559

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

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
15
Flavobacterial Diseases: Columnaris
Disease, Cold-water Disease and
Bacterial Gill Disease
E.B. Shotts, Jr
1*
and C.E. Starliper
2
1
Department of Medical Microbiology, University of Georgia, Athens, Georgia
30602, USA;
*
Present address: Office of the Director, National Fish Health
Research Laboratory, 1700 Leetown Road, Kearneysville, West Virginia 25430,
USA;
2
National Fish Health Research Laboratory, 1700 Leetown Road,
Kearneysville, West Virginia 25430, USA.
INTRODUCTION
The genus Flavobacterium contains organisms which have been assigned at
various times to the genera Chondrococcus (Ordal and Rucker, 1944), Cyto-
phaga (Garnjobst, 1945) or Flexibacter (Leadbetter, 1974). Most recently,
members of this group which cause disease in fish were placed into the genus
Flavobacterium, with the exception of the marine counterpart (Flexibacter
maritimus) to Flavobacterium columnare, which was left in the genus
Flexibacter (Bernardet et al., 1996). It is generally accepted that Flavobacterium
columnare causes columnaris disease and Flavobacterium psychrophilum
causes cold-water disease. Flavobacterium branchiophilum causes bacterial
gill disease (BGD), while Flexibacter maritimus causes marine columnaris.
Other poorly defined Flavobacterium-like organisms have a wide range of
virulence and have been implicated as opportunistic fish pathogens (Lien, 1988;
Shotts and Teska, 1989; Starliper, 1992).
A common feature among these organisms is a yellow- to yellowish-brown-
or orange-pigmented growth on solid media, which turns pink to pinkish red in
the presence of alkali (Reichenbach et al., 1981). These organisms belong to a
larger group often called the yellow-pigmented bacteria (YPB) (Shotts et al.,
1983). A unique characteristic of most of those causing fish disease is a
spreading or gliding motility, which differs dramatically from ordinary flagellar
motility.
Generally, these bacteria have been described as Gram-negative rods, with
flexuous filaments, which are about > 5 m in length and < 1 m in width. There
are no resting cells, fruiting bodies, spores or microcysts associated with any of
these organisms (Leadbetter, 1974; Bernardet et al., 1996). Colonies are usually
pigmented (yellow to orange), varying in shape from flat and spreading to a
560
E.B. Shotts Jr and C.E. Starliper
discrete entire convex shape, depending upon the respective isolate and the
growth medium.
Usually, these bacteria are oxidase-, phosphatase- and ribonuclease-
positive. However, despite numerous attempts to group them phenotypically, the
differentiation and classification of the spreading (gliding) YPB remain in an
ambiguous state of flux (Borg, 1960; Bullock, 1972; McMeekin et al., 1971;
Shewan and McMeekin, 1983; Bernardet and Grimont, 1989; Bernardet et al.,
1996). This inability to group them phenotypically could be attributed, in part, to
their nutritional requirements. There are no standardized biochemical,
enzymatic, immunological, or genetic methods that can be used economically to
study these bacteria in clinical situations and this has further deterred our
understanding of this complex group of organisms.
The ability of these organisms to cause both acute and chronic disease and
their ubiquitous nature in the environment have made them one of the most
devastating groups of bacterial pathogens of fish. The estimated annual losses
from columnaris disease, cold-water disease and BGD are staggering.
CLINICAL SIGNS AND DISTRIBUTION OF DISEASE
All fish worldwide are susceptible to some form of disease caused by
Flavobacterium spp. The host and disease aetiology encountered usually follows
a geographical and temperature gradient. These microorganisms are considered
ubiquitous in the aquatic environment and are usually found causing disease as a
result of fish stress due to mechanical and/or environmental insults.
The first documented occurrences of this group of organisms causing
disease were in a number of species of fishes in the Mississippi River valley
(Davis, 1922), where high mortality was reported. Although the bacterium was
not cultured, it was named Bacillus columnaris and described as a rod found in
columnar masses, and the disease was called columnaris disease. It was
isolated and named Chondrococcus columnaris by Ordal and Rucker (1944).
Since then, it has been isolated from a wide variety of both warm- and cool-
water fishes with clinical signs. Typically, disease is not spontaneous but
requires some type of physical or environmental insult; normal, healthy fish are
usually resistant to F. columnare (Vogel, 1958). The infection with F. columnare
begins at the mouth, fins and gills as primary sites, but injuries elsewhere on the
body may provide a primary infection site. These infection sites increase the
numbers of organisms in the water column, facilitating the spread of the disease,
until it is treated or there is a dramatic fish mortality. The incubation period
varies with strain, physical status of the fish and route of exposure. The disease
condition is affected by temperature and is usually seen between 10 and 30C.
Stocking and transport of fish, while a stressing influence, are not usually major
factors in the spread of the disease.
The disease may first be noted as an increase in mucus on the head and upper
body, which gives way to circular areas of greyish opalescent growth, having a
slight lemon-yellow tint. Gills usually have whitish spots on filament tips,
followed by overgrowth on the filaments. As the condition progresses, wide-
561 Flavobacterial Diseases
spread erythemic spots appear, which may result in extensive necrosis,
bacteraemia and ultimately death (Fig. 15.1). Morbidity in crowded or poorly
managed situations may reach 100% and mortality approach 70%, usually made
up of young and/or highly stressed fish. In wild ranging fish, morbidity may vary
from 1% to 30%, depending upon stressful environmental parameters and the
species at risk.
Another member of this genus is associated with disease in cooler waters
and was first distinguished from columnaris by Borg (1960) in 1948 and
eventually named cold-water disease. The organism was first called Cytophaga
psychrophila and subsequently named Flavobacterium psychrophilum (Bernardet
et al., 1996). This organism usually causes disease below 12C and does not
survive at temperatures over 25C (Pacha, 1968; Anderson and Conroy, 1969).
Disease is first noted as tissue necrosis associated with the fins and is often
called fin rot at this stage (Fig. 15.2). As the disease moves to a more chronic
form, with tissue involvement at the base of the caudal fin (the caudal peduncle),
it is often referred to as peduncle disease (Van Duijn, 1973). This condition is
most often seen at water temperatures between 4 and 10C. The disease occurs
most frequently in cultured salmon species, brook trout (Salvelinus fontinalis),
rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), lake trout
(Salmo namaycush) and several species of whitefish, carp, dace and suckers.
Early signs of the disease are subtle, consisting of the presence of rough skin
and loss of fin-tip integrity. These early signs may precede infection and suggest
Fig. 15.1. Channel catfish showing lesions of Flavobacterium columnare. Note skin sloughing
on posterior third of body of middle fish primarily between dorsal and caudle fins. On bottom fish,
a lesion is just beginning at the end of pectoral fin; top fish does not show signs of infection.
562
E.B. Shotts Jr and C.E. Starliper
other predisposing conditions, including changes in water pH, malnutrition and
toxic components in the water column. These factors may lead to bacterial
colonization, noted as a line of whitish material along the fin margin, perhaps
leading to separation of fin rays. As the infection continues, a line of necrosis,
with a line of reddened tissue, develops at the site of bacterial activity. If this is
unchecked, it spreads and necrosis in the peduncle may continue and, in extreme
cases, the caudal vertebrae may be exposed.
Morbidity may range from 1% to 50%, particularly where water temper-
atures are below 10C and there is a low physiological resistance to disease. In
many of these instances, mortality may approach 75% (Post, 1983).
Chronic and unusual forms of cold-water disease result in spiral or erratic
swimming behaviour, while the fish are void of typical external lesions (Kent
et al., 1989; Blazer et al., 1996). This occurs in fish that have recently recovered
from epizootics of the typical form of infection (Kent et al., 1989) or in
populations that have no recent history of typical cold-water disease (Blazer et
al., 1996). Chronic periostitis, osteitis, meningitis and ganglioneuritis are
observed histologically. Infections are seen in the cranial area and anterior
vertebrae; inflammation and cartilage necrosis can be observed along the
vertebral column. Long, thin, Gram-negative bacteria can be seen histologically
in areas of inflammation and may also be isolated from internal tissues,
including kidney, spleen and heart. Treatment of these forms of cold-water
disease using antimicrobials is often not efficacious.
A third organism, F. branchiophilum, also causes high mortalities in cool-
and cold-water fishes. The disease was first noted and reported by Davis (1926).
In the ensuing years, it was seen that a nutritional gill disease (pantothenic acid
deficiency) was noted in the western USA, a second type of haemorrhagic gill
disease from aquatic toxicants with secondary mycotic involvement, and a third
Fig. 15.2. Fish showing fin rot or fin necrosis of the tail clinically diagnostic of Flavobacterium
psychrophilum infection.
563 Flavobacterial Diseases
type caused by an infectious agent. Although noted in 1926, it was not until 1978
(Kimura et al. 1978) that the causative agent was isolated. Subsequently,
Wakabayashi et al. (1989) studied isolates from the USA and Japan and
characterized the organism, naming it Flavobacterium branchiophila. In 1990,
von Graevenitz proposed a name change to F. branchiophilum. Lien (1988)
demonstrated genetic relationships between fish isolates of Flavobacterium and
Flexibacter.
The disease caused by F. branchiophilum appears to be closely related to
environmental gill disease, as both contribute significantly to a sharp reduction
in the respiratory capacity of salmonids. In infectious gill disease, the first
noticeable signs are anorexia and the fish lining up at the fresh-water inlet.
Concomitantly, small white to grey spots may appear on the gills and may be
seen as the fish swims or turns. These lesions may be either unilateral or
bilateral, proliferate rapidly and are firmly attached masses of bacterial growth
on the lamellae and filaments of the gill. Morbidity in severe cases of BGD may
approach 100% if untreated.
A fourth organism causing disease in fish is Flexibacter maritimus. It is a
halophilic organism and is in many respects similar in disease and characteriza-
tion to Flavobacterium columnare. This organism will not be discussed in detail
here, since our primary emphasis is on freshwater species. Flexibacter
maritimus was reported as a salt-water-requiring organism producing infections
characterized by eroded mouth and fins and skin lesions (Wakabayashi et al.,
1984, 1986). Infections follow physical trauma or malnutrition, with morbidities
approaching 100% and mortalities of 70% (Wakabayashi, 1993).
AETIOLOGICAL AGENTS
The agents causing columnaris disease, cold-water disease and BGD are closely
related, although their phenotypic characteristics vary widely (Bader, 1995;
Bernardet et al., 1996). Bader (1995) suggested that the pathogenic members of
the flavobacteria are grouped on a single branch of the phylogenetic tree with
members of the BacteroidesFlavobacterium. Other genera on this branch
include Capnocytophaga, Flexibacter and Cytophaga (Krieg and Holt, 1984).
They require reduced nutrient media for primary isolation and growth. The
three media which are used routinely for isolation are: Ordals (Ordal and
Rucker, 1944); tryptoneyeast extractgelatin medium (TYG) and HsuShotts
medium (Bullock et al., 1986).
The morphology of the colony and phenotypic characteristics of the
organisms vary. Flavobacterium columnare colonies grown at 2025C are
yellowish, flat, rhizoid and spreading to discrete, convex colonies. These depend
upon the medium used for primary isolation. The bacterium is a Gram-negative
rod, approximately 5 m in length, which forms flexuous filaments, with a width
of about 1 m. Their yellow pigment changes to pink in 3% potassium hydroxide
(KOH). The pathogen does not grow on conventional microbiological media,
hydrolyses elastin and chondroitin sulphate, utilizes glucose and adheres tightly
to agar. There are four other groups of bacteria which grossly on agar appear to
564
E.B. Shotts Jr and C.E. Starliper
be F. columnare, but they do not break down chondroitin sulphate and may be
grouped into unspeciated groups, utilizing a battery of sugars and their ability to
grow on conventional media (Shotts and Teska, 1989; Bader, 1995). These latter
groups produce infection on damaged tissues as opportunistic pathogens and may,
without detailed laboratory study, be mistakenly diagnosed as F. columnare.
Flavobacterium psychrophilum colonies are spreading, non-diffusible
yellow-pigmented colonies. Microscopic preparations show Gram-negative
rods, from 1 m to 7.5 m in length and 0.75 m in width. Generally, growth is
between 5C and 25C and they (like F. columnare) do not tolerate 2% sodium
chloride (NaCl). They are aerobic, will growth sparsely on conventional media,
will hydrolyse elastin and starch, but not chondroitin sulphate. They do not
adhere tightly to agar.
The causal bacterium of BGD, F. branchiophila, was described by
Wakabayashi et al. (1989), using strains isolated from the USA Japan and
Hungary (Kimura et al., 1978; Wakabayashi et al., 1980; 1989; Farkas, 1985).
Flavobacterium branchiophilum is the current nomenclature (von Graevenitz,
1990). These cells are Gram-negative, non-motile rods (0.5 m 58 m),
which grow aerobically in the temperature range of 1025C. Colonies on
Cytophaga agar (Anacker and Ordal, 1959) are light yellow in colour, non-
spreading and small, 0.51.0 mm in diameter after 5 days incubation at 18C;
they are smooth, round, raised and have an entire border. Strains produce
catalase and cytochrome oxidase; utilize gelatin, casein and starch; and produce
acid from glucose, fructose, sucrose, maltose, trehalose, cellobiose, melibiose,
raffinose and inulin. The deoxyribonucleic acid (DNA) guanine plus cytosine
(G + C) content ranges from 29% to 31%. Strains isolated from Ontario and
Korea are very similar in their biochemical and physiological properties;
however, these strains demonstrated cell lengths of up to 15 m (Ostland et al.,
1994; Ko and Heo, 1997). Ostland et al. (1994) also reported 35 mm colony
diameters after 67 days incubation at 18C. This bacterium is very difficult to
culture on primary isolation, because it is relatively slow-growing. This
increases the possibility for overgrowth from other non-BGD bacteria, which
may be present on diseased gill tissue.
DIAGNOSTIC METHODS
A clinical diagnosis of columnaris disease is made visually, by observing an
erosive or necrotic lesion of the skin or gill. These are commonly noted on the
head/body anterior to the dorsal fin. This lesion may have an erythemic edge and
present as whitish plaques, which may develop rapidly into ulcers. Often, in the
early stages, there may be pale yellowish areas at the outer edge of the lesion,
where the organism is growing in large numbers. As the ulcer deepens, a
bacteraemia may occur. Infections of the gill may also occur, starting on the tips
of the lamellae and progressing inward, with necrosis of epithelium. Diagnosis is
usually done by microscopic examination of scrapings from an eroded area.
Classically, small tufts or haystack-like colonies of the organism may be seen,
giving rise to the name columnaris disease (Fig. 15.3). This presumptive
565 Flavobacterial Diseases
diagnosis is sufficient in routine situations, and cultures are not necessary,
because of the predictability of management for this disease. Should it become
necessary because of treatment failure or in case of in-depth studies, isolates
may usually be obtained with minimal contamination by culturing on Hsu
Shotts medium at 37C. Subsequent identification of isolates may be done by
screening for: (i) adherence to agar; (ii) chromoshift from yellow to pink in the
presence of 3% sodium hydroxide (NaOH); and (iii) production of
chondroitinase (Griffin, 1992). Further grouping of other F. columnare-like
organisms may be done using biochemical criteria (Shotts and Teska, 1989;
Bader, 1995).
Diagnosis of F. psychrophilum in cold- or cool-water species follows much
the same format as described for F. columnare, except that lesions for evaluation
would be better taken from fin tips or obvious greyish material on the edge of the
fins. The caudal fin should also be examined for small, thin mats of flexuous
bacteria. If culture is desired, probably TYG, Ordals, or Cytophaga agar would
be desirable, with incubation temperatures in the 1517C range. Preliminary
identification of isolates would include: (i) chromoshift; and (ii) proteolytic
activity. Subsequent detailed studies should be based on biochemical
characteristics (Krieg and Holt, 1984; Cipriano et al., 1996).
The polymerase chain reaction (PCR) has been developed for the differ-
entiation of F. columnare, F. psychrophilum and Flexibacter maritimus. This
technique, developed around a toothpick transfer from a culture, affords
definitive diagnosis within a single workday (Bader, 1995). Other studies with
Fig. 15.3. Microscopic picture (approximately 450 ) of material from a skin scraping showing
two masses (centre) of bacterial cells or haystacks presumptively diagnostic, with other signs, of
columnaris disease.
566
E.B. Shotts Jr and C.E. Starliper
PCR to amplify Flavobacterium psychrophilum DNA have proved useful in
diagnostic applications (Toyama et al., 1996).
An accurate diagnosis of clinical BGD can be made using clinical signs and
behaviour, along with the presence of filamentous bacterial cells observed in wet
mounts, and by Gram staining (Bullock, 1990). In wet mounts of excised gill
tissue, severe hyperplasia is often present, with cells evident on the periphery
(Fig. 15.4). Gram-stained gill-tissue smears show long, thin (0.5 m 515 m)
Gram-negative cells. There is also an indirect fluorescent antibody test (IFAT)
for diagnostic and research purposes (Huh and Wakabayashi, 1987; Bullock
et al., 1994; Ostland et al., 1994). Enzyme-linked immunoassays have also been
developed to identify a specific F. branchiophilum antigen or host-produced
antibody in response to F. branchiophilum (MacPhee et al., 1985; Lumsden
et al., 1993). Other serodiagnostic techniques have also been employed,
primarily on a need basis as tools in research projects; these include
immunodiffusion, immunoelectrophoresis and macroscopic slide and microtitre
agglutination assays (Huh and Wakabayashi, 1989; Ostland et al., 1989, 1994;
Ko and Heo, 1997). Toyama et al. (1996) applied PCR technology to amplify a
segment of the 16S ribosonal DNA (rDNA) of F. branchiophilum, demonstrating
specificity, as the constructed DNA probes did not react with DNA from other
bacterial species, including the related fish-pathogenic species Flexibacter
maritimus and Flavobacterium columnare. Bacterial culture is not routinely
attempted with BGD as a confirmatory diagnosis, because of the relative ease of
diagnosis by other means and because the bacterium is extremely difficult to
isolate and culture.
Fig. 15.4. A wet mount (approximately 450 ) of excised gill tissue showing severe hyperplasia
and necrosis often seen from fish with bacterial gill disease.
567 Flavobacterial Diseases
CONTROL AND TREATMENT
The epizootiology and control of both columnaris and cold-water disease have
similarities. They involve water, physiological and environmental stress,
vertical transmission (Brown et al., 1997) and the apparent ubiquitousness of
this group of organisms in the environment.
Pacha and Ordal (1970) suggest that temperatures below 1213C are not
conducive to outbreaks of columnaris disease, even with highly virulent
organisms. Similarly, F. psychrophilum is not a potential problem at 3C
(Bernardet and Keronault, 1989). In general, the temperature window of growth
for cold-water disease is from 4C to 30C, while the temperature range for
columnaris disease is about 1037C.
It is generally accepted that adverse physiological stresses enhance
susceptibility, as do adverse environmental parameters. Water in the pH 6.0
range and softer water (10 p.p.m. calcium carbonate (CaCO
3
) or less) are not
conducive to persistence of F. columnare in the water column (Fijan, 1968).
Often, slight changes downward in water temperature, with a reduction of
population density, will stop pending outbreaks of columnaris.
Treatments for both columnaris and cold-water disease have been directed
toward the use of improved water-management practices and of chemicals, such
as Roccal, Cyncal and Hyamine (benzalkonium chloride), or other quaternary
ammonia compounds. Both of these organisms are susceptible to the common
antibiotics, however, none have Food and Drug Administration (FDA) approval
for use in fish. Antibiotics are expensive compared with other alternatives
(Pacha, 1968; Amend, 1970; Bernardet and Keronault, 1989).
Experimental vaccines against both F. columnare and F. psychrophilum are
available and both have provided protective immunity against challenge in
laboratory studies (Schachte and Mora, 1973; Holt, 1987; Moore et al., 1990).
Avoidance of BGD in hatcheries involves reducing those stressors which
either are known or suspected to be important in predisposing fish to disease or
are advantageous for the bacteriums proliferation (Piper et al., 1983; Bullock,
1990; Bullock et al., 1991). Primary suspected factors include increased density,
feeding and compromised sanitary conditions, i.e. accumulation of excrement
and uneaten food. The latter is especially true with small fish, because of more
dust in starter and granular feeds and because it is inherently more difficult to
maintain cleanliness in fry. Directly related to the primary factors are reduced
dissolved oxygen and increased total ammonia.
Successful treatment of BGD depends on early intervention in the
progression of disease. Mortalities can be significantly reduced if treatment is
administered as soon as initial signs (e.g. loss of appetite, diagnosis of
filamentous bacteria on gills) are evident. If treatment is begun at the peak of an
epizootic, many fish will not survive the disease and treatment, as the gill tissue
is already severely damaged. Therapy is achieved with chemicals, and these not
only cleanse gill tissue of F. branchiophilum cells but also kill viable cells in the
water column to prevent further reinfection. Currently, no chemicals are
approved by the FDA to treat BGD. It is important to have a raceway or tanks
free of debris in order to minimize organic material, which can reduce the
568
E.B. Shotts Jr and C.E. Starliper
efficacy of some chemicals. Chloramine-T (sodium p-toluene-sulphon-
chloramide), a disinfectant, is frequently used as a flow-through treatment
exposure for 1 h at 6.58.5 mg l
1
(From, 1980; Bullock et al., 1991). An
additional treatment may be necessary if the initial one was administered well
into the disease progression. Benzalkonium chlorides are also used to treat BGD
(Piper et al., 1983), at 12 mg l
1
active ingredient for 1 h, delivered in a static
bath or as flow through. Commonly used forms of these compounds are Roccal
(10% or 50% active) and Hyamine 1622 or 3500. Roberts (1994) had success
using hydrogen peroxide to treat cutthroat (Oncorhynchus clarki) and rainbow
trout, recommending 50 mg l
1
as a 30-min bath or 100 mg l
1
for a 1 h drip for
fish smaller than 110 kg
1
, and 50 mg l
1
as a 30-min bath for larger fish. Heo et
al. (1990a) successfully treated rainbow trout fingerlings by immersion for 2
min in a 5% NaCl bath. Kudo and Kimura (1983a) also had success treating
rainbow trout fingerlings for BGD using a 5% NaCl bath, but they used a 1 min
treatment. There is no vaccine against F. branchiophilum, and probably none
would be required, because disease involves external infection and effective
treatments are available.
None of these bacterial species are known to produce disease in humans;
they are known to infect only aquatic species and primarily fishes.
PATHOGENESIS AND IMMUNITY
The mechanisms of pathogenesis of both F. columnare and F. psychrophilum are
sketchy at best. This is probably related to the difficulties of primary isolation of
the organisms (Dalsgaard, 1993; Thune et al., 1993).
Attachment to the host is a necessary prerequisite. As early as 1967, Pate
and Ordal noted a capsular outer glycocalyx of mucopolysaccharide associated
with F. columnare and attributed its presence to adherence. DelCorral (1988),
correlating the activity of this layer with the presence of hemagglutinating (HA)
activity, using several species of red blood cells, found it of doubtful importance
in cellular adherence. Work by DelCorral showed a virulence relationship
between HA activity and F. columnare infectivity in channel catfish (Ictalurus
punctatus). Subsequently, the HA activity was blocked by N-acetyl-galacto-
samine and by fibrinogen, and sensitivity to L-galactosidase suggested that the
HA activity was related to an -linked galactoside. In further work, it was noted
that a positive relation existed between in vitro epithelial cell adherence and
isolate virulence (DelCorral, 1988). It would appear that similar findings of
attachment are known for F. psychrophilum (Dalsgaard, 1993).
At present it seems that these organisms have a probable two-component
glycocalyx of an acid mucopolysaccharide (Thune et al., 1993) and galacto-
samine (Johnson and Chilton, 1966), which act to adhere the organism to the fish
surface. It would appear that the galactosamine glucan has more of a role in
attachment than more conventional attachment structures, such as pili or
fimbriae (Holt, 1987).
Increased iron (Fe
2+
) appears to enhance the virulence of F. columnare (Kou
et al., 1981). It has also been shown that these organisms are resistant to the
569 Flavobacterial Diseases
bactericidal activity of normal catfish serum, although the classic antibody
complement cascade is effective in killing the organism (Ourth and Bachinski,
1987). There is also evidence of bactericidin activity and typing suggests at least
13 groups (Becker and Fujihara, 1987). Additionally, it has been established that
these organisms can lyse a number of competitive Gram-negative bacteria on
fish surfaces (Pacha and Porter, 1968).
Proteases have been demonstrated in these two organisms (Garnjobst, 1945)
and there has been a great deal of work directed to this area (Dalsgaard, 1993;
Bertolini et al., 1994; Nomura and OHara, 1994). These have been found in
some cases to be virulence factors and to contribute to direct tissue damage and/
or invasiveness. Although several plasmids have been reported, there is no
evidence of exotoxins being involved in the disease process (Holt, 1987). A
bacteriophage is known to be associated with F. columnare, but there is no
evidence of transduction virulence (Anacker and Ordal, 1955). Lipopoly-
saccharide may play a role as an endotoxin in the disease process in both
organisms. However, the study needs further refinement (Dalsgaard, 1993).
There are a number of reports of specific, high agglutinating antibody titres
in fish (Pacha, 1968; Bullock, 1972; Schachte and Mora, 1973; Becker and
Fujihara, 1987; Holt, 1987). Similarly, numerous attempts at vaccination, with
equivocal to doubtful to encouraging results, have been reported (Fujihara and
Nakatani, 1971; Holt, 1987; Moore et al., 1990; Obach and Baudin-Laurencin,
1991). Despite these findings, there is no commercial vaccine available for
either of these diseases.
A number of studies indicate that pathogenicity of F. branchiophilum is
largely due to extracellular products (ECP), either by fimbriae (pili) for
attachment or other virulence factors in the ECP. Flavobacterium branchio-
philum possesses pili and these are important for attachment to the gill surface
(Ototake and Wakabayashi, 1985; Heo et al., 1990b); the pili are a 23 kDa
protein (Heo et al., 1990a). However, fimbriae were not detected on bacterial
cells from rainbow trout gills from natural outbreaks of BGD, but glycocalyx
was, which can also facilitate adhesion (Speare et al., 1991a). Ototake and
Wakabayashi (1985) isolated ECP from an F. branchiophilum growth culture
and demonstrated HA and bacterioagglutinating activities; they also detected
protease, phosphatase and phosphoamidase activities, but no haemolysin or
endotoxin. They further demonstrated the significance of ECP in vivo by
mechanically removing them by agitation in a whirling blender (washed cells).
Although this did not cause a substantial decrease in the number of viable cells
compared with the unwashed, there was a dramatic reduction in infectivity.
Following a water-borne challenge, no washed cells were detected on gills of
rainbow trout just 24 h after exposure. In contrast, with unwashed cells, there
were 8.4 10
6
colony-forming units per gram of gill tissue at 3 days post-
exposure. Furthermore, juvenile rainbow trout immersed in cell-free ECP
developed clubbing of gill filaments, followed by fusing of lamellae and even
mortality at the higher doses. Kudo and Kimura (1983b) also exposed fingerling
rainbow trout to a bacterial extract and induced fusion of lamellae and/or
filaments, hypertrophy and hyperplasia in these fish. They noted that the induced
lesions were histopathologically similar to those in natural BGD but less
570
E.B. Shotts Jr and C.E. Starliper
advanced than previously noted following water-borne challenge (Kudo and
Kimura, 1983a).
Speare et al. (1991b) studied the ultrastructural relation of the BGD
bacterium to the gill tissue prior to and during natural outbreaks in rainbow
fingerlings. Briefly, bacterial colonization is preceded by degeneration of
microridges of the filament epithelium and slight alterations of the filament tips.
Bacteria then spread to proximal regions of filaments and lamella. Extensive
colonization takes place, in addition to other gill lesions and, ultimately, clinical
signs and death.
Wakabayshi and Iwado (1985) demonstrated an increased lactate/pyruvate
ratio in muscle tissue of rainbow trout near death that were bath-exposed,
although muscle lactate levels were actually lower than in uninfected controls.
They concluded this to be a result of breakdown in gas exchange in the gills,
which impedes the ability to provide oxygen and this leads to increased
anaerobic metabolism (which reduces muscle lactate). On the other hand, Byrne
et al. (1991) concluded that a decrease in muscle lactate of the BGD-infected
fish of Wakabayashi and Iwado (1985) would indicate hypoxia is not the primary
cause of death. According to Byrne et al. (1991), hypoxia is a primary cause of
death by BGD. They suggest that the bacteria cause damage to respiratory cell
membranes, resulting in loss of osmotically active serum Na
+
and Cl

, causing
fluid shifts from extracellular to intracellular compartments. Serum proteins and
packed cell volume increase and the result is haemoconcentrated blood. This
circulatory disturbance is the primary cause of death, exacerbated by hypoxia.
They further propose that the greater severity of BGD in smaller fish, compared
with larger, reflects smaller ionic reserves.
Bullock (1972) was able to induce a BGD outbreak in fish only after
severely compromising them by maintaining them in crowded conditions with
low dissolved oxygen and increased ammonia. He was, however, not successful
in transmitting a BGD infection in (stressed or control) trout, using various
myxobacterial cultures or by horizontal transmission, using live or dead BGD-
infected fish. In contrast, other workers have been successful in transmitting
BGD by using bacterial cultures in a water-borne challenge, as the bacteria
readily attached to gills (Kimura et al., 1978; Wakabayashi et al., 1980; Kudo
and Kimura, 1983c; Ferguson et al., 1991), via horizontal transmission from
diseased to healthy fish in clean water and across different fish species and ages
(Ferguson et al., 1991). MacPhee et al. (1995) demonstrated that feeding of fish,
following bath exposure to F. branchiophilum, produced clinical disease and
death by BGD, whereas unfed fish developed only mild clinical signs of disease.
They surmised that it was due to physiological changes to the fish, which
facilitated bacterial colonization of the gills. Wakabayashi et al. (1980) rarely
had mortality in juvenile rainbow trout; however, Ferguson et al. (1991)
produced clinical disease and death within 24 h of exposure, and in good water
quality.
There is no vaccine to protect against BGD. The use of the immune response
to control BGD is confronted by several factors that may preclude its feasibility.
First, epizootics are more severe in fry and fingerling fish, which have immature
immune systems, and fish vaccinated at an age prior to when epizootics are
571 Flavobacterial Diseases
anticipated may be too young for them to become immunocompetent. A second
factor is that BGD epizootics are relatively rapid, so high mortality occurs before
a secondary response can be activated to confer protection. Also, BGD is
considered primarily an external disease and chemical therapy is used for
treatment; the effects of chemicals on gill-associated or mucosal antibody-
producing cells are not known.
Lumsden et al. (1993, 1995) demonstrated gill-associated antibody response
in rainbow and brook trout exposed to BGD. Lumsden et al. (1993) exposed 2-
year-old brook trout to BGD, initially by horizontal challenge with infected
rainbow trout. Survivors were bath-exposed to 1.0 10
6
F. branchiophilum 200
days later. A slight response in both serum and gill antibody to F.
branchiophilum was detected following both the primary and secondary
challenges, but only the increase in gill antibody after the second challenge was
significant, with a maximum titre of 1 : 16, 57 days after the second challenge.
Lumsden et al. (1995) also used acetone-killed F. branchiophilum cells as a
vaccine. Rainbow trout (about 50 g) were exposed (6 weeks apart) by bath and
intraperitoneal injection; they demonstrated gill and serum antibody response by
both routes. Gill antibody response was highest and for a longer duration
following the second exposure. The group immersed in the highest
concentration of antigen had the greatest protection, with 11.7% mortality
compared with 45.3% for the group not exposed previously to antigen. However,
protection was not associated with a reduction in the number of bacteria on the
gill surface. Heo et al. (1990a) challenged rainbow fingerlings with 4.0 10
7
colony-forming units (cfu) ml
1
live F. branchiophilum by immersion, followed
by a 2-min dip in 5% NaCl. This was done three times at 6 day intervals. No
antibody was detected, nor were they able to demonstrate serum antibody in
rainbow trout that had survived several natural infections.
TOPICS FOR FUTURE STUDY
Some of the key areas of research which need further resolution are as follows.
1. Delineation of the mechanism(s) of disease in infected fishes.
2. Relationships between high agglutinating titre and protection in the face of
vaccination.
3. Development of either a genus vaccine or a subunit vaccine which is targeted
towards bacterial adherence to the fish.
4. Studies to explain the lack of detectable inflammatory response during
infection.
5. Investigations directed toward disease management via water-quality
management.
6. Two of the more important topics are the elucidation of the role of
environmental factors, or, more importantly, the combination of them that results
in optimum conditions for the pathogens.
7. Improved techniques for bacterial culture of the causative bacteria.
572
E.B. Shotts Jr and C.E. Starliper
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