Atypical bacterial gill disease: a new form of bacterial gill

disease affecting intensively reared salmonids

V E Ostland
1
, P J Byrne
1
, J S Lumsden
1,2
, D D MacPhee
1,3
, J A Derksen
1,4
, M Haulena
1,5
,
K Skar
6
, E Myhr and H W Ferguson
1,8
1 Fish Pathology Laboratory, Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph,
Ontario, Canada
2 Institute of Veterinary, Animal and Biomedical Sciences, College of Science, Massey University, Palmerston North,
New Zealand
3 Maritime Veterinary Services, St George, New Brunswick, Canada
4 Environmental Science Department, Lethbridge Community College, Lethbridge, Alberta, Canada
5 The Marine Mammal Center, Marin Headlands, GGNRA, Sausalito, California, USA
6 Veterinary Institute, Trondheim, Norway
7 National Vetinary Institute, Oslo, Norway
8 Institute of Aquaculture, University of Stirling, Stirling, UK
Abstract
An unusual form of bacterial gill disease (BGD) was
identified which affected five species of cultured
salmonids from Canada (i.e. rainbow trout, chi-
nook salmon and Atlantic salmon), Norway (i.e.
brown trout) and Chile (i.e. coho salmon). All
outbreaks occurred at low water temperatures
(< 10 C) and with clinical presentations distinct
from classical BGD, which is caused by Flavobac-
terium branchiophilum. In contrast to classical
BGD, fish did not show marked respiratory distress
with flaring of the opercula, the animals did not
orientate at the surface of the water column near
inflow water or at the margins of the tanks, and the
feed response of the fish was varied. While mortality
was increased, it was not precipitous as in classical
BGD. Eight outbreaks were examined in greater
detail using histopathology, scanning electron
microscopy, bacteriology and immunohistochemis-
try. Large numbers of small bacterial rods were seen
adhering to the lamellar epithelium of affected gills
from all outbreaks. Histologically, the lamellar
epithelium appeared swollen, often with evidence
of single cell degeneration and exfoliation. In more
severe instances, the formation of lamellar synechiae
was seen, usually associated with sequestration of
bacteria between fused lamellae. By contrast with
typical BGD, overt epithelial hyperplasia, lamellar
fusion and filamental clubbing were not common
sequelae to infection; instead, the end result was
shortened and somewhat stubby lamellae covered
with swollen epithelial cells. The predominant
bacterium recovered from affected gills was a small,
Gram-negative, motile, fluorescent pigment-produ-
cing rod that shared phenotypic characteristics with
Pseudomonas fluorescens. Polyclonal antisera pre-
pared against three representative isolates indicated
a weak antigenic similarity among them. Immuno-
histochemistry corroborated this finding, in that the
antisera reacted strongly with gill sections contain-
ing the homologous bacteria, but not against
morphologically similar bacteria in heterologous
sections. A Gram-negative, yellow pigmented
bacterium (YPB), identified as Flavobacterium
psychrophilum, was also recovered, but only from
the gills in the Ontario outbreaks. Antiserum
prepared against this YPB indicated an antigenic
similarity among isolates recovered from the
Journal of Fish Diseases 1999, 22, 351358
Correspondence Professor H Ferguson, Institute of Aqua-
culture, University of Stirling, Stirling FK9 4LA, UK.
E-mail: HWF1@stir.ac.uk
351
1999
Blackwell Science Ltd.
Ontario outbreaks, but immunohistochemistry
failed to recognize antigenically related bacteria on
the gills of fish from the other outbreaks. Based on
the unusual clinical presentation and the histo-
pathological appearance of the gills, in conjunction
with the absence of filamentous bacteria associated
with and recovered from affected gills, the present
authors have called this condition `atypical bacterial
gill disease' or ABGD.
Introduction
Infectious gill diseases, which are caused by yellow-
pigmented bacteria (YPB), are common production
diseases of intensively cultured fish (Wakabayashi,
Egusa & Fryer 1980; Daoust & Ferguson 1983;
Farkas 1985; Speare & Ferguson 1989; Toranzo &
Barja 1993). Most YPB have similar cell morphol-
ogy, but only a few produce distinctive branchial
lesions. For example, Flavobacterium columnare
produces marked and grossly visible necrosis of
the gill filaments, while Flavobacterium branchio-
philum produces a more diffuse epithelial prolifera-
tion in the absence of overt necrosis, leading to
branchial pallor and clubbing of the filaments
(Speare & Ferguson 1989; Thune, Stanley &
Cooper 1993; Toranzo & Barja 1993).
During routine diagnostic histopathology, an
unusual form of bacterial gill disease (BGD) was
recognized in intensively cultured salmonids from a
variety of geographical locations. These outbreaks
were considered `atypical' because outbreaks oc-
curred at water temperatures < 10 C, there was an
absence of obvious respiratory distress and histo-
pathology indicated the presence of large numbers of
short bacilli adhering to the lamellar epithelium. The
morphological appearance of the bacteria and the
response of the gills to infection, combined with the
unusual clinical presentation, led the present authors
to believe that this condition was quite distinct from
the `typical' form of BGD. It has become apparent
that this unusual form of BGD is more widespread
than was previously thought, and in this paper, the
present authors describe some of the clinical history,
the pathology and the bacteriology of this condition
in order to provide a description and definition of
this novel form of BGD. The present authors have
called this type of bacterial gill disease `atypical
bacterial gill disease' or ABGD.
Materials and methods
Background
From 1992 to 1997, an unusual form of BGD was
identified in a variety of salmonids from diverse
geographical locations (Table 1). Details of the
history of these fish were often limited, except that
fish were submitted for examination because of
increased mortality with or without evidence of
morbidity. All fish were reared in freshwater
environments; fish from Ontario, Canada, origi-
nated from two net pen sites (outbreaks C, E and F)
moored in Lake Huron or from a land-based
hatchery (outbreak G) which received groundwater.
The brown trout from Norway (outbreak A) were
reared in outdoor tanks which received surface
water from a nearby river. The coho salmon
(outbreak B) were from a hatchery in Chile, while
the chinook and Atlantic salmon (outbreaks D and
H) originated from hatcheries in British Columbia,
Canada, which utilized non-recirculated ground-
water. The common denominator among the
outbreaks was that disease occurred at water
temperatures < 10 C. The Ontario and Norwe-
gian outbreaks typically occurred during the winter
months when water temperatures were less than 3
4 C. In Ontario and Norway, farmers reported
that affected fish appeared dark, were lethargic
and usually showed no interest in feed. Fish were
often observed resting on the bottom, effectively
giving the impression that the animals were
`sleeping'. Respiratory distress with tachybranchia
Table 1 Origin of atypical bacterial gill disease outbreaks
Outbreak Year Species Location
A 1992 Brown trout Norway
B 1994 Coho salmon Chile
C 1994 Rainbow trout Ontario, Canada
D 1994 Chinook salmon British Columbia, Canada
E 1995 Rainbow trout Ontario, Canada
F 1996 Rainbow trout Ontario, Canada
G 1996 Rainbow trout Ontario, Canada
H 1997 Atlantic salmon British Columbia
Journal of Fish Diseases 1999, 22, 351358 V E Ostland et al. Atypical bacterial gill disease
352
1999
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was not reported. However, when disturbed, the
fright response of the fish was weak and they
would eventually sink to the bottom again.
Mortality rates were not as explosive or as great as
those reported for fish with typical BGD, but
nonetheless, some farmers reported total mortality
reaching 20%.
Histopathology and electron microscopy
Formalin and/or Bouin's preserved tissues from
brown trout, Salmo trutta L., coho salmon,
Oncorhynchus kisutch (Walbaum), chinook salmon,
Oncorhynchus tshawytscha (Walbaum), and Atlantic
salmon, Salmo salar L., were submitted to the Fish
Pathology Laboratory, Department of Pathobiol-
ogy, Ontario Veterinary College, University of
Guelph, Guelph, Ontario, Canada, for routine
diagnostic examination. Live moribund rainbow
trout from Ontario were submitted directly from
the affected farms. The Ontario fish were preserved
overnight in Bouin's, transferred to 70% isopropa-
nol and submitted for routine histopathology.
Representative tissues from all outbreaks were
embedded in paraffin wax and 5-mm sections were
stained with haematoxylin and eosin.
For scanning electron microscopy (SEM), repre-
sentative gill tissue from outbreaks A and C was
preserved overnight in 2.5% glutaraldehyde in
0.1 m phosphate buffer (at 4 C). The following
day, the tissues were transferred to the same buffer
and held for several days at 4 C until processing
for SEM.
Bacteriology
Gill tissue from moribund rainbow trout (the
Ontario outbreaks) was placed in pre-weighed vials
containing 1 mL of ice-cold, filtered tap water
(0.1 mm syringe filter, Gelman Sciences, Ann
Arbour, MI, USA). After weighing the vials to the
nearest 0.1 mg, gill tissue was homogenized briefly
and the number of bacterial colony forming units
per gram (CFUs g
1
) of gill tissue was estimated by
streaking 20-mL aliquots (in duplicate) of serial 10-
fold dilutions (prepared in filtered tap water) on
tryptic soy agar (TSA, Difco, Detroit, MI, USA),
cytophaga agar containing 1.5% agar (CA; Anacker
& Ordal 1959) and Pseudomonas-selective agar
(PsA, Becton Dickinson and Co., Cockeysville,
MD, USA). All media were incubated aerobically at
10 C for 12 days before enumeration. Addition-
ally, kidney tissue was streaked onto CA and TSA
to help rule out the presence of other bacterial
pathogens. The most common bacteria recovered
from each outbreak were subcultured onto the same
medium for further phenotypic characterization
using standard methods and criteria (MacFaddin
1980; Holt, Krieg, Sneath, Stanley & Williams
1994). Commercially available identification strips
were also utilized to corroborate the biochemical
characteristics (API 20E strips, bioMerieux Vitek
Inc., Hazelwood, MO, USA). The most common
bacteria recovered from diseased brown trout and
coho salmon gills (outbreaks A and B, respectively)
were generously donated for comparison with those
recovered from the rainbow trout gills in Ontario.
Antiserum production
Polyclonal antiserum was raised against four
representative gill isolates recovered from rainbow
trout in Ontario, brown trout in Norway and coho
salmon in Chile. Three isolates (1 Ga, TSC and
B63-A) were grown in tryptic soy broth (TSB,
Difco, Detroit, MI, USA), while the YPB isolate
(B63-E) was grown in cytophaga broth (CB;
Anacker & Ordal 1959). All were incubated at
18 C for 72 h, killed by the addition of 0.1%
formalin (v/v) and held at 10 C. The following
day, the cells were harvested by centrifugation,
washed twice in phosphate-buffered saline (PBS,
pH 7.2) and adjusted to an optical density (OD,
550 nm) of 1.0. One-millilitre aliquots of the
washed whole cell preparations were frozen at
70 C until required.
For immunization, the cells were thawed and
combined with an equal volume of Freund's
incomplete adjuvant. Following homogenization,
1 mL of this suspension was injected subcuta-
neously into the shoulder region of each of two
female New Zealand white rabbits. The rabbits
were boosted at 2-week intervals for a total of five
immunizations. One week after the last immuniza-
tion, the rabbits were anaesthetized and exsangui-
nated by cardiac puncture, and the blood was
allowed to clot overnight at 4 C. Serum was
collected the following day, dispensed into aliquots
and stored at 20 C. All serum was heat-
inactivated at 56 C for 30 min before use.
Immunohistochemistry
Immunohistochemistry was used to determine
Journal of Fish Diseases 1999, 22, 351358 V E Ostland et al. Atypical bacterial gill disease
353
1999
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whether the bacteria recovered from the outbreaks
of `atypical' BGD were antigenically related to those
observed in the gill sections. Paraffin-wax-em-
bedded sections from each case were mounted on
Superfrost Plus glass slides (Fisher Scientific,
Pittsburgh, PA, USA) and heated overnight at
37 C before these were deparaffinized as previously
described (Ostland, Lumsden, MacPhee & Fergu-
son 1994) and held submerged in distilled water for
staining. Representative gill and other tissue sections
were probed with anti-whole cell serum raised from
the Ontario (B63-A and B63-E), the Norwegian
(1 Ga) and the Chilean (TSC) isolates. Optimal
dilutions for each antiserum (prepared in PBS) were
determined empirically and ranged from 1:100 to
1:500. Bacteria were visualized using a commer-
cially available biotinylated goat-antirabbit perox-
idase system (Histostain SP Kit, Zymed
Laboratories, San Francisco, CA, USA). Non-
immune rabbit serum served as a control.
Bacterial agglutination
The antigenic relationships among the bacteria
recovered from outbreaks A, B, C, E, F and G were
examined using whole cell agglutination in a 96-
well, round-bottom microtitre plate assay (Ostland
et al. 1994). Bacteria were grown, harvested and
killed as described above. After adjusting the cells to
an OD of 0.8, 50 mL of this suspension was placed
into duplicate wells containing two-fold serial
dilutions of antiserum in PBS and the plates were
incubated at room temperature for 2 days. The
reciprocal of the highest dilution that gave a positive
agglutination was recorded as the titre. The
following bacteria were used to test the specificity
of the antisera: ATCC 7965 Aeromonas hydrophila,
ATCC 19264 Listonella (Vibrio) anguillarum,
ATCC 33660 Pseudomonas anguilliseptica, ATCC
10145 Pseudomonas aeruginosa, ATCC 13525
Pseudomonas fluorescens, ATCC 49510 Flavobacter-
ium psychrophilum (formerly Flexibacter psychrophi-
lus), ATCC 49513 Flavobacterium columnare
(formerly Flexibacter columnaris) and NCMB
1947
T
Flavobacterium psychrophilum (formerly
Flexibacter psychrophilus).
Results
Histopathology and ultrastructure
Histologically, the gills of all species of affected
salmonids had moderate to large numbers of short
Gram-negative rods adherent to the lamellar
epithelium as well as free within the interlamellar
spaces (Fig. 1). Overt necrosis of the branchial
tissues was not a common feature; branchial
responses associated with the presence of the
bacteria included multifocal to diffuse lamellar
epithelial swelling, degeneration and sloughing.
Bacteria could often be seen on the surface of the
exfoliated cells, with attenuation of the adjacent
epithelium a common response to exfoliation. In
some instances, chloride cells appeared to be
particularly involved; this observation was more
prevalent in the Atlantic and Pacific salmon.
Interlamellar epithelial hyperplasia was evident in
outbreaks which were felt to be chronic. This
response to infection resulted in lamellae which
appeared shorter than normal, essentially giving
these a stubby appearance. However, the occasional
formation of lamellar synechiae, usually with a focal
to multifocal distribution and bacteria present
between the fused lamellae, was of note. Unlike
Figure 1 Histological presentation of atypical bacterial gill
disease in brown trout from
Norway. Large numbers of bacteria (arrows) were associated with
the surface of the swollen lamellar epithelium and were free
within interlamellar space.
Journal of Fish Diseases 1999, 22, 351358 V E Ostland et al. Atypical bacterial gill disease
354
1999
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the more `typical' form of BGD, diffuse filamental
clubbing was not a significant feature of this dis-
ease. Branchial inflammation as a consequence of
infection was considered minimal, but was com-
posed of both macrophages and neutrophils within
the filamental interstitium.
The Ontario outbreaks also had focal aggregates
of thin, medium length Gram-negative bacteria
associated with the epithelium at the base of the
lamellae. These often coincided with the presence of
lamellar synechiae and morphologically similar
bacteria could be seen between the fused lamellae.
Similar bacteria were not seen associated with
affected gills from the other outbreaks.
Scanning electron microscopy of affected brown
and rainbow trout gills demonstrated the presence
of moderate to large numbers of short bacilli (0.4
0.5 1.31.8 mm) distributed as focal aggregates
over the surface of the lamellar epithelium (Fig. 2).
The formation of lamellar synechiae was common
along entire gill filaments; in some instances, the
lamellae were distinctly shorter in appearance. The
degree of epithelial swelling, degeneration and
occasionally exfoliation associated with the presence
of the bacteria appeared to be a reflection of the
numbers of bacteria. Usually, there was a distinct
thickening of the epithelial microridges, and in
severely affected regions, this gave the lamellar
surface an almost smooth appearance.
In addition to the short bacilli present on the gills
of the brown and rainbow trout, SEM demon-
strated the presence of significant numbers of long,
thin bacteria (0.380.53 4.87.2 mm) associated
with the gill lesions of the rainbow trout. These
bacteria had tapered ends, giving them an almost
fusiform appearance; their presence on the respira-
tory surfaces also produced similar morphological
alterations to the lamellar epithelial surfaces. Of
interest was the fact that these bacteria never seemed
to colonize regions containing the short bacilli; they
were most frequently observed at the base of the
lamellar synechiae, often seen between those
lamellae which were undergoing fusion.
Bacteriology
Large numbers of a mixed bacterial microflora were
recovered from the gills of affected rainbow trout in
Ontario (Table 2). Compared to PsA and TSA, CA
yielded the greatest total colony forming units per
gram gill tissue, ranging from 4.59 10
5
to
494.5 10
5
CFUs g
1
. Slightly fewer bacteria were
recovered on TSA, with amounts ranging from
2.72 10
5
to 446.5 10
5
CFUs g
1
. The fewest
bacteria were recovered on PsA (0.04 10
5

0.45 10
5
CFUs g
1
), probably because this med-
ium was designed for the selective isolation of
Pseudomonas aeruginosa. Yellow-pigmented bacteria
were recovered on both CA and TSA. These
Figure 2 Scanning electron micrograph of brown trout gills with
the small, rod-shaped bacteria associated with the thickened
microridges of the swollen lamellar epithelium.
Table 2 Total bacterial colony-forming units per gram of gill
tissue (CFUs g
1
) recovered from the gills of rainbow trout with
atypical bacterial gill disease using tryptic soy agar (TSA),
Pseudomonas-selective agar (PsA) and cytophaga agar (CA)
Number Isolation
medium
Total CFUs
g
1
( 10
5
)
Yellow-pigmented
bacteria (%)
Outbreak C
3 TSA 2.72 22
3 PsA 0.17 0
3 CA 4.59 35
Outbreak E
3 TSA 4.26 45
3 PsA 0.04 0
3 CA 55.46 73
Outbreak F
2 TSA 446.5 18
2 PsA 0.45 0
2 CA 494.9 58
Journal of Fish Diseases 1999, 22, 351358 V E Ostland et al. Atypical bacterial gill disease
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1999
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bacteria comprised 3573% of the total bacterial
flora isolated from the diseased rainbow trout gills.
The YPB were not recovered on PsA (Table 2).
Bacteria were not recovered from the kidneys of
affected fish in Ontario.
Incubation of the rainbow trout gill homogenates
on TSA resulted in the recovery of large numbers of
small, cytochrome-c oxidase positive, motile Gram-
negative bacilli (B63-A). These colonies produced a
mucoid substance which collected on the Petri dish
lid during extended incubation. Similar colony
types were recovered on PsA, usually in pure
culture. Yellow-pigmented, non-motile bacterial
rods were also recovered on TSA, but these re-
presented a low proportion of the total flora
recovered. Growth on the PsA plates yielded
mucoid bacterial colonies similar in morphology
to those recovered on TSA. On PsA, these Gram-
negative, oxidase-positive, motile bacilli secreted a
greenish pigment which diffused into the surround-
ing agar. However, the dominant isolate recovered
on CA was a long, Gram-negative, oxidase-positive,
non-motile, yellow pigmented rod (B63-E) which
did not exhibit colonial spreading.
The Gram-negative bacilli which were recovered
from diseased fish in Norway (1 Ga), Chile (TSC)
and Ontario (B63-A) were short, motile rods (0.4
0.7 23 mm) which grew at 4 C, but not at
37 C, produced a diffusable fluorescent pigment,
and possessed gelatinolytic activity, traits shared by
strains of P. fluorescens (Holt et al. 1994). The API
20E profiles supported this finding because these
were identified as P. fluorescens. Whole-cell agglu-
tination indicated that the Ontario isolates were
weakly related to those from Norway and Chile
because heterologous titres ranged from 2 to 8 (data
not shown). Rabbit anti-B63-A, anti-1 Ga and
anti-TSC sera weakly agglutinated ATCC 13525,
the P. fluorescens reference strain; titres were 8, 4
and 4, respectively (data not shown). The remain-
ing reference strains were not agglutinated by these
antisera (data not shown).
The most common YPB recovered from each of
the Ontario outbreaks were cytochrome-c oxidase-
positive, weakly catalase-positive, non-fermentative,
non-motile rods (0.50.7 57 mm). All possessed
flexirubin-type cell wall pigments, exhibited growth
from 5 to 25 C and were able to hydrolyze gelatin
and casein, but not starch, agar or cellulose. None
of the YPB could degrade simple or complex
carbohydrates. Based on these phenotypic charac-
teristics, these YPB were identified as F. psychrophi-
lum (Holt et al. 1994). The four Ontario isolates all
displayed antigenic similarities with isolate B63-E
because heterologous titres ranged from 16 to 512
(data not shown). Furthermore, anti-B63-E serum
produced agglutination titres of 64 and 512,
respectively, against NCMB 1947
T
and ATCC
49510, the F. psychrophilum (formerly Flexibacter
psychrophilus) reference strains (data not shown).
Anti-B63-E serum did not agglutinate the other
reference strains (data not shown).
Immunohistochemistry
Antisera raised against the P. fluorescens isolates
recovered from brown trout (Norway) and coho
salmon (Chile) gills reacted weakly with the bacteria
present in the respective homologous sections.
These antisera did not cross-react with morpholo-
gically similar bacteria present on the gills of the
chinook or Atlantic salmon (British Columbia) nor
the rainbow trout from Ontario. Similarly, rabbit
anti-B63-A serum reacted against the morphologi-
cally similar bacteria on the rainbow trout gills from
the Ontario outbreaks, but it did not recognize
morphologically similar bacteria present on the gills
of the other species of salmonids. Neither the 1 Ga,
TSC nor B63-A antiserum cross-reacted with the
other bacteria seen in section.
Rabbit anti-B63-E serum raised against F.
psychrophilum recovered from rainbow trout gills
in Ontario recognized only the thin, long bacteria
on the gills associated with the Ontario outbreaks;
other tissues were all negative. This antiserum did
not recognize the short bacilli present on the gills of
the affected species from any of the outbreaks. Anti-
B63-E serum failed to demonstrate antigenically
related bacteria on the gills of the brown trout
(outbreak A), coho salmon (outbreak B), chinook
salmon (outbreak D) and Atlantic salmon (outbreak
H).
Discussion
The present results indicate that a non-filamentous
bacterium is associated with a novel form of
bacterial gill disease affecting intensively reared
salmonids from geographically diverse locations.
While these findings are preliminary, comparative
histopathology has demonstrated that affected gills
of all species of salmonids harboured large numbers
of short Gram-negative bacilli present on the
lamellar epithelium. From a bacteriological per-
Journal of Fish Diseases 1999, 22, 351358 V E Ostland et al. Atypical bacterial gill disease
356
1999
Blackwell Science Ltd.
spective, the gills of Ontario-reared rainbow trout
with this `atypical' form of BGD had total bacterial
CFUs g
1
of gill tissue roughly two to four orders of
magnitude greater than those of healthy rainbow
trout gills (Ostland, Ferguson, Prescott, Stevenson
& Barker 1990). The most consistent bacterium
recovered from the diverse microflora associated
with the diseased gills was a short, Gram-negative
rod which shared phenotypic characteristics with P.
fluorescens. Immunohistochemistry has provided
evidence that the P. fluorescens-like bacteria recov-
ered from diseased gills were antigenically related to
those seen histologically. Since the morphology of
the bacteria associated with this condition is distinct
from the filamentous bacteria present on the gills of
salmonids with `typical' BGD, and since the
histopathology and clinical presentations are also
quite different, the present authors propose the
name `atypical bacterial gill disease' or ABGD as a
more appropriate descriptor for this novel presenta-
tion of BGD.
The genus Pseudomonas contains a large number
of species which are widely distributed in nature,
inhabiting water and soil, plant surfaces and the
mucosal surfaces of healthy animals, including
humans (Palleroni 1984). Some species are obligate
animal pathogens while others are considered to be
saprophytic and/or opportunistic pathogens of both
plants and animals. For example, P. aeruginosa is an
opportunistic pathogen capable of causing respira-
tory infections in a variety of animal species;
however, it can also produce both localized and
systemic forms of disease (Gyles 1986; Saiman,
Cacalano, Gruenert & Prince 1992; Govan &
Deretic 1996). Four fish-pathogenic Pseudomonas
species (P. anguilliseptica, P. chlororaphis, P.
fluorescens and P. pseudoalcaligenes) have been
isolated from diseased fish (Hatai, Egusa, Nakajima
& Chikahata 1975; Miyazaki, Kubota & Miyashita
1984; Wiklund & Bylund 1990; Austin & Stobie
1992; Berthe, Michel & Bernardet 1995). These
species of Pseudomonas seem to produce predomi-
nantly systemic disease at water temperatures
ranging from 9 to 22 C (Hatai et al. 1975;
Wiklund & Bylund 1990; Thune et al. 1993;
Berthe et al. 1995). However, some strains of P.
fluorescens have been recovered from haemorrhagic
skin and muscle lesions, but these fish were also
septicaemic (Meyer & Collar 1964; Bullock 1965).
In contrast, the P. fluorescens strains recovered from
the gills of fish with ABGD appear to be obligate
mucosal pathogens because bacteria were found
only on the gills, there was no histological evidence
of systemic disease and bacteria were not recovered
from the kidney of affected fish in Ontario. To the
best of the present authors' knowledge, this is the
first report of P. fluorescens associated with a
bacterial gill disease affecting salmonids at low
water temperatures.
Scanning electron microscopy demonstrated that
the Ontario outbreaks of ABGD also had long,
thin, fusiform-shaped bacteria present on the gills of
rainbow trout. Morphologically similar bacteria
were not seen on the gills of the brown trout from
Norway. Histologically, it was difficult to discern
whether the thin, long, Gram-negative bacteria
evident on the gills from Ontario outbreaks were
indeed fusiform, although light microscopy sug-
gested that one YPB isolate (B63-E) appeared
fusiform (unpublished observations). Agglutination
studies with anti-B63-E serum indicated that this
YPB shared antigenic similarities with F. psychro-
philum. However, it is not known whether the
fusiform bacteria observed with SEM were indeed
those recovered from affected gills or observed
histologically. A fusiform-shaped, Gram-negative
bacterium has been associated with BGD outbreaks
of Pacific salmon (Hoskins 1976) and a similar
morphology has been described for F. psychrophilum
recovered from coldwater disease or rainbow trout
fry syndrome (RTFS; Lorenzen, Dalsgaard &
Bernardet 1997). Nevertheless, F. psychrophilum is
not commonly recovered from fish gills (Holt,
Rohovec & Fryer 1993) and this is probably the
first report of F. psychrophilum being recovered
from a bacterial gill disease in rainbow trout,
although it was recovered only from the Ontario
outbreaks. It is worth noting that there was no
indication, either from culture results or from
immunohistochemistry, of systemic involvement of
the isolate, as is seen in RTFS. A more in-depth
examination of a larger number of outbreaks from a
variety of geographic locations would help ascertain
the significance of F. psychrophilum in ABGD.
The cause of ABGD remains unknown. The
present authors have attempted to reproduce the
disease under controlled laboratory conditions
using the isolates they have recovered, but have
been unsuccessful, even under conditions normally
successful for the experimental reproduction of the
`typical' form of BGD (Ferguson, Ostland, Byrne
& Lumsden 1991). However, the present authors
did not try to reproduce the disease at low water
temperatures.
Journal of Fish Diseases 1999, 22, 351358 V E Ostland et al. Atypical bacterial gill disease
357
1999
Blackwell Science Ltd.
The treatment of ABGD appears to be difficult,
and clinical responses following a 1-h formalin or
chloramine-T bath treatment were often varied.
Some farmers reported a good response, regardless
of the therapeutant, while others reported no
response at all. Furthermore, most farmers felt that
antibiotic administered in the feed was not effective
for controlling ABGD. Treatment of ABGD in net
pens was not tried, but when ambient water
temperatures rose above 56 C, some farmers
reported that the fish appeared to recover sponta-
neously. This suggests that raising the ambient
water temperature may be an effective management
strategy, although this is probably not possible for
most groundwater facilities.
Acknowledgments
The Fish Pathology Laboratory received much of its
funding from the Ontario Ministry of Agriculture,
Food and Rural Affairs. P.J.B. and J.S.L. were
Fellows of the Medical Research Council of
Canada. H.W.F. was in receipt of a Norwegian
Government Fellowship.
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