine. Bulletin of the Aquaculture Association of Canada 92,11–15.
Emmerich, R. and Weibel, E. (1894) Ueber eine durch Bakterien erengte Seuche unterden Forellen. Archives fur Hygiene und Bakteriologie 21, 1–21.
Enger, Ø. (1997) Survival and inactivation of Aeromonas salmonicida outside the host –a most superficial way of life. In: Bernoth, E.-M., Ellis, A.E., Midtlyng, P.J., Olivier,G. and Smith, P. (eds) Furunculosis: Multidisciplinary Fish Disease Research.Academic Press, London, pp. 159–177.
Enger, Ø. and Thorsen, B.K. (1992) Possible ecological implications of the high cellsurface hydrophobicity of the fish pathogen Aeromonas salmonicida. CanadianJournal of Microbiology 38, 1048–1052.
Erdal, J.I. and Reitan, L.J. (1992) Immune response and protective immunity aftervaccination of the Atlantic salmon (Salmo salar L.) against furunculosis. Fish andShellfish Immunology 2, 99–108.
Evelyn, T.P.T. (1971) An aberrant strain of the bacterial fish pathogen Aeromonassalmonicida isolated from a marine host, the sablefish (Anoplopoma fimbria) andfrom two species of cultured pacific salmon. Journal of the Fisheries ResearchBoard of Canada 28, 1629–1634.
Evenberg, D. and Lugtenberg, B. (1982) Cell surface of the fish pathogenic bacteriumAeromonas salmonicida. II. Purification and characterization of a major cellenvelope related to autoagglutination, adhesion and virulence. Biochemica etBiophysica Acta 684, 249–254.
Evenberg, D., VanBoxtel, R., Lugtenberg, B., Schurer, F., Blommaert, J. and Bootsma,R. (1982) Cell surface of the fish pathogenic bacterium Aeromonas salmonicida: 1.Relationship between autoagglutination and the presence of a major cell envelopeprotein. Biochemica et Biophysica Acta 684, 241–248.
Evenberg, D., Versluis, R. and Lugtenberg, B. (1985) Biochemical and immunological
409Furunculosis
characterization of the cell surface of the fish pathogenic bacterium Aeromonassalmonicida. Biochemica et Biophysica Acta 815, 233–244.
Evenberg, D., de Graaff, P., Fleuren, W. and Van Muiswinkel, W.B. (1986) Bloodchanges in carp (Cyprinus carpio) induced by ulcerative Aeromonas salmonicidainfections. Veterinary Immunology and Immunopathology 12, 321–330.
Evenberg, D., de Graaff, P., Lugtenberg, B. and van Muiswinkel, W.B. (1988) Vaccineinduced protective immunity against Aeromonas salmonicida tested inexperimental carp erythrodermatitis. Journal of Fish Diseases 11, 337–350.
Ewing, W.H., Hugh, R. and Johnson, J.G. (1961) Studies on the Aeromonas Group.Department of Health, Education and Welfare, Public Health Service,Communicable Disease Centre, Atlanta, Georgia, USA, 37 pp.
Ezura, Y., Tajima, K., Yoshimizu, M. and Kimura, T. (1980) Studies on the taxonomy andserology of causative organisms of fish vibriosis. Fish Pathology 14, 167–179.
Ferguson, H.W. (1977) Furunculosis in brown trout. Bulletin de l’Office Internationaldes Epizooties 87, 465–467.
Fernández, A.I.G., Pérez, M.J., Rodríguez, L.A. and Nieto, T.P. (1995) Surfacephenotypic characteristics and virulence of Spanish isolates of Aeromonassalmonicida after passage through fish. Applied and Environmental Microbiology61, 2010–2012.
Ford, L.A. (1994) Detection of Aeromonas salmonicida from water using a filtrationmethod. Aquaculture 122, 1–7.
Ford, L.A., Cipriano, R.C. and Penniston, T.K. (1994) Isolation of Aeromonassalmonicida from paddlefish, Polyodon spathula. Journal of Wildlife Diseases 30,447–449.
Frerichs, G.N. and Holliman, A. (1991) Isolation of a brown pigment-producing strain ofPseudomonas fluorescens cross-reacting with Aeromonas salmonicida diagnosticantisera. Journal of Fish Diseases 14, 599–601.
Frerichs, G.N. and Roberts, R.J. (1989) The bacteriology of teleosts. In: Roberts, R.J.(ed.) Fish Pathology. Baillière Tindall, London, pp. 289–319.
Frerichs, G.N., Miller, S.D. and McManus, C. (1992) Atypical Aeromonas salmonicidaisolated from healthy wrasse (Ctenolabrus rupestris). Bulletin of the EuropeanAssociation of Fish Pathologists 12, 48–49.
Fryer, J.L., Hedrick, R.P., Park, J.W. and Hah, Y.C. (1988) Isolation of Aeromonassalmonicida from Masu salmon in the Republic of Korea. Journal of WildlifeDiseases 24, 364–365.
Fuller, D.W., Pilcher, K.S. and Fryer, J.L. (1977) A leukocytolytic factor isolated fromcultures of Aeromonas salmonicida. Journal of the Fisheries Research Board ofCanada 34, 1118–1125.
Garduño, R.A. and Kay, W.W. (1992a) A single structural type in the regular surfacelayer of Aeromonas salmonicida. Journal of Structural Biology 108, 202–208.
Garduño, R.A. and Kay, W.W. (1992b) Interaction of the fish pathogen Aeromonassalmonicida with rainbow trout macrophages. Infection and Immunity 60, 4612–4620.
Garduño, R.A., Thornton, J.C. and Kay, W.W. (1993) Aeromonas salmonicida grown invivo. Infection and Immunity 61, 3854–3862.
Garduño, R.A., Phipps, B.M. and Kay, W.W. (1995) Physical and functional S-layerreconstitution in Aeromonas salmonicida. Journal of Bacteriology 177, 2684–2694.
Gilroy, D. and Smith, P. (1995) Laboratory validation of an ELISA detection system forAeromonas salmonicida. Abstract. In: Proceedings of Diseases of Fish andShellfish, 7th International Conference of the European Association of FishPathologists, Palma de Mallorca.
410 M. Hiney and G. Olivier
Gilroy, D. and Smith, P. (1997) Detection of cell associated epitopes followingvaccination of Atlantic salmon pre-smolts with an oil-based anti-Aeromonassalmonicida vaccine. Abstract. In: Proceedings of Diseases of Fish and Shellfish,8th International Conference of the European Association of Fish Pathologists,Edinburgh, Scotland.
Gjedrem, T. (1997) Breeding to raise resistance. In: Bernoth, E.-M., Ellis, A.E.,Midtlyng, P.J., Olivier, G. and Smith, P. (eds) Furunculosis: Multidisciplinary FishDisease Research. Academic Press, London, pp. 405–422.
Gjedrem, T., Salte, R. and Gjoen, H.M. (1991) Genetic variation in susceptibility ofAtlantic salmon to furunculosis. Aquaculture 97, 1–6.
Graham (née Chung), S., Jeffries, A.H. and Secombes, C.J. (1988) A novel assay todetect macrophage bactericidal activity in fish: factors influencing the killing ofAeromonas salmonicida. Journal of Fish Diseases 11, 389–396.
Griffin, P.J., Snieszko, S.F. and Friddle, S.B. (1953) A more comprehensive descriptionof Bacterium salmonicida. Transactions of the American Fisheries Society 82, 129–138.
Griffiths, S.G. and Lynch, W.H. (1990) Characterization of Aeromonas salmonicidavariants with altered cell surfaces and their use in studying surface proteinassembly. Archives of Microbiology 154, 308–312.
Groberg, W.J., Jr, McCoy, R.H., Pilcher, K.S. and Fryer, J.L. (1978) Relation of watertemperature to infections of Coho salmon (Oncorhynchus kisutch), Chinook salmon(O. tshawytscha), and Steelhead trout (Salmo gairdneri) with Aeromonassalmonicida and A. hydrophila. Journal of the Fisheries Research Board of Canada35, 1–7.
Groman, D., Tweedie, D. and Shaw, D. (1992) Experiences with atypical furunculosis inNewfoundland: an overview. Bulletin of the Aquaculture Association of Canada92(1), 36–39.
Gudmundsdóttir, B.K. (1996) Comparison of extracellular proteases produced byAeromonas salmonicida strains, isolated from various fish species. Journal ofApplied Bacteriology 80, 105–113.
Gudmundsdóttir, B.K., Hastings, T.S. and Ellis, A.E. (1990) Isolation of a new toxicprotease from a strain of Aeromonas salmonicida subspecies achromogenes.Diseases of Aquatic Organisms 9, 199–208.
Gudmundsdóttir, S., Magnadóttir, B. and Gudmundsdóttir, B.K. (1995) Effects ofantigens from Aeromonas salmonicida ssp. achromogenes on primed and unprimedleucocytes from Atlantic salmon (Salmo salar L.). Fish and Shellfish Immunology.5, 493–504.
Gudmundsdóttir, B.K., Jonsdóttir, H., Steinthorsdóttir, B., Magnadóttir, B. andBudmundsdóttir, S. (1997) Survival and humoral antibody response of Atlanticsalmon, Salmo salar L., vaccinated against Aeromonas salmonicida spp.achromogenes. Journal of Fish Diseases 20, 351–360.
Gustafson, C.E., Thomas, C.J. and Trust, T.J. (1992) Detection of Aeromonassalmonicida from fish by using polymerase chain reaction amplification of thevirulence surface array protein gene. Applied and Environmental Microbiology 58,3816–3825.
Hackett, J.L., Lynch, W.H., Paterson, W.D. and Coombs, D.H. (1984) Extracellularprotease, extracellular haemolysis, and virulence in Aeromonas salmonicida.Canadian Journal of Fisheries and Aquatic Sciences 41, 1354–1360.
Hahnel, G.B., Gould, R.W. and Boatman, E.S. (1983) Serological comparison ofselected isolates of Aeromonas salmonicida ssp. salmonicida. Journal of FishDiseases 6, 1–11.
411Furunculosis
Hänninen, M.-L. and Hirvelä-Koski, V. (1997) Molecular and phenotypic methods forthe characterization of atypical Aeromonas salmonicida. Veterinary Microbiology56, 147–158.
Hänninen, M.-L., Ridell, J. and Hirvelä-Koski, V. (1995) Phenotypic and molecularcharacteristics of Aeromonas salmonicida subsp. salmonicida isolated in southernand northern Finland. Journal of Applied Bacteriology 79, 12–21.
Hardie, L.J., Fletcher, T.C. and Secombes, C.J. (1994) Effect of temperature onmacrophage activation and the production of macrophage activating factor byrainbow trout (Oncorhynchus mykiss) leucocytes. Developmental and ComparativeImmunology 18, 57–66.
Harmon, P.R., Knox, D.E., Cornick, J.W., Zwicker, B.W. and Olivier, G. (1991) Atypicalfurunculosis in Atlantic salmon. Bulletin of the Aquaculture Association of Canada91(3), 67–68.
Håstein, T. and Lindstad, T. (1991) Diseases in wild and cultured salmon: possibleinteraction. Aquaculture 98, 277–288.
Håstein, T., Jakob Saltveit, S. and Roberts, R.J. (1978) Mass mortality among minnowsPhoxinus phoxinus (L.) in Lake Tveitevatn, Norway, due to an aberrant strain ofAeromonas salmonicida. Journal of Fish Diseases 1, 241–249.
Hastings, T.S. (1997) Chemotherapy of furunculosis. In: Bernoth, E.-M., Ellis, A.E.,Midtlyng, P.J., Olivier, G. and Smith, P. (eds) Furunculosis: Multidisciplinary FishDisease Research, Academic Press, London, pp. 423–432.
Hastings, T.S. and Ellis, A.E. (1985) Differences in the production of haemolytic andproteolytic activities by various isolates of Aeromonas salmonicida. In Ellis, A.E.(ed.) Fish and Shellfish Pathology. Academic Press, London, pp. 69–77.
Hellberg, H., Mokssness, E. and Høie, S. (1996) Infection with atypical Aeromonassalmonicida in farmed common wolffish, Anarhichas lupus L. Journal of FishDiseases 19, 329–332.
Hennigan, M., Vaughan, L.M., Foster, T.J., Smith, P.R. and Gannon, F.X. (1989)Characterization of Aeromonas salmonicida strains using DNA probe technology.Canadian Journal of Fisheries and Aquatic Sciences 46, 877–879.
Heo, G.-J. and Seo, J.-J. (1996) Efficacy of ciprofloxacin against selected fish pathogensin cultured fish. Bulletin of the European Association of Fish Pathologists 17, 8012.
Herman, R.E. (1968) Fish furunculosis 1952–1966. Transactions of the AmericanFisheries Society 97, 221–230.
Hiney, M.P. (1994) Development and validation of detection techniques for the fishpathogen Aeromonas salmonicida. PhD thesis, National University of Ireland.
Hiney, M. (1995) Detection of stress inducible furunculosis in salmonids vaccinatedwith water and oil-based furunculosis vaccines. Bulletin of the EuropeanAssociation of Fish Pathologists 15, 105–106.
Hiney, M. (1997) How to test a test: methods of field validation. Bulletin of the EuropeanAssociation of Fish Pathologists 17, 245–250.
Hiney, M.P. and Smith, P.R. (1998) Validation of polymerase chain reaction-basedtechniques for proxy detection of bacterial fish pathogens: framework, problemsand possible solutions for environmental applications. Aquaculture 162, 41–68.
Hiney, M., Dawson, M.T., Heery, D.M., Smith, P.R., Gannon, F. and Powell, R. (1992)DNA probe for Aeromonas salmonicida. Applied and Environmental Microbiology58, 1039–1042.
Hiney, M.P., Kilmartin, J.J. and Smith, P.R. (1994) Detection of Aeromonas salmonicidain Atlantic salmon with asymptomatic furunculosis infections. Diseases of AquaticOrganisms 19, 161–167.
Hiney, M., Samuelsen, O.B., Kerry, J., Coyne, R., Purcell, L. and Smith, P. (1995)
412 M. Hiney and G. Olivier
Failure of Flumisol bath treatments during commercial transport of salmon smoltsto cure stress inducible furunculosis. Aquaculture 136, 31–42.
Hiney, M., Gilroy, D., Padley, D., Powell, R. and Smith, P. (1997a) Applicability of non-culture-based diagnostic techniques: a case study of furunculosis at a salmonidproduction unit on the west coast of Ireland. Abstract. In: Proceedings of 22ndAnnual Eastern Fish Health Workshop, Atlantic Beach, North Carolina.
Hiney, M., Smith, P. and Bernoth, E.-M. (1997b) Covert Aeromonas salmonicidainfections. In: Bernoth, E.-M., Ellis, A.E., Midtlyng, P.J., Olivier, G. and Smith, P.(eds) Furunculosis: Multidisciplinary Fish Disease Research. Academic Press,London, pp. 54–97.
Hirst, I.D. and Ellis, A.E. (1994) Iron regulated outer membrane proteins of Aeromonassalmonicida are important protective antigens in Atlantic salmon againstfurunculosis. Fish and Shellfish Immunology 4, 29–45.
Hirst, I.D. and Ellis, A.E. (1996) Utilization of transferrin and salmon serum as sourcesof iron by typical and atypical strains of Aeromonas salmonicida. Microbiology142, 1543–1550.
Hirst, I.D., Hastings, T.S. and Ellis, A.E. (1991) Siderophore production by Aeromonassalmonicida. Journal of General Microbiology 137, 1185–1192.
Hirst, I.D., Hastings, T.S. and Ellis, A.E. (1994) Utilization of haem compounds byAeromonas salmonicida. Journal of Fish Diseases 17, 365–373.
Hirvelä-Koski, V., Koski, P. and Kuoppamäki, T. (1988) Enrichment in tryptic soy brothfor the detection of Aeromonas salmonicida from the kidney of carrier fish. Bulletinof the European Association of Fish Pathologists 8, 85.
Hirvelä-Koski, V., Koski, P. and Niiranen, H. (1994) Biochemical and drug resistance ofAeromonas salmonicida in Finland. Diseases of Aquatic Organisms 20, 191–196.
Hjeltnes, B., Bergh, Ø., Wergeland, H. and Holm, J.C. (1995) Susceptibility of Atlanticcod Gadua morhua, halibut Hippoglossus hippoglossus and wrasse (Labridae) toAeromonas salmonicida subsp. salmonicida and the possibility of transmission offurunculosis from farmed salmon Salmo salar to marine fish. Diseases of AquaticOrganisms 23, 25–31.
Hodgkinson, J.L., Bucke, D. and Austin, B. (1987) Uptake of the fish pathogenAeromonas salmonicida by rainbow trout (Salmo gairdneri L.). FEMSMicrobiology Letters 40, 207–210.
Høie, S. (1995) The suitability of PCR to detect bacterial pathogens in fish. Abstract. In:Proceedings of Diseases of Fish and Shellfish, 7th International Conference of theEuropean Association of Fish Pathologists, Palma de Mallorca, p. 56.
Høie, S., Heum, M. and Thoresen, O.F. (1996) Detection of Aeromonas salmonicida bypolymerase chain reaction in Atlantic salmon vaccinated against furunculosis. Fishand Shellfish Immunology 6, 199–206.
Høie, S., Heum, M. and Thoresen, O.F. (1997) Evaluation of a polymerase chainreaction-based assay for the detection of Aeromonas salmonicida ssp. salmonicidain Atlantic salmon Salmo salar. Diseases of Aquatic Organisms 30, 27–35.
Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T. and Williams, S.T. (1994) The genusAeromonas. In: Bergey’s Manual of Determinative Bacteriology. Williams andWilkins, Baltimore, pp. 175–289.
Horne, J.H. (1928) Furunculosis in trout and the importance of carriers in the spread ofthe disease. Journal of Hygiene, Cambridge 28, 67–78,
Houghton, G., Wiergetjes, G.F., Groeneveld, A. and Van Muiswinkel, W.B. (1991)Difference in resistance of carp, Cyprinus carpio L., to atypical Aeromonassalmonicida. Journal of Fish Diseases 14, 333–341.
Humphrey, J.D. and Ashburner, L.D. (1993) Spread of the bacterial fish pathogen
413Furunculosis
Aeromonas salmonicida after importation of infected goldfish, Carassius auratus,into Australia. Australian Veterinary Journal 70, 453–454.
Iida, T., Sakata, C., Kawatsu, H. and Fukuda, Y. (1997) Atypical Aeromonas salmonicidainfection in cultured marine fish. Fish Pathology 32, 65–66.
Inglis, V., Ferichs, G.N., Millar, S.D. and Richards, R.H.(1991) Antibiotic resistance ofAeromonas salmonicida isolated from Atlantic salmon, Salmo salar L., in Scotland.Journal of Fish Diseases 14, 353–358.
Inglis, V., Roberts, R.J. and Bromage, N.R. (1993) Bacterial Diseases of Fish. BlackwellScientific Publications, Oxford.
Inglis, V., Colquhoun, D., Perason, M.D., Miyata, M. and Aoki, T. (1996) Analysis ofDNA relationships among Aeromonas species by RAPD (randomly amplifiedpolymorphic DNA) typing. Aquaculture International 4, 43–53.
Ishiguro, E.E., Kay, W.W., Ainsworth, T., Chamberlain, J.B., Austen, R.A., Buckley, J.T.and Trust, T.J. (1981) Loss of virulence during culture of Aeromonas salmonicida athigh temperatures. Journal of Bacteriology October, 148, 333–340.
Ishiguro, E.E., Ainsworth, T., Shaw, D.H., Kay, W.W. and Trust, T.J. (1983) Alipopolysaccharide-specific bacteriophage for Aeromonas salmonicida. CanadianJournal of Microbiology 29, 1458–1461.
Ishiguro, E.E., Ainsworth, T., Kay, W.W. and Trust, T.J. (1986) Heme requirement forgrowth of fastidious atypical strains of Aeromonas salmonicida. Applied andEnvironmental Microbiology 51, 668–670.
Izumikawa, K. and Ueki, N. (1997) Atypical Aeromonas salmonicida infection incultured Schlegel’s black rockfish. Fish Pathology 32, 67–68.
Jençiç, V. and Piano, J.Z. (1997) Detection of Aeromonas salmonicida in salmonid fishesin Slovenia by PCR technique. Abstract. In: Proceedings of Diseases of Fish andShellfish, 8th International Conference of the European Association of FishPathologists, Edinburgh, Scotland.
Jensen, N.J. (1977) The diagnosis of furunculosis in salmonids. Bulletin de l’OfficeInternational des Epizooties 87, 469–473.
Jensen, N.J. and J.L. Larsen (1980) Seasonal occurrence of Aeromonas salmonicidacarriers. In: Ahne, W. (ed.) Fish Diseases, Springer-Verlag, Berlin, pp. 87–93.
Johansson, N. (1977) Studies on diseases in hatchery reared Atlantic salmon (Salmosalar L.) and sea-trout (Salmo trutta L.) in Sweden. PhD thesis, Uppsala University,Uppsala, Sweden.
Johnsen, B.O. and Jensen, A.J. (1994) The spread of furunculosis in salmonids inNorwegian rivers. Journal of Fish Diseases 45, 47–55.
Kay, W.W. and Trust, T.J. (1997) The surface of Aeromonas salmonicida: what does itlook like and what does it do? In: Bernoth, E.-M., Ellis, A.E., Midtlyng, P.J., Olivier,G. and Smith, P. (eds) Furunculosis: Multidisciplinary Fish Disease Research,Academic Press, London, pp. 235–247.
Kay, W.W., Buckley, J.T., Ishiguro, E.E., Phipps, D.M., Monette, J.P.L. and TrustT.J. (1981) Purification and disposition of a surface protein associated withvirulence of Aeromonas salmonicida. Journal of Bacteriology 147, 1007–1084.
Kay, W.W., Phipps, B.M., Ishiguro, E.E., Olafson, R.W. and Trust, T.J. (1984) Surfacelayer virulence A-proteins from Aeromonas salmonicida strains. Canadian Journalof Biochemistry and Cell Biology 62, 1064–1071.
Kay, W.W., Ishiguro, E.E., Phipps, B.M., Belland, R.J. and Trust, T.J. (1986) Properties,organisation and role in virulence of the surface protein array of Aeromonassalmonicida. In: Vivarès, C.P., Bonami, J.-R. and Jaspers, E. (eds) Pathology inMarine Aquaculture. European Aquaculture Society, Bredene, Belgium.
Kimura, T. (1969) A new subspecies of Aeromonas salmonicida as an etiological agent
414 M. Hiney and G. Olivier
of furunculosis on ‘Sakuamasu’ (Oncorhynchus masou) and pink salmon (O.gorbuscha) rearing from maturity. I. On the morphological and physiologicalproperties. Fish Pathology 3, 34–44.
Kitao, T., Yoshida, T., Aoki, T. and Fukudone, M. (1984) Atypical Aeromonassalmonicida, the causative agent of an ulcer disease of eel occurred in KagoshimaPrefecture. Fish Pathology 19, 113–117.
Kitao, T., Yoshida, T., Aoki, T. and Fukudone, M. (1985) Characterization of an atypicalAeromonas salmonicida strain causing epizootic ulcer disease in cultured eel. FishPathology 20, 107–114.
Klontz, G.W. (1968) Oral Immunization of Coho Salmon against Furunculosis.Resource Publication of the Bureau of Sport Fisheries and Wildlife 81–82.
Klontz, G.W. and Wood, J.W. (1972) Observations on the Epidemiology of FurunculosisDisease in Juvenile Coho Salmon (Oncorhynchus kisutch). Food and AgricultureOrganization of the United Nations: European Inland Fisheries AdvisoryCommission 1–8.
Krantz, G.E. and Heist, C.E. (1970) Prevalence of naturally acquired agglutinatingantibodies against Aeromonas salmonicida in hatchery trout in centralPennsylvania. Journal of the Fisheries Research Board of Canada 27, 969–973.
Krovacek, K., Faris, A., Eriksson, L., Janson, E., Ljungberg, O. and Månsson, I. (1987)Virulence, cytotoxic and inflammatory activities of Vibrio anguillarum andAeromonas salmonicida isolated from cultured salmonid fish in Sweden. ActaVeterinaria Scandinavica 28, 47–54.
Kwon, M.G., Lee, J.-Y., Park, S., Iida, T., Horono, I. and Aoki, T. (1997) RAPDanalysis of atypical Aeromonas salmonicida isolated in Japan. Fish Pathology 32,109–115.
Lallier, R., LeBlanc, L.. Desbiens, M. and Ouellet, G. (1990) Detection of pathogens insubclinically and clinically infected salmonids. Canadian Technical Reports inFisheries and Aquatic Science 1761, 31–39.
Lamas, J. and Ellis, A.E. (1994a) Atlantic salmon (Salmo salar) neutrophil responses toAeromonas salmonicida. Fish and Shellfish Immunology 4, 201–219.
Lamas, J. and Ellis, A.E. (1994b) Electron microscopic observations of the phagocytosisand subsequent fate of Aeromonas salmonicida by Atlantic salmon neutrophils invitro. Fish and Shellfish Immunology 4, 539–546.
Lee, K.-K. and Ellis, A.E. (1989) The quantitative relationship of lethality betweenextracellular protease and extracellular haemolysin of Aeromonas salmonicida onAtlantic salmon (Salmo salar L.). FEMS Microbiology Letters 61, 127–132.
Lee, K.-K. and Ellis, A.E. (1990) Glycerophospholid cholesterol acyltransferasecomplexed with lipopolysaccharide (LPS) is a major lethal exotoxin and cytolycinof Aeromonas salmonicida: LPS stabilizes and enhances toxicity of the enzymes.Journal of Bacteriology 172, 5382–5392.
Lee, K.-K. and Ellis, A.E. (1991a) Interaction between salmonid serum components andthe extracellular haemolytic toxin of Aeromonas salmonicida. Diseases of AquaticOrganisms 11, 207–216.
Lee, K.K. and Ellis, A.E. (1991b) The role of the lethal extracellular cytolysin ofAeromonas salmonicida on the pathology of furunculosis. Journal of Fish Diseases14, 453–460.
Le Tendre, G.C., Schneider, C.P. and Ehlinger, N.F. (1972) Net damage and subsequentmortality from furunculosis in smallmouth bass. New York Fish and Game Journal19, 73–82.
Liu, P.V. (1961) Observations on the specificities of extra-cellular antigens of the generaAeromonas and Serratia. Journal of General Microbiology 24, 145–153.
415Furunculosis
Ljungberg, O. and Johansson, N. (1977) Epizootiological studies on atypical Aeromonassalmonicida infection of salmonids in Swedish fish farms, 1967–1977. Bulletin del’Office International des Epizooties 87, 475–478.
Lund, V., Jørgensen, T., Holm, K.O. and Eggset, G. (1991) Humoral immune response inAtlantic salmon, Salmo salar L., to cellular and extracellular antigens of Aeromonassalmonicida. Journal of Fish Diseases 14, 443–452.
Lund, T., Gjedrem, T., Bentsen, H.B., Eide, D.M., Larsen, H.J.S. and Røed, K.H. (1995)Genetic variation in immune parameters and associations to survival in Atlanticsalmon. Journal of Fish Biology 46, 748–758.
Lunder, T. and Håstein, T. (1990) Infeksjoner med Aeromonas salmonicida. In: Poppe, T.(ed.) Tiskehelse. Sykdommer-behandling-Foregygging. John Greig, Oslo, pp.140–148.
McCarthy, D.H. (1975) Fish furunculosis. Journal of the Institute of FisheriesManagement 6, 13–17.
McCarthy, D.H. (1977a) Some ecological aspects of the bacterial fish pathogenAeromonas salmonicida. In: Skinner, F.A. and Shewan, J.M. (eds) AquaticMicrobiology. Academic Press, London, pp. 299–324.
McCarthy, D.H. (1977b) The identification and significance of atypical strains ofAeromonas salmonicida. Bulletin de Office International des Epizooties 87,459–463.
McCarthy, D.H. (1978) A study of the taxonomic status of some bacteria currentlyassigned to the genus Aeromonas. PhD thesis, Portsmouth Polytechnic, Council forNational Academic Awards, UK.
McCarthy, D.H. and Roberts, R.J. (1980) Furunculosis of fish – the present state of ourknowledge. In: Droop, M.A. and Jannasch, H.W. (eds) Advances in AquaticMicrobiology. Academic Press, London, pp. 293–341.
McCormick, W.A., Stevenson, R.M.W. and MacInnes, J.I. (1990) Restrictionendonuclease fingerprinting analysis of Canadian isolates of Aeromonassalmonicida. Canadian Journal of Microbiology 36, 24–32.
McCraw, B.M. (1952) Furunculosis of Fish. United States Fish and Wildlife Service,Special Scientific Report No. 84, 84 pp.
McDermott, L.A. and Berst, A.H. (1968) Experimental plantings of brook trout(Salvelinus fontinalis) from furunculosis-infected stock. Journal of the FisheriesResearch Board of Canada 25, 2643–2649.
McFadden, T.W. (1970) Furunculosis in non-salmonids. Journal of the FisheriesResearch Board of Canada 27, 2365–2370.
McGee, Z.A. (1986) Interaction of cell wall defective bacteria with host defences. In:Madoff, S. (ed.) The Bacterial L-forms. Dekker, New York, pp. 277–285.
McIntosh, D. and Austin, B. (1990) Recovery of cell-wall deficient forms (L-forms) ofthe fish pathogens Aeromonas salmonicida and Yersinia ruckeri. Systematic andApplied Microbiology 13, 378–381.
McIntosh, D. and Austin, B. (1991) The role of cell-wall deficient bacteria (L-forms;spheroplasts) in fish diseases. Journal of Applied Bacteriology – SymposiumSupplement 70, 1S–7S.
MacInnes, J.I., Trust, T.J. and Crosa, J.H. (1979) Deoxyribonucleic acid relationshipamong members of the genus Aeromonas. Canadian Journal of Microbiology 25,579–586.
McIntosh, D., Gibb, A. and Austin, B. (1991) L-forms of Aeromonas salmonicida andYersinia ruckeri. Abstract. In: Proceedings of European Association of FishPathologists, Regional Meeting on Salmon Diseases, Marine Laboratory,Aberdeen.
416 M. Hiney and G. Olivier
Mackie, T.J. and Menzies, W.J.M. (1938) Investigations in Great Britain of furunculosisof the Salmonidae. Journal of Comparative Pathology and Therapeutics 51, 225.
Mackie, T.J., Arkwright, J.A., Pyrce-Tannatt, T.E., Mottram, J.C., Johnston, W.D. andMenzies, W.J. (1930) Interim Report of the Furunculosis Committee. HMSO,Edinburgh.
Mackie, T.J., Arkwright, J.A., Pyrce-Tannatt, T.E., Mottram, J.C., Johnston, W.D. andMenzies, W.J. (1933) Interim Report of the Furunculosis Committee. HMSO,Edinburgh..
Mackie, T.J., Arkwright, J.A., Pyrce-Tannatt, T.E., Mottram, J.C., Johnston, W.D. andMenzies, W.J. (1935) Final Report of the Furunculosis Committee. HMSO,Edinburgh.
McLoughlin, M. (1993) EC fish health controls on the movement of fish species. In:Meldon, J. (ed.) Aquaculture in Ireland – Towards Sustainability. An Taisce, Dublin.
Malnar, L., Teskeredzic, E. and Coz-Rakovac, R. (1988) Epizootiology, pathogenesis,diagnosis, treatment and prophylaxis of furunculosis. Ichthyologia 20, 67–76.
Manning, M.J., Grace, M.F. and Secombes, C.J. (1982) Developmental aspects ofimmunity and tolerance in fish. In: Roberts, R.J. (ed.) Microbial Diseases of Fish.Academic Press, London, pp. 31–46.
Markestad, A., Grave, K. and Sunde, M. (1995) [Sales of antimicrobials for use infarmed fish 1994]. Salgstall for antibakterielle midler brukt til oppdrettsfisk 1994.[in Norwegian]. Norsk Veterinaertidsskrift 107, 222.
Markwardt, N.M. and Klontz, G.W. (1989a) Evaluation of four methods to establishasymptomatic carriers of Aeromonas salmonicida in juvenile spring chinooksalmon, Oncorhynchus tshawytscha, (Walbaum). Journal of Fish Diseases 12,311–315.
Markwardt, N.M. and Klontz, G.W. (1989b) A method to eliminate the asymptomaticcarrier state of Aeromonas salmonicida in salmon. Journal of Fish Diseases 12,317–322.
Markwardt, N.M., Gocha, Y.M. and Klontz, G.W. (1989) A new application ofCoomassie brilliant blue agar: detection of Aeromonas salmonicida in clinicalsamples. Diseases of Aquatic Organisms 6, 231–233.
Marsden, M.J., Cox, D. and Secombes, C.J. (1994) Antigen-induced release ofmacrophage activating factor from rainbow trout Oncorhynchus mykiss leucocytes.Veterinary Immunology and Immunopathology 42, 199–208.
Marsden, M.J., Freeman, L.C., Cox, D. and Secombes, C.J. (1996) Non-specific immuneresponses in families of Atlantic salmon, Salmo salar, exhibiting differentialresistance to furunculosis. Aquaculture 146, 1–16.
Marsh, M.C. (1902) Bacterium truttae, a new bacterium pathogenic to trout. Science 16,706.
Maule, A.G., Schreck, C.B. and Kaattari, S.L. (1986) Changes in the immune system ofcoho salmon (Oncorhynchus kisutch) during the parr-to-smolt transformation andafter implantation of cortisol. Canadian Journal of Fisheries and Aquatic Sciences44, 161–166.
Mettam, A.E. (1915) Report on the Outbreak of Furunculosis in the River Liffey, in 1913.Scientific Investigations of the Fisheries Branch, Department of Agriculture forIreland.
Michel, C. and Dubois-Darnaudpeys, A. (1980) Persistence of the virulence ofAeromonas salmonicida strains kept in river sediment. Annales de RecherchesVétérinaires 11, 375–380.
Midtlyng, P.J. (1996) A field study on vaccination of Atlantic salmon (Salmo salar L)against furunculosis. Fish and Shellfish Immunology 6, 553–565.
417Furunculosis
Midtlyng, P.M. (1997) Vaccination against furunculosis. In: Bernoth, E.-M., Ellis, A.E.,Midtlyng, P.J., Olivier, G. and Smith, P. (eds) Furunculosis: Multidisciplinary FishDisease Research. Academic Press, London, pp. 382–404.
Midtlyng, P.J., Reitan, L.J., Lillehaug, A. and Ramstad, A. (1996) Protection, immuneresponses and side effects in Atlantic salmon (Salmo salar L) vaccinated againstfurunculosis by different procedures. Fish and Shellfish Immunology 6, 599–613.
Mitchell, H.M. (1992) Furunculosis treatment, control and chemotherapeutic regulatoryapproaches to salmon aquaculture in the United States. Bulletin of the AquacultureAssociation of Canada 92(1), 22–28.
Miyata, M., Aoki, T., Inglis, V., Yoshida, T. and Endo, M. (1995) RAPD analysis ofAeromonas salmonicida and Aeromonas hydrophila. Journal of AppliedBacteriology 79, 181–185.
Miyata, M., Inglis, V. and Aoki, T. (1996) Rapid identification of Aeromonassalmonicida subspecies salmonicida by the polymerase chain reaction. Aquaculture141, 13–24.
Miyazaki, T. and Kubota, S.S. (1975) Histopathological studies on furunculosis inAmago. Fish Pathology 9, 213–218.
Mooney, J., Powell, E., Clabby, C. and Powell, R. (1995) Detection of Aeromonassalmonicida in wild Atlantic salmon using a specific DNA probe test. Diseases ofAquatic Organisms 21, 131–135.
Morgan, J.A.W., Rhodes, G. and Pickup, R.W. (1993) Survival of nonculturableAeromonas salmonicida in lake water. Applied and Environmental Microbiology59, 874–880.
Munro, A.L.S. and Gauld, J.A. (1996) Scottish Fish Farms Annual Production Survey1995. SOAEFD Marine Laboratory, Aberdeen, 35 pp.
Munro, A.L.S. and Hastings, T.S. (1993) Furunculosis. In: Inglis, V., Roberts, R.J. andBromage, N.R. (eds) Bacterial Diseases of Fish. Blackwell Scientific Publications,London, pp. 122–142.
Munro, A.L.S., Hastings, T.S., Ellis, A.E. and Liversidge, J. (1980) Studies on anichthyotoxic material produced extracellularly by the furunculosis bacteriumAeromonas salmonicida. In: Ahne, W. (ed.) Fish Diseases. Springer-Verlag, pp.99–106.
Nakai, T., Miyakawa, M., Muroga, K. and Kamito, K. (1989) The tissue distribution ofatypical Aeromonas salmonicida in artificially infected Japanese eels, Anguillajaponica. Fish Pathology 24, 23–28.
Nakatsugawa, T. (1994) Atypical Aeromonas salmonicida isolated from cultured shottedhalibut. Fish Pathology 29, 193–198.
Nerland, A.H., Høgh, B.T., Olsen, A.B. and Jensen, H.B. (1993) Aeromonas salmonicidaspp. salmonicida requires exogenous arginine and methionine for growth. Journalof Fish Diseases 16, 605–608.
Nielsen, B., Olsen, J.E. and Larsen, J.L. (1993) Plasmid profiling as an epidemiologicalmarker within Aeromonas salmonicida. Diseases of Aquatic Organisms 15, 129–135.
Nielsen, B., Olsen, J.E. and Larsen, J.L. (1994) Ribotyping of Aeromonas salmonicidasubsp. salmonicida. Diseases of Aquatic Organisms 18, 155–158.
Noga, E.J. and Berkhoff, H.A. (1990) Pathological and microbiological features ofAeromonas salmonicida infection in the American eel (Anguilla rostrata). FishPathology 25, 127–132.
Nomura, T., Yoshimizu, M. and Kimura, T. (1991a) Prevalence of Aeromonassalmonicida in chum salmon (Oncorhynchus keta), pink salmon (O. gorbuscha),and masu salmon (O. masou) returning to rivers in Hokkaido. Gyobyo Kenkyu 26,139–147.
418 M. Hiney and G. Olivier
Nomura, T., Yoshimizu, M. and Kimura, T. (1991b) Prevalence of Aeromonassalmonicida in the kidney of chum salmon (Oncorhynchus keta) and masu salmon(O. masou) at various life stages. Gyobyo Kenkyu 26, 149–153.
Nomura, T., Yoshimizu, M. and Kimura, T. (1992) Detection of Aeromonas salmonicidain the coelomic fluid and kidney of mature chum salmon (Oncorhynchus keta), pinksalmon (O. gorbuscha) and masu salmon (O. masou). Gyobyo Kenkyu 27, 69–72.
Nomura, T., Yoshimizu, M. and Kimura, T. (1993) An epidemiological study of furun-culosis in salmon propagation in Japanese rivers. Fisheries Research 17, 137–146.
Noonan, B. and Trust, T.J. (1995) Molecular characterization of an Aeromonassalmonicida mutant with altered surface morphology and increased systemicvirulence. Molecular Microbiology 15, 65–75.
Nougayrede, P., Sochon, E. and Vuillaume, A. (1990) Isolation of Aeromonassalmonicida subspecies salmonicida in farmed turbot (Psetta maxima) in France.Bulletin of the European Association of Fish Pathologists 10, 139–140.
Novotny, A.J. (1978) Vibriosis and furunculosis in marine cultures salmon in PugetSound, Washington. Marine Fisheries Review 40, 52–55.
O’Brien, D., Mooney, J., Ryan, D., Powell, E., Hiney, M., Smith, P.R. and Powell, R.(1994) Detection of Aeromonas salmonicida, causal agent of furunculosis insalmonid fish, from the tank effluent of hatchery-reared Atlantic salmon smolts.Applied and Environmental Microbiology 60, 3874–3877.
O’Grady, P. and Smith, P. (1992) Use of flumisol bath treatments to eliminate stressinducible furunculosis in salmon smolts. Bulletin of the European Association ofFish Pathologists 12, 201–203.
O’Grady, P., Palmer, R., Rodger, H. and Smith, P. (1987) Isolation of Aeromonassalmonicida strains resistant to the quinolone antibiotics. Bulletin of the EuropeanAssociation of Fish Pathologists 7, 43–46.
O’Grady, P., Moloney, M. and Smith, P.R. (1988) Bath administration of the quinolineantibiotic flumequine to brown trout Salmo trutta and Atlantic salmon, S. salar.Diseases of Aquatic Organisms 19, 193–199.
O’hIci, B., Olivier, G. and Powell, R. (1998) Interspecific genetic diversity of the fishpathogen Aeromonas salmonicida as derived from random amplified polymorphicDNA and pulsed-field gel electrophoresis analysis. Applied and EnvironmentalMicrobiology (in press).
Ohtsuka, H., Nakai, T., Muroga, K. and Jo, Y. (1984) Atypical Aeromonas salmonicidaisolated from diseased eels. Fish Pathology 19, 101–107.
Olivier, G. (1990) Virulence of Aeromonas salmonicida: lack of relationship withphenotypic characteristics. Journal of Aquatic Animal Health 2, 119–127.
Olivier, G. (1992) Furunculosis in the Atlantic provinces: an overview. Bulletin of theAquaculture Association of Canada 92(1), 4–10.
Olivier, G. (1997) Effect of nutrition on furunculosis. In: Bernoth, E.-M., Ellis, A.E.,Midtlyng, P.J., Olivier, G. and Smith, P. (eds) Furunculosis: Multidisciplinary FishDisease Research. Academic Press, London, pp. 327–344.
Olivier, G., Evelyn, T.P.T. and Lallier, R. (1985) Immunogenicity of vaccines from avirulent and an avirulent strain of Aeromonas salmonicida. Journal of Fish Diseases8, 43–55.
Olivier, G., Moore, A.R. and Fildes, J. (1990) Taxonomy and virulence of atypicalstrains of A. salmonicida isolated from salmonid and non-salmonid fish. Abstract.In: International Conference on Bacterial Diseases of Fish, Stirling, Scotland.
Ossiander, F.J. and Wedemeyer, G. (1973) Computer programme for sample sizesrequired to determine disease incidence in fish population. Journal of the FisheriesResearch Board of Canada 30, 1383–1384.
419Furunculosis
Ostland, V.E., Hicks, B.D. and Daly, J.G. (1987) Furunculosis in baitfish and itstransmission to salmonids. Diseases of Aquatic Organisms 2, 163–166.
Pace, N.R. (1997) Opening the door onto the natural microbial world: molecularmicrobial ecology. The Harvard Lectures 91, 178–202.
Padley, D., Gallagher, T., Cotter, D. and Powell, R. (1997) Detection of Aeromonassalmonicida in a freshwater lake catchment: a comparison of bacteriology and DNAprobe analysis. Abstract. In: Proceedings of Diseases of Fish and Shellfish, 8thInternational Conference of the European Association of Fish Pathologists,Edinburgh, p. P-179.
Paterson, W.D., Dovey, D. and Desautels, D. (1980) Relationships between selectedstrains of typical and atypical Aeromonas salmonicida, Aeromonas hydrophila andHaemophilis piscium. Canadian Journal of Microbiology 26, 588–598.
Pedersen, K., Kofod, H., Dalsgaard, I. and Larsen, J.L. (1994) Isolation of oxidase-negative Aeromonas salmonicida from diseased turbot Scophthalmus maximus.Diseases of Aquatic Organisms 18, 149–154.
Pérez, M.J., Fernández, A.I.G., Rodríguez, L.A. and Nieto, T.P. (1996) Differentialsusceptibility to furunculosis of turbot and rainbow trout and release of thefurunculosis agent from furunculosis-affected fish. Diseases of Aquatic Organisms26, 133–137.
Pickering, A.D. (1986) Changes in blood composition of the brown trout, Salmo truttaL., during the spawning season. Journal of Fish Biology 29, 335–347.
Pickering, A.D. (1997) Husbandry and stress. In: Bernoth, E.-M., Ellis, A.E., Midtlyng,P.J., Olivier, G. and Smith, P. (eds) Furunculosis: Multidisciplinary Fish DiseaseResearch. Academic Press, London, pp. 178–202.
Pickup, R.W., Rhodes, G., Cobban, R.J. and Clarke, K.J. (1996) The postponement ofnon-culturability in Aeromonas salmonicida. Journal of Fish Diseases 19, 65–74.
Plehn, M. (1911) Die Furunculose der Salmoniden. Zentralblatt für Bakteriologie,Parasitenkunde, Infektionskrankheiten und Hygiene, Orignale, Abteilung I 60,609–624.
Pol, J.M.A., Bootsma, R. and Berg-Blommaert, J.M. (1981) Pathogenesis of carperythrodermatitis (CE): role of bacterial endo- and exotoxin. In: Ahne, W. (ed.)Proceeding of Fish Diseases, Third COPRAQ Session. Springer-Verlag, Berlin, pp.120–125.
Popoff, M. (1971) Studies on Aeromonas salmonicida. II. Characterization and typing ofbacteriophages that act on Aeromonas salmonicida. Annales de RecherchesVétérinaires 2, 33–45.
Popoff, M. (1984) Genus III Aeromonas Kluyver and van Niel 1936, 398 AL. In: Krieg,N.R. and Holt, J.G. (eds) Bergey’s Manual of Systematic Bacteriology Vol. 1.Williams & Wilkins, Baltimore, pp. 545–548.
Popoff, M. and Lallier, R. (1984) Biochemical and serological characteristics ofAeromonas. In: Bergan, T. (ed.) Methods in Microbiology. Academic Press, London,pp. 127–145.
Pourreau, C.N., Evenberg, E., De Raadt, W.M., Van Mechelen, J.A.N. and vanMuiswinkel, W.B. (1986) Does Aeromonas salmonicida affect the immune systemof carp, Cyprinus carpio L.? Veterinary Immunology and Immunopathology 12,331–338.
Pourreau, C.N., Koopman, M.B.H., Hendriks, G.F.R., Evenberg, D. and vanMuiswinkel, W.B. (1987) Modulation of the mitogenic PHA response of carp,Cyprinus carpio L., by extracellular products of Aeromonas salmonicida. Journalof Fish Biology (Supplement A) 31, 133–143.
Powell, M.C., Wright, B.M. and Burka, J.F. (1991) Degranulation of eosinophilic
420 M. Hiney and G. Olivier
granule cells induced by capsaicin and substance P in the intestine of the rainbowtrout (Oncorhynchus mykiss Walbaum). Cell and Tissue Research 266, 469–474.
Power, P., Dunne, S. and Smith, P.R. (1987) Failure of tryptone soya agar to support thegrowth of Aeromonas salmonicida. Bulletin of the European Association of FishPathologists 7, 75.
Price, N.C., Banks, R.M., Campbell, C.M., Duncan, D. and Stevens, L. (1990) Thespecificity of the major (70K Da) protease secreted by Aeromonas salmonicida.Journal of Fish Diseases 13, 49–58.
Rabb, L. and McDermott, L. (1962) Bacteriological studies of freshwater fish. II.Furunculosis in Ontario fish in natural waters. Journal of the Fisheries ResearchBoard of Canada 19, 989–995.
Real, F., Acosta, B., Déniz, S., Oros, J. and Rodriguez, E. (1994) Aeromonassalmonicida infection in Sparus aurata in the Canaries. Bulletin of the EuropeanAssociation of Fish Pathologists 14, 153–155.
Reitan, L.J. and Thuvander, A. (1991) In vitro stimulation of salmonid leucocytes withmitogens and with Aeromonas salmonicida. Fish and Shellfish Immunology 1, 297–307.
Rintamäki, P. and Valtonen, E.T. (1991) Aeromonas salmonicida in Finland:pathological problems associated with atypical and typical strains. Journal of FishDiseases 14, 323–331.
Rockey, D.D., Dungan, C.F., Lunder, T. and Rohovec, J.S. (1991) Monoclonalantibodies against Aeromonas salmonicida lipopolysaccharide identify differencesamong strains. Diseases of Aquatic Organisms 10, 115–120.
Rodgers, C.J., Pringle, J.H., McCarthy, D.H. and Austin, B. (1981) Quantitative andqualitative studies of Aeromonas salmonicida bacteriophage. Journal of GeneralMicrobiology 125, 335–345.
Rose, A.S., Ellis, A.E. and Adams, A. (1989) An assessment of routine Aeromonassalmonicida carrier detection by ELISA. Bulletin of the European Association ofFish Pathologists 9, 65–67.
Roszak, D.B. and Colwell, R.R. (1987) Survival strategies of bacteria in the naturalenvironment. Microbiological Reviews 51, 365–379.
Rudden, S. and Smith, P. (1995) The use of differential inhibition by Pseudomonas sp. asa sub-typing technique for the fish pathogen Aeromonas salmonicida. Abstract. In:Proceedings of Diseases of Fish and Shellfish, 7th International Conference of theEuropean Association of Fish Pathologists, Palma de Mallorca.
St Jean, B. (1992) Practical experience with furunculosis in an Atlantic salmon hatcheryin Washington State. Bulletin of the Aquaculture Association of Canada 1, 47–48.
Sakai, D.K. (1984) The non-specific activation of rainbow trout, Salmo gairdneriRichardson, complement by Aeromonas salmonicida extracellular products andcorrelation of complement activity with the inactivation of lethal toxicity products.Journal of Fish Diseases 7, 329–338.
Sakai, D.K. (1985) Loss of virulence in a protease-deficient mutant of Aeromonassalmonicida. Infection and Immunity 48, 146–152.
Sakai, D.K. (1986) Electrostatic mechanism of survival of virulent Aeromonas salmoni-cida strains in river water. Applied and Environmental Microbiology 51, 1343–1349.
Sakai, D.K. and Kimura, T. (1985) Relationship between agglutinative properties ofAeromonas salmonicida strains isolated from fish in Japan and their resistance tomechanisms of host defence. Fish Pathology 20, 9–21.
Sakai, M., Atsuta, S. and Koboyashi, M. (1986) Comparative sensitivities of severaldiagnostic methods used to detect fish furunculosis. Kitasato Archives ofExperimental Medicine 59, 43–48.
421Furunculosis
Sakazaki, P. and Balows, A. (1981) The genus Vibrio, Plesimonas and Aeromonas. In:Starr, M.P., Stolp, H., Tüper, H.G., Balows, A. and Schlegel, H.G. (eds) TheProkaryotes: a Handbook on Habitats, Isolation and Identification of Bacteria.Springer Verlag, New York, pp. 1272–1301.
Sako, H. and Hara, T. (1981) Effect of water temperature on the growth of Aeromonassalmonicida inoculated into yamame, Oncorhynchus masou. Bulletin of theNational Research Institute of Aquaculture 2, 73–81.
Salte, R., Norberg, K., Arnesen, J.A., Ødegaard, O.R. and Eggset, G. (1992) Serineprotease and glycerophospholipid:cholesterol acyltransferase of Aeromonassalmonicida work in concert in thrombus formation; in vitro the process iscounteracted by plasma antithrombin and α2-macroglobulin. Journal of FishDiseases 15, 215–227.
Sandaa, R.-A. and Enger, Ø. (1996) High frequency transfer of a broad host rangeplasmid present in an atypical strain of the fish pathogen Aeromonas salmonicida.Diseases of Aquatic Organisms 24, 71–75.
Scallan, A. (1983) Investigations into asymptomatic carriers of furunculosis. PhD thesis,National University of Ireland.
Scallan, A. and Smith, P.R. (1984) Investigations into seasonal variation of latentfurunculosis. Abstract. In: Proceedings of the 4th Session EIFAC/COPRAQ on FishDiseases, Cadiz.
Scallan, A. and Smith, P.R. (1985) Control of asymptomatic carriage of Aeromonassalmonicida in Atlantic salmon smolts with flumequine. In: Ellis, A.E. (ed.) Fishand Shellfish Pathology. Academic Press, London, pp. 119–127.
Scallan, A. and Smith, P. (1993) Importance of sampling time in detecting stressinducible furunculosis in Atlantic salmon smolts. Bulletin of the EuropeanAssociation of Fish Pathologists 13, 77–78.
Scallan, A., Hickey, C. and Smith, P. (1993) Evidence for the transient nature of stressinducible asymptomatic Aeromonas salmonicida infections of Atlantic salmon.Bulletin of the European Association of Fish Pathologists 13, 210–212.
Schiewe, M.H., Novotny, A.J. and Harrell, L.W. (1988) Furunculosis of salmonids. In:Sindermann, C.J. and Lightner, D.V. (eds) Developments in Aquaculture andFisheries Science Vol. 17: Disease Diagnosis and Control in North AmericanMarine Aquaculture. Elsevier, Amsterdam, pp. 328–331.
Schubert, R.H.W. (1974) Genus II. Aeromonas Kluyver and van Neil 1936. In:Buchanan, R.E. and Gibbons, N.E. (eds) Bergey’s Manual of DeterminativeBacteriology, 8th edn. Williams and Wilkins, Baltimore, pp. 345–348.
Secombes, C.J. (1994a) Cellular defences of fish: an update. In: Pike, A.W. and Lewis,J.W. (eds) Parasitic Diseases of Fish. Salmara Publishing, Tresaith, Dyfed, pp.209–224.
Secombes, C.J. (1994b) The phylogeny of cytokines. In: Thomson, A. (ed.) The CytokineHandbook. Academic Press, London, pp. 567–594.
Secombes, C.J. and Fletcher, T.C. (1992) The role of phagocytes in the protectivemechanisms of fish. Annual Review of Fish Diseases 2, 53–71.
Secombes, C.J. and Oliver, G. (1997) Host–pathogen interactions in salmonids. In:Bernoth, E.-M., Ellis, A.E., Midtlyng, P.J., Olivier, G. and Smith, P. (eds)Furunculosis: Multidisciplinary Fish Disease Research. Academic Press, London,pp. 269–296.
Sheeran, B. and Smith, P.R. (1981) A second extracellular proteolytic activity associatedwith the fish pathogen Aeromonas salmonicida. FEMS Microbiology Letters 11,73–76.
Shieh, H.S. (1985) Correlation between cell virulence and protease toxicity of
422 M. Hiney and G. Olivier
Aeromonas salmonicida isolates. Microbios Letters 28, 69–72.Shotts, E.B.J. (1994) Furunculosis. In: Thoesen J.C. (ed.) Blue Book, Version 1.
Suggested Procedures for the Detection and Isolation of Certain Finfish andShellfish Pathogens. American Fisheries Society, Fish Health Section, Bethesda,Maryland.
Shotts, E.B.J., Talkington, F.D., Elliot, D.G. and McCarthy, D.H. (1980) Aetiology of anulcerative disease in goldfish, Carassius auratus (L.): characterization of thecausative agent. Journal of Fish Diseases 3, 181–186.
Slack, H.D. (1937) Notes on the viability of Bacillus salmonicida Emmerich and Weibel.Annals of Applied Biology 24, 665–672.
Slater, C.H. and Schreck, C.B. (1993) Testosterone alters the immune response ofchinook salmon, Oncorhynchus tshawytscha. General and ComparativeEndocrinology 89, 291–298.
Smith, I.W. (1963) The classification of Bacterium salmonicida. Journal of GeneralMicrobiology 33, 263–274.
Smith, P. (1997) The epizootiology of furunculosis: the present state of our ignorance.In: Bernoth, E.-M., Ellis, A.E., Midtlyng, P.J., Olivier, G. and Smith, P. (eds)Furunculosis: Multidisciplinary Fish Disease Research. Academic Press, London,pp. 25–53.
Smith, P.R. (1992) Antibiotics and the alternatives. In: DePauw, N. and Joyce, J. (eds)Aquaculture and the Environment. European Aquaculture Society, Ghent, Belgium,pp. 223–234.
Smith, P.R. and Davey, S. (1993) Evidence for the competitive exclusion of Aeromonassalmonicida from fish with stress inducible furunculosis by fluorescentpseudomonads. Journal of Fish Diseases 16, 521–524.
Smith, P.R., Brazil, G.M., Drinan, E.M., O’Kelly, J., Palmer, R. and Scallan, A. (1982)Lateral transmission of furunculosis in sea water. Bulletin of the EuropeanAssociation of Fish Pathologists 3, 41–42.
Smith, P., Hiney, M.P. and Samuelsen, O.B. (1994) Bacterial resistance to antimicrobialagents used in fish farming: a critical evaluation of method and meaning. AnnualReview of Fish Diseases 4, 273–313.
Snieszko, S.F. (1957) Genus IV. Aeromonas Kluyver and van Niel 1936. In: Breed, R.S.,Murray, E.G.D. and Smith, N.R. (eds) Bergey’s Manual of DeterminativeBacteriology, 7th edn. Williams and Wilkins, Baltimore, pp. 189–193.
Snieszko, S.F., Griffin, P.J. and Friddle, S.B. (1950) A new bacterium (Haemophiluspiscium n. sp.) from ulcer disease of trout. Journal of Bacteriology 59, 699–710.
Sørum, H., Sandersen, K. and Heum, M. (1998) Development of a polymerase chainreaction based on plasmid DNA for detection of Aeromonas salmonicida subspeciessalmonicida from healthy carrier fish. Diseases of Aquatic Organisms (in press).
Sövényi, J.F. (1986) Applicability of coagglutination test for detecting Aeromonassalmonicida antigens from extracts of bacterial cultures and experimentallyinfected tissues of the common carp (Cyprinus carpio L.). Acta VeterinariaHungarica 34, 151–156.
Sövényi, J.F., Elliot, D.G., Csaba, G.Y., Olah, J. and Majnarich, J.J. (1984) Cultural,biochemical and serological characteristics of bacterial isolates from carperythrodermatitis in Hungary. Revue Scientifique Technique Office Internationaldes Epizooties 3, 597–609.
Sövényi, J.F., Bercsényi, M. and Bakos, J. (1988) Comparative examination ofsusceptibility of two genotypes of carp (Cyprinus carpio L.) to infection withAeromonas salmonicida. Aquaculture 70, 301–308.
Sutherland, D.W. and Inglis, V. (1992) Multiple antibiotic sensitivity patterns of
423Furunculosis
Aeromonas salmonicida within diagnostic specimens from outbreaks offurunculosis. Bulletin of the European Association of Fish Pathologists 12,163–165.
Tajima, K., Takahashi, T., Ezura, Y. and Kimura, T. (1983) Studies on the virulencefactors produced by Aeromonas salmonicida, causative agent of furunculosis inSalmonidae. II. Studies on the pathogenicity of the protease of Aeromonassalmonicida Ar-4 (EFDL) on yamabe (Oncorhynchus masou f. ishikawai) andgoldfish (Carassius auratus) and the substance which exhibits cytotoxic effect onRTG-2 (rainbow trout gonad) cells. Bulletin of the Faculty of Fisheries, HokkaidoUniversity 34, 111–123.
Teska, J.D. and Cipriano, R.C. (1993) Nonselective nature of Coomassie brilliant blueagar for the presumptive identification of Aeromonas salmonicida in clinicalspecimens. Diseases of Aquatic Organisms 16, 239–242.
Teska, J.D., Cipriano, R.C. and Schill, W.B. (1992) Molecular and geneticcharacterization of cytochrome oxidase-negative Aeromonas salmonicida isolatedfrom coho salmon (Oncorhynchus kisutch). Journal of Wildlife Diseases 28,515–520.
Thoesen, J.C. (1994) A standard inspection procedure for detection for certain pathogensin salmonid fish. In: Thoesen, J.C. (ed.) Blue Book, Version 1. SuggestedProcedures for the Detection and Identification of Certain Finfish and ShellfishPathogens. American Fisheries Society, Fish Health Section, Bethesda, Maryland.
Thornton, J.C., Garduño, R.A., Carlos, S.J. and Kay, W.W. (1993) Novel antigensexpressed by Aeromonas salmonicida grown in vivo. Infection and Immunity 61,4582–4589.
Thrusfield, M. (1986) Veterinary Epidemiology. Butterworth, London.Toranzo, A.E. and Barja, J.L. (1992) First report of furunculosis in turbot reared in
floating cages in northwest of Spain. Bulletin of the European Association of FishPathologists 12, 147–149.
Toranzo, A.E., Barja, J.L., Colwell, R.R. and Hetrick, F.M. (1983) Characterization ofplasmids in bacterial fish pathogens. Infection and Immunity 39, 184–192.
Traxler, G.S. and Bell, G.R. (1988) Pathogens associated with impounded Pacificherring Clupea harengus pallasi, with emphasis on viral erythrocytic necrosis(VEN) and atypical Aeromonas salmonicida. Diseases of Aquatic Organisms 5,93–100.
Treasurer, J. and Cox, D. (1991) The occurrence of Aeromonas salmonicida in wrasse(Labridae) and implications for Atlantic salmon farming. Bulletin of the EuropeanAssociation of Fish Pathologists 11, 208–210.
Treasurer, J. and Laidler, L.A. (1994). Aeromonas salmonicida infection in wrasse(Labridae) used as cleaner fish, on an Atlantic salmon, Salmo salar L., farm.Journal of Fish Diseases 17. 155–161.
Trust T.J. (1986) Pathogenesis of infectious disease of fish. Annual Review ofMicrobiology 40, 479–502.
Trust, T.J., Howard, P.S., Chamberlain, J.B., Ishiguro, E.E. and Buckley, J.T. (1980a)Additional surface protein in auto-aggregating strains of atypical Aeromonassalmonicida. FEMS Microbiology Letters 9, 35–38.
Trust, T.J., Khouri, A.G., Austen, R.A. and Ashburner, L.D. (1980b) First isolation inAustralia of atypical Aeromonas salmonicida. FEMS Microbiology Letters 9,315–318.
Trust, T.J., Ishiguro, E.E., Chart, H. and Kay, W.W. (1983) Virulence properties ofAeromonas salmonicida. Journal of the World Mariculture Society 14, 193–200.
Tsoumas, A., Alderman, D.J. and Rodgers, C.J. (1989) Aeromonas salmonicida:
424 M. Hiney and G. Olivier
development of resistance to 4-quinolone antimicrobials. Journal of Fish Diseases12, 493–507.
Udey, L.R. (1982) A differential medium for distinguishing Alr+ from Alr– phenotypesin Aeromonas salmonicida. Abstract. In: Proceedings of the 13th AnnualConference and Workshop and 7th Eastern Fish Health Workshop. InternationalAssociation for Aquatic Animal Medicine, Baltimore, Maryland, p. 41.
Udey, L.R. and Fryer, J.L. (1978) Immunization of fish with bacterins of Aeromonassalmonicida. Marine Fisheries Review 40, 12–17.
Vallejo, A.N.J. and Ellis, A.E. (1989) Ultrastructural study of the response of eosinophilgranule cells to Aeromonas salmonicida extracellular products and histamineliberators in rainbow trout, Salmo gairdneria Richardson. Developmental andComparative Immunology 13, 133–148.
Vaughan, L.M. (1997) Aeromonas salmonicida subspecies salmonicida – a moleculargenetic perspective. In: Bernoth, E.-M., Ellis, A.E., Midtlyng, P.J., Olivier, G. andSmith, P. (eds) Furunculosis: Multidisciplinary Fish Disease Research. AcademicPress, London, pp. 297–321.
Vaughan, L.M., Smith, P. and Foster, T.J. (1993) An aromatic-dependent mutant of thefish pathogen Aeromonas salmonicida is attenuated in fish and is effective as a livevaccine against the salmonid disease furunculosis. Infection and Immunity 61,2172–2181.
Warr, G.W. and Cohen, N. (1991) Phylogenesis of Immune Functions. CRC Press, BatonRouge.
Whittington, R.J. and Cullis, B. (1988) The susceptibility of salmonid fish to an atypicalstrain of Aeromonas salmonicida that infects goldfish, Carassius auratus (L.), inAustralia. Journal of Fish Diseases 11, 461–470.
Whittington, R.J., Gudkovs, N., Carrigan, M.J., Ashburner, L.D. and Thurstan, S.J.(1987) Clinical, microbiological and epidemiological findings in recent outbreaksof goldfish ulcer disease due to atypical Aeromonas salmonicida in south-easternAustralia. Journal of Fish Diseases 10, 353–362.
Whittington, R.J., Djordjevic, S.P., Carson, J. and Callinan, R.B. (1995) Restrictionendonuclease analysis of atypical Aeromonas salmonicida isolates from goldfish,Carassius auratus, silver perch Bidyanus bidyanus, and greenback flounderRhombosolea tapirina in Australia. Diseases of Aquatic Organisms 22, 185–191.
Wichardt, U.-P., Johansson, N. and Ljungberg, O. (1989) Occurrence and distribution ofAeromonas salmonicida infections on Swedish fish farms, 1951–1987. Journal ofAquatic Animal Health 1, 187–196.
Wiklund, T. (1990) Atypical Aeromonas salmonicida isolated from ulcers of pike, Esoxlucius L. Journal of Fish Diseases 13, 541–544.
Wiklund, T. (1995a) Survival of ‘atypical’ Aeromonas salmonicida in water andsediment microcosms of different salinities and temperatures. Diseases of AquaticOrganisms 21, 137–143.
Wiklund, T. (1995b) Virulence of ‘atypical’ Aeromonas salmonicida isolated fromulcerated flounder Platichthys flesus. Diseases of Aquatic Organisms 21, 145–150.
Wiklund, T. and Bylund, G. (1991) A cytochrome oxidase negative bacterium(presumptively an atypical Aeromonas salmonicida) isolated from ulceratedflounders (Platichthys flesus L.) in the northern Baltic sea. Bulletin of the EuropeanAssociation of Fish Pathologists 11, 74–76.
Wiklund, T. and Dalsgaard, I. (1995) Atypical Aeromonas salmonicida associated withulcerated flatfish species in the Baltic Sea and the North Sea. Journal of AquaticAnimal Health 7, 218–224.
Wiklund, T., Sazonov, A., L’Iniova, G.P., Pugaewa, V.P., Zoobaha, S.V. and Bylund, G.
425Furunculosis
(1992) Characteristics of Aeromonas salmonicida subsp. salmonicida isolated fromwild Pacific salmonids in Kamchatka, Russia. Bulletin of the European Associationof Fish Pathologists 12, 76–79.
Wiklund, T., Lönnström, L. and Niiranen, H. (1993) Aeromonas salmonicida spp.salmonicida lacking pigment production, isolated from farmed salmonids inFinland. Diseases of Aquatic Organisms 15, 219–223.
Wiklund, T., Dalsgaard, I., Eerola, E. and Olivier, G. (1994) Characteristics of ‘atypical’,cytochrome oxidase-negative Aeromonas salmonicida isolated from ulceratedflounders (Platichthys flesus (L.)). Journal of Applied Bacteriology 76, 511–520.
Wildavsky, A. (1995) But is it True? A Citizen’s Guide to Environmental Health andSafety Issues. Harvard University Press, Cambridge, Massachusetts, p. 574.
Willumsen, B. (1990) Aeromonas salmonicida subsp. salmonicida isolated from Atlanticcod and coalfish. Bulletin of the European Association of Fish Pathologists 10,62–63.
Wilson, B.W. and Holliman, A. (1994) Atypical Aeromonas salmonicida isolated fromulcerated chub Leuciscus cephalus. Veterinary Record 135, 185–186.
Wilson, I.G. (1997) Inhibition and facilitation of nucleic acid amplification. Applied andEnvironmental Microbiology 63, 3741–3751.
Yamaguchi, N., Teshima, C., Kurashige, S., Saito, T. and Mitsuhashi, S. (1980) Seasonalmodulation of antibody formation in rainbow trout (Salmo gairdneri). In: Solomon,J.B. (ed.) Aspects of Development and Comparative Immunology – I. Pergamon,Oxford/New York, pp. 483–484.
Yoshimizu, M., Direkbusarakom, S., Nomura, T., Ezura, Y. and Kimura, T. (1992)Detection of antibody against Aeromonas salmonicida in the serum of salmonid fishby the enzyme linked immunosorbent assay. Gyobyo Kenkyu 27, 73–82.
