Fish 13

479
CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
13
Edwardsiella Septicaemias
J.A. Plumb
Department of Fisheries and Allied Aquacultures, International Center for
Aquaculture and Aquatic Environments, Auburn University, Alabama 36849,
USA.
INTRODUCTION
The genus Edwardsiella includes two species of bacteria that cause major
diseases in fish: Edwardsiella tarda (Ewing et al., 1965) infects fish and
other animals and Edwardsiella ictaluri (Hawke, 1979) infects fish only. A
third species, Edwardsiella hoshinae (Grimont et al., 1980), infects birds
and reptiles. Edwardsiella tarda produces the disease commonly known as
fish gangrene, emphysematous putrefactive disease of catfish or red disease
of eels and hereafter known in this text as Edwardsiella septicaemia (ES),
and E. ictaluri causes enteric septicaemia of catfish (ESC). Because E.
tarda and E. ictaluri produce distinctively different diseases, they are
discussed separately.
EDWARDSIELLA SEPTICAEMIA (EDWARDSIELLA
TARDA)
Edwardsiella septicaemia (ES) is a serious systemic bacterial disease of
cultured Japanese eels (Anguilla japonica) in Japan and Taiwan (Egusa,
1976), Japanese flounder (Paralichthys olivaceus) (Nakatsugawa, 1983) and
occasionally other cultured fish in Asia and elsewhere. In the USA, E. tarda
causes a septicaemia infection in cultured channel catfish (Ictalurus
punctatus) and occasionally in other species of fish. Edwardsiella tarda
sometimes produces a subclinical infection in fish intended for human
consumption, where it may create problems during the cleaning process
(Meyer and Bullock, 1973). When channel catfish with undetected muscle
lesions are skinned, the equipment becomes contaminated, which requires
processing interruption, cleaning of equipment and disposing of infected
fish.
480
J.A. Plumb
Host range and geographical distribution
Edwardsiella tarda infects a wide variety of fish (Table 13.1). The most
prominent hosts are eels (Anguillidae) and catfish (Ictaluridae), but many other
fish groups are also susceptible. The most notable are flounders (Pleuronectidae)
and tilapias (Cichlidae). Most diseases associated with E. tarda occur in cultured
fish in either fresh or marine waters; at least 21 species of fish are known to have
been infected and probably all species of fish are susceptible under certain
conditions. Edwardsiella tarda generally causes disease only in warm-water
fish, but on at least two occasions produced infections of salmonids. The first
occurred in migrating chinook salmon (Oncorhynchus tshawytscha) in the
Rogue River, Oregon (Amandi et al., 1982), and the second was in a small
number of Atlantic salmon (Salmo salar) on a spawning run in Nova Scotia,
Canada (Martin, 1984).
Edwardsiella tarda infections are not limited to fish. This organism may
infect reptiles (snakes, turtles and alligators) (Wallace et al., 1966; Nagel et al.,
1982; Sugita and Deguchi, 1983), birds (White et al., 1969, 1973), cattle (Ewing
et al., 1965), swine (Arambulo et al., 1967), marine mammals (Coles et al.,
1978) and other warm-blooded animals. In many instances, E. tarda is part of the
normal intestinal microflora of aquatic animals (Wyatt et al., 1979; Van Damme
and Vandepitte, 1980; Kanai et al., 1988). Bauwens et al. (1983) found E. tarda
Table 13.1. Species of fish from which Edwardsiella tarda has been isolated.
Species of fish Reference
Atlantic salmon, Salmo salar Martin, 1984
Australian eel, Anguilla reinhardti Eaves et al., 1990
Betta, Betta splendens Vladick et al., 1983
Channel catfish, Ictalurus punctatus Meyer and Bullock,
1973
Chinook salmon, Oncorhynchus tshawytscha Amandi et al., 1982
Colored carp, Cyprinus carpio Sae-Oui et al., 1984
Crimson sea bream, Evynnis japonica Kusuda et al., 1977
European eel, Anguilla anguilla Taksdal et al., 1989
J apanese eel, Anguilla japonica Hoshinae, 1962
J apanese flounder, Paralichthys olivaceus Nakatsugawa, 1983
Largemouth bass, Micropterus salmoides White et al., 1973
Mullet, Mugil cephalus Kusuda et al., 1976
Rosy barb, Puntius conchonius Humphrey et al.,
1986
Red sea bream, Pagrus major Yasunaga et al.,
1982
Sand goby, Oxyeleotris marmoratus Supamataya, 1988
Sea bass, Dicentrarchus labrax Blanch et al., 1990
Striped bass, Morone saxatilis Herman and Bullock,
1986
Tilapia, Oreochromis niloticus Aoki and Kitao, 1981
Tilapia, Tilapia mossambica Kaige et al., 1986
Yellowtail, Seriola gaingu Yasunaga et al.,
1982
481 Edwardsiella Septicaemias
in the intestine of a variety of zoo animals, especially fish-eaters and water-
loving species. Edwardsiella tarda has also been associated with several
different manifestations in humans (King and Adler, 1964; Jordan and Hadley,
1969; Bockemuhl et al., 1971; Van Damme and Vandepitte, 1980).
Edwardsiella tarda is a ubiquitous organism, having been isolated from
animals or the environment of most continents. The list of countries in which
E. tarda has been found is lengthy but includes predominantly the USA, Japan,
Taiwan, Thailand, Israel and many Third World countries (Table 13.2).
The disease
Edwardsiella septicaemia, caused by E. tarda, is a mild to severe condition, but
clinical signs of infection differ slightly between species of fish. In channel
catfish, E. tarda initially produces small, 15 mm, cutaneous lesions, which are
located dorsolaterally in the muscle (Meyer and Bullock, 1973). These small
lesions progress to larger necrotic abscesses within the flank muscle or caudal
Table 13.2. Countries in which Edwardsiella tarda and E. ictaluri have been
found either in infected fish, in other animals or from environmental sources.
E. tarda E. ictaluri
Australia (How et al., 1983) Australia (How et al., 1983)
Bangladesh (Chowdhury and Taiwan (Chung and Kou, 1983)
Wakabayashi, 1990) Thailand (Kasornchandra et al., 1987)
Belgium (Bauwens et al., 1983) United States (Hawke, 1979)
Canada (Martin, 1984)
China (Zheng, 1987)
Czecholslovakia (Vladick et al., 1983)
Germany (Bockemuhl et al., 1983)
India (Koshi and Lalitha, 1976)
Israel (Sechter et al., 1983)
Italy (Maserati et al., 1985)
J apan (Hoshinae, 1962)
Korea (Kokuska, 1973)
Madagascar (Fourquet et al., 1975)
Mali (Vandepitte et al., 1980)
Nigeria (Gugnani et al., 1986)
Norway (Bergen et al., 1988)
Panama (Kourany et al., 1977)
Philippines (Tacal and Menez, 1968)
Singapore (Tan and Lim, 1977)
Spain (Marinez, 1987)
Taiwan (Liu and Tsai, 1980)
Thailand (Boonyaratpalin, 1983)
USA (Meyer and Bullock, 1973)
Former USSR (Kalina, 1980)
Zare (Van Damme and Vandepitte,
1980)
482
J.A. Plumb
peduncle, where they form obvious convex swollen areas and the skin loses its
pigmentation (Fig. 13.1). Lesions in the muscle, which contain large amounts of
necrotized tissue, emit a putrid odour when incised. As infection progresses,
affected fish lose mobility of the caudal portion of the body and a generalized
Fig. 13.1. (A) Edwardsiella tarda infection in channel catfish. The muscle tissue is necrotic, has
lost its firmness and has open lesions (arrow). (Photo by Ahmad Darwish.) (B) A juvenile
largemouth bass with necrotic E. tarda lesion on the caudal peduncle (arrow).
internal hyperaemia, similar to other bacterial septicaemias, is evident. The
kidney, in particular, is enlarged and the liver is mottled; both organs are soft.
Initially, it was believed that E. tarda caused disease in only larger channel
catfish (i.e. over 0.4 kg); however, there seems to be no distinct size or age
differential in susceptibility of any species of fish. Edwardsiella septicaemia of
catfish in the USA occurs mostly during the warm summer months.
Eels with acute infection of E. tarda develop severe hyperaemia, with
bloody congestion of fins (Fig. 13.2A), ecchymotic or petechial haemorrhage on
various body surfaces, gas-filled pockets in the skin and necrosis of the muscle
(Fig. 13.2B). The anal region is swollen and hyperaemic. Internally, there is a
general hyperaemia of the peritoneum; the liver is mottled, oedematous and
abscessed. Although Egusa (1976) reported that E. tarda infections of eels in
Japan were more prevalent during the summer, Liu and Tsai (1980) found that
infections of eels in Taiwan were most common when water temperatures were
1018C, during JanuaryApril.
A variety of clinical signs occur in other species of fish. For example,
E. tarda causes exophthalmia and cataracts in tilapia, as well as abscesses in
internal organs (Kubota et al., 1981). Japanese flounders, naturally infected with
E. tarda, develop ulcerative lesions and loss of skin, which expose underlying
muscle, haemorrhage in fins, rectal protrusion and swelling of the spleen.
Infected cage-cultured largemouth bass (Micropterus salmoides) developed
necrotic lesions on the caudal peduncle (see Fig. 13.1).
The source of E. tarda is presumably the intestinal contents of carrier animals,
but it may be a common inhabitant of the aquatic environment. In the USA. E.
tarda was isolated from 75% of water samples, 64% of pond-mud samples and
100% of frogs, turtles and crayfish from catfish ponds (Wyatt et al., 1979).
Edwardsiella tarda was also isolated from as high as 88% of fillets of dressed
cultured catfish and from 30% of imported fish fillets. In an ecological study of E.
tarda in a salt-water flounder farm, Rashid et al. (1994a) isolated the bacterium
from 86% of water samples from one pond and 22% of water samples from a
second pond. The organism was present in 44% of sediment samples and 14% of
fish from the first pond, and 0% of sediment samples and 2% of fish from the
second pond. Ironically, clinical ES did not occur in the fish in either pond, but the
data indicate that the incidence of E. tarda in fish is associated with its presence in
the environment. On the other hand, the high incidence in the environment may
have been a function of the organisms presence in the fish. Hidaka et al. (1983)
showed that E. tarda cell counts in eel-pond water at 2226C were four times
higher when clinical disease was present than when there was no disease.
In spite of the apparent extensive presence of E. tarda in fertile channel
catfish ponds, infections in these fish are not common. When mortality does
occur, it seldom exceeds 5%; however, if the fish are moved into confined
holding tanks, the rate of infection may quickly accelerate to 50%, with
concomitant deaths (Meyer and Bullock, 1973). Mortality data of E. tarda-
infected cultured eels in Asia indicate potentially high losses. Ishihara and
Kusuda (1981) induced up to 60% mortality in 100 g eels experimentally
infected by immersion. In a naturally infected Japanese eel population, the
mortality was 80% (Kodoma et al., 1987).
484
J.A. Plumb
Although environmental stressors are not essential precursors to E. tarda
infections in fish, high temperature, poor water quality and high organic fertility
probably contribute to the onset and severity of the disease. Juvenile channel
catfish that were experimentally infected with Aeromonas hydrophila and then
exposed to environmental stressors (low dissolved oxygen, high ammonia and
high carbon dioxide) developed E. tarda infections in 2550% of the fish
(Walters and Plumb, 1980). This compared to 4.512.5% E. tarda infections in
Fig. 13.2. (A) Edwardsiella tarda infection in J apanese eel with haemorrhagic and congested
anal fin (arrow). (B) Cross-sections of body of J apanese eel with inflamed and necrotic muscle
lesions (arrows).
the non-A. hydrophila-injected or otherwise non-stressed fish. Indications were
that environmentally induced stress and other bacterial infections could
predispose channel catfish to naturally present E. tarda. On several occasions,
the author has seen intensively cultured (recirculating systems) Nile tilapia
(Oreochromis niloticus) that were environmentally stressed or had a moderate to
heavy infection of Trichodina (protozoan parasite) also being infected with
E. tarda and Streptococcus spp. (J.A. Plumb, unpublished). While stressed,
neither bacterial infection responded to chemotherapy, but, as soon as the
stressor was relieved and parasites were eliminated, the E. tarda infection
disappeared. Many epizootics of E. tarda occur in fish predisposed to fluctuating
water temperatures (Liu and Tsai, 1980; Amandi et al., 1982) or fish in highly
enriched waters (Meyer and Bullock, 1973).
Edwardsiella tarda is a health threat to other animals, including humans
(Bockemuhl et al., 1971; Boehrer et al., 1977; Kourany et al., 1977; Clarridge et
al., 1980). In fact, some of the first isolates of E. tarda were from human faeces
(Ewing et al., 1965). In humans, the bacterium usually causes diarrhoea and
gastroenteritis (King and Adler, 1964; Jordan and Hadley, 1969; Bockemuhl et
al., 1971; Van Damme and Vandepitte, 1980), while extraintestinal infections
may produce a typhoid-like illness, peritonitis with sepsis and cellulitis (Fields
et al., 1967). Occasionally, E. tarda-induced abscesses have been seen in liver
(Zighelboim et al., 1992). Several other clinical conditions in humans have been
associated with E. tarda, including meningitis (Sonnenwirth and Kallus, 1968;
Sachs et al., 1974). Funada et al. (1988) found E. tarda septicaemia
complicating an acute leukaemia patient in Japan while Gilman et al. (1971)
thought that the organism was involved with jungle diarrhoea and possibly
associated with Entamoeba histolytica (protozoan) infection in Thailand. Van
Damme and Vandepitte (1980) reported that sporadic cases of tropical diarrhoea
in humans with E. tarda were traced to consumption of freshwater fish in Zare.
Serious and/or life-threatening infections of E. tarda in the muscle, which
resulted from wounds received while fishing or puncture wounds caused by
catfish spines, have been described in humans (Clarridge et al., 1980; Hargraves
and Lucey, 1990). Although Wyatt et al. (1979) could not correlate, or
substantiate, E. tarda in aquatic animals to human infections, there is sufficient
evidence to indicate that the organism can be a public-health problem, as well as
a threat to other animals.
The microorganism
Isolates used to establish the genus Edwardsiella, described by Sakazaki and
Murata (1962), were originally from human faeces in the USA and snakes in
Japan, simultaneously in 1959 (Ewing et al., 1965; Sakazaki, 1967). Species of
Edwardsiella, a member of the Enterobacteriaceae, are small, straight rods of
about 1 m in diameter and 23 m in length (Farmer and McWhorter, 1984).
They are Gram-negative, usually motile, with peritrichous flagella, and are
facultatively anaerobic.
Hoshinae (1962) isolated an enteric bacterium from eels in Japan, which he
486
J.A. Plumb
named Paracolabacterium anguillimortiferum; however, the biochemical and
biophysical characteristics of this organism are identical to those of E. tarda
(ATCC 15947), which holds taxonomic precedence (Table 13.3) (Ewing et al.,
1965). Since no cultures of P. anguillimortiferum are available (Egusa, 1976), and
to prevent confusion, tarda is the specific epithet (Farmer and McWhorter, 1984).
Edwardsiella tarda is catalase-positive, cytochrome oxidase-negative and
Table 13.3. Biochemical and biophysical characteristics of Edwardsiella
tarda, E. ictaluri and E. hoshinae. Key characteristics for presumptive
identification are noted by *. (Grimont et al., 1980; Hawke et al., 1981; Farmer
and McWhorter, 1984; Waltman et al., 1986a,b; Plumb and Vinitnantharat,
1989).**
Characteristic E. tarda E. ictaluri E. hoshinae
Motility at:
25C* + + +
37C* + +
Growth at 40C + +
Tolerance of NaCl
1.5%* + + +
4.0%* +
Cytochrome oxidase*
Indole* +
Methyl red + +
Citrate (Christensens) + +
H
2
S production:
Triple sugar iron* +
Peptone iron sugar + +
Lysine decarboxylase + + +
Ornithine decarboxylase + + +
Malonate utilization +
Gas from glucose + +
Acid production from:
D-mannose, maltose + + +
D-mannitol, sucrose +
Trehalose
L-Arabinose
J ordans tartrate
Nitrite from nitrate + + +
Tetrathionate reductase + ? +
Edwardsiella isolation Black centres Green translucent Unknown
media
Mol % G +C of DNA 5558 5657 53
+, Positive for 90100% of isolates; , negative for 90100% of isolates; ,
mixed reactions; G +C, guanine plus cytosine; DNA, deoxyribonucleic acid.
**All isolates of E. tarda, E. ictaluri and E. hoshinae tested are negative for
VogesProskauer, Simmons citrate, urea, phenlyalanine deaminase, arginine
dihydrolase, gelatin hydrolysis, growth in KCN; acid production from glycerol,
salacin, adonitol, D-arabilol, cellobiose, dulcitol, erythritol, lactose, inositol,
melibiose, -methyl-D-glucoside, raffinose, L-rhamnose, D-xylose and mucate;
aesculin hydrolysis, acetate utilization, deoxyribonuclease, lipase, -
galactosidase (o-nitrophenol-b-D-galactopyranoside, ONPG), pectate
hydrolysis, pigment production and tyrosine clearing.
glucose-fermentative, reduces nitrate to nitrite, is lactose-negative and indole-
positive and produces an alkaline slant, acid butt and hydrogen sulphide (H
2
S)
on triple-sugar iron (TSI) agar (Shotts and Teska, 1989). Waltman et al. (1986a)
compared 116 isolates of E. tarda and found little variation in their biochemical
and biophysical characteristics, although isolates from Taiwan differed slightly
from those in the USA.
Farmer and McWhorter (1984) listed a wild-type E. tarda and a second
biogroup 1 by biochemical and biophysical characteristics. Strains of E. tarda
which are negative for D-mannitol, sucrose and L-arabinose are more common
than biogroup 1, and therefore are designated E. tarda wild type. Edwardsiella
tarda may also be divided into serological groups, based on the O-agglutination
test; Park et al. (1983) used this test to identify 61% (270) of 445 E. tarda
isolates from kidneys of infected eels, rectum of eels and other fish and water
and sediments from eel ponds, which were classified into four serotypes (A, B, C
and D). Of the isolates from eel kidneys, 72% were serotype A, indicating that it
may be the predominant type causing fish disease. Rashid et al. (1994b) also
determined that 28 strains of E. tarda from diseased flounder were identical to
serotype A.
Edwardsiella tarda survives in water from 14 to 45C, with highest survival
at 3037C, at pH 4.010.0 (optimum 7.58.0) and at 04% sodium chloride
(NaCl) (optimum 0.51.0%) (Ishihara and Kusuda, 1982). The organism can be
isolated for over 76 days in pond water (20C), indicating that it can survive for
long periods. As indicated elsewhere, E. tarda occurs in both fresh and salt
water (Fourquet et al., 1975; Ishihara and Kusuda, 1982; Chowdhury and
Wakabayashi, 1990), mud (White et al., 1973; Wyatt et al., 1979) and fouling
material in nets (Kanai et al., 1988).
Diagnostic methods
Clinical signs of E. tarda infections vary between species of fish; therefore,
these signs are generally of little use, except to indicate the likely presence of a
bacterial infection. Also, Nishibuchi et al. (1980) pointed out that, for diagnostic
purposes, it is necessary to isolate bacteria from diseased eels, as well as other
species of fish, because clinical signs of E. tarda, A. hydrophila, Vibrio
anguillarum and Pseudomonas anguilliseptica infections generally cannot
otherwise be differentiated. Some other definitive method of detection, such as
serological (fluorescent antibody technique (FAT), enzyme-linked immuno-
sorbent assay (ELISA)), may be satisfactory for diagnosis. Edwardsiella tarda
can be isolated on brainheart infusion (BHI) agar or trypton soya agar (TSA)
with inocula from internal organs or lesions in the muscle of clinically infected
fish and identified using conventional bacteriological and/or serological
methods (Meyer and Bullock, 1973; Amandi et al., 1982). When incubated at
2630C, small, round, convex transparent colonies of approximately 0.5 mm in
diameter are visible in 2448 h. Amandi et al. (1982) improved isolation
incidence (from 2% to 19%) from brain of chinook salmon by first inoculating
thioglycolate media and, after incubation, transferring an inoculum to BHI agar.
488
J.A. Plumb
Edwardsiella tarda forms small green colonies with black centres on
Edwardsiella isolation media (EIM) (Shotts and Waltman, 1990).
Important characteristics of E. tarda that are of presumptive diagnostic
value are motility, indole production, reaction on TSI agar, citrate and methyl red
media, and salt and temperature tolerance (Table 13.3). Using the Minitek
numerical identification system, Taylor et al. (1995) correctly identified E. tarda
100% of the time compared with 83% positive identification with the API 20E
system. However, both of these systems are applicable for E. tarda identification
in most instances. Positive identification can be made using specific serum
agglutination or FAT. There is no evidence of serological cross-reactivity
between E. tarda and E. ictaluri (Rogers, 1981; Klesius et al., 1991).
Transmission of the disease
Edwardsiella tarda is transmitted through the water from an infected source
(carrier animal faeces, water, mud) to susceptible fish. Fish can be
experimentally infected with E. tarda by injection (intraperitoneal (i.p.) and
intramuscular (i.m.)), stomach gavage or immersion in a bath containing the
pathogen; however, infection is not guaranteed simply by introducing the fish to
the pathogen. Huang and Liu (1986) killed 100% of i.p. injected eels when
E. tarda was mixed with A. hydrophila, but water-borne exposure to the same
mixture failed to induce mortality unless sublethal concentrations of nitrogenous
compounds were present. A disease identical to natural infections in tilapia was
also reproduced by i.m. injection by Miyashita (1984). Most probably, fish
become naturally infected via injuries to the epithelium or through the intestine.
Miyazaki et al. (1992) injured the intestine with hydrogen peroxide prior to
introducing E. tarda into the lumen via a silicon tube. This procedure resulted in
death 523 days later and the infected fish developed pathological lesions in
kidneys and livers that were nearly identical to those observed in natural
infections.
Transmission of E. tarda in catfish, eels and most other fish species appears
to be enhanced at water temperatures from 20 to 30C. Japanese flounder were
most susceptible at 2025C by i.m. injection, in which a median lethal dose
(LD
50
) of 7.1 10
1
colony-forming units (cfu) was established (Mekuchi et al.,
1995a). This compared with an LD
50
of 1.7 10
2
cfu for i.p. injection and
3.6 10
6
cfu ml
1
and 1.3 10
6
cfu fish
1
for immersion and oral exposure,
respectively, at the same temperatures.
Treatment and protection
The first step in controlling most infectious diseases in the aquatic environment
is through health management, which includes attempts to avoid contact
between pathogen and host, management of the environment to favour increased
host resistance, reduction of stressful conditions and removal of sick and dead
fish as soon as possible, and E. tarda infections are no exception. Also,
utilization of prophylactic treatments and implementation of sanitary
aquacultural practices, judicious use of legal drugs and chemicals when
infections occur, application of vaccines, if available, and use of genetically
improved stocks are aids in health management (Plumb, 1994). Because E. tarda
is a non-obligate pathogen, it is not possible to completely eliminate or totally
prevent the organisms presence in most instances. For example, preventing
infected animals (e.g. undesirable fish, turtles, snakes, etc.) from coming into
contact with the aquaculture species is impractical, except under certain
circumstances, such as closed recirculating systems. Maintaining a suitable
oxygen concentration and low carbon dioxide and ammonia, reducing water
enrichment and prevention of wide temperature fluctuations are basic to the
health-management approach. Although these goals are difficult to attain in
intensive or commercial aquaculture, they should be pursued.
Therapy of ES is by oral application of drugs in feeds of cultured fish. Two
drugs, oxytetracycline and a potentiated sulphonamide (sulphadimethoxine
ormetoprim in a 5 : 1 ratio), are approved by the US Food and Drug
Administration (FDA) for some bacterial diseases of some fish. Bearing in mind
that neither of these drugs is approved by the FDA for E. tarda infections in any
species of fish, oxytetracycline is fed at 50 mg of drug kg
1
of fish day
1
for
1214 days, followed by a 21-day withdrawal period before fish are processed
for human consumption. Sulphadimethoxineormetoprim is also used as a feed
additive to deliver 50100 mg of drug kg
1
day
1
for 5 days, requiring only 3
days withdrawal for fish that are skinned prior to being sold (catfish), but up to
42 days is required if fish are not skinned during processing. Because of a
possible lack of palatability of feed with the higher dose of sulphadimethoxine
ormetoprim, it was suggested by Johnson and Smith (1994) that the amount of
drug be reduced and feed level increased. Both of these drugs are generally
effective against E. tarda infections as long as treatment is initiated prior to the
disease advancing to a point when fish stop feeding. Some countries have a
wider range of antibiotics available with which to treat bacterial infections.
Resistance of bacteria to antibiotics is a constant problem in cultured fishes.
Fifty-four strains of E. tarda from cultured eels in Taiwan were tested in vitro for
their susceptibility to a large number of antibiotics (Chen et al., 1984). They
found a high rate of susceptibility of E. tarda to gentamicin sulphate and
nalidixic acid and a high rate of resistance to erythromycin, and all sulpha drugs.
Waltman and Shotts (1986a) tested 116 isolates of E. tarda from the USA and
Taiwan for their susceptibility to 37 antimicrobials and found a higher number of
resistant isolates from Taiwan. Antibiotic sensitivity testing of only five E. tarda
isolates from catfish and tilapia in the USA by Plumb et al. (1995) showed that
all were sensitive to sulphadimethoxineormetoprim and four were sensitive to
oxytetracycline.
Antibiotic resistance of some E. tarda isolates from Taiwan was plasmid-
mediated (Aoki et al., 1977; Aoki and Kitao, 1981). Aoki et al. (1986) detected
13 transferable R plasmids in E. tarda, two of which encoded for resistance to
chloramphenicol, tetracycline and sulphonamide. Subsequent studies showed
that 20% of 152 antibiotic-resistant strains of E. tarda possessed transferable
antibiotic-resistant R plasmids; therefore, the function of some of these
490
J.A. Plumb
structures in the bacterium are unknown (Aoki et al., 1987). When R plasmid
resistance to tetracycline and sulphonamides occurs, Aoki et al. (1989)
recommend that a potentiated sulphonamide at 25 mg kg
1
day
1
, oxalinic acid at
12.5 mg kg
1
day
1
or miloxacin at 6.2 mg kg
1
day
1
be fed. Liu and Wang (1986)
reported that nearly 92% of E. tarda isolates from the water of eel-culture ponds
showed some degree of antibiotic resistance. Edwardsiella tarda has a long
history of causing disease in South-East Asian aquaculture, where the pathogen
has had greater exposure to a variety of antibiotics; therefore, higher resistance
may be encountered (Aoki et al., 1989).
Immunization has become a popular theme for controlling and preventing
numerous diseases of fish (Ellis, 1988; Anderson, 1992; Mekuchi et al. 1995b;
Press and Lillehaug, 1995). Salati (1988) reviewed the techniques and
procedures for vaccinating eels against E. tarda and stated that the two basic
types of vaccines are whole-cell bacterins and bacterial extracts. Immunization
of eels against E. tarda was first investigated by Song and Kou (1981). Song et
al. (1982) reported that a single immersion of 6 g elvers was effective, but that
two or three exposures to the vaccine were more effective in eliciting immunity
and protection, which lasted 10 weeks. Salati et al. (1983) immunized eels with
lipopolysaccharide (LPS), culture filtrates and formalin-killed whole cells
(FKC). They concluded that LPS was the immunogenic component, but it was
later shown that polysaccharide without the lipid component was more antigenic
than other preparations (Salati and Kusuda, 1985). In a more recent study,
immunization of eels by injection of FKC and LPS from E. tarda showed only
slight to moderate protection from challenge by injection with virulent E. tarda
21 days after vaccination (Gutierrez and Miyazaki, 1994).
Japanese flounder were vaccinated with formalin-killed E. tarda and
extracellular and intracellular components by i.m. injection, immersion and
orally (Rashid et al., 1994b). While the extracellular and intracellular
components were lethal to the flounder, the serum agglutinating antibody titres
against FKC rose in all other immunized groups, except those vaccinated by
immersion. Clear protection was not demonstrated with the FKC, but death of
challenged fish was delayed in the fish immunized by immersion and injection.
It was concluded by Salati (1988) that bacterins which are simple and cheap to
produce on a large scale do provide protection to anguillettes and while the
protection is not complete, it may prove useful enough to farms with a severe
Edwardsiella problem.
Pathogenesis
Most pathogenesis and pathology studies of E. tarda have been in Japanese eels.
This is in contrast to a paucity of histopathological information in channel
catfish; however, there are some reports describing E. tarda infections in other
fish. Egusa (1976) described E. tarda infections of eels that spread from lesions
in visceral organs into the musculature and then to the dermis. Miyazaki and
Egusa (1976a, b) described histopathology of the suppurative interstitial
nephritis forms of edwardsiellosis in adult eels, in which the haematopoietic
tissue of the kidney has masses of neutrophils, which contain phagocytized
bacteria. Small abscesses, which develop from primary foci of neutrophils in
haemopoietic tissue and nephrons, are present in the early stages of infection.
Enlarged abscesses become liquefied, from which bacteria spread to surround-
ing tissues, where blood-vessels form emboli, which produce additional
abscesses. Peripheral abscesses progress into ulcers in the epidermis. General-
ized infections show ulcerative and necrotic (serousexudative and liquefactive)
lesions in the spleen, liver, epicardium, stomach, gill and musculature (Fig.
13.3).
In the hepatitis form, microabscesses, which contain bacteria-laden macro-
phages, develop in the liver (Fig. 13.3). As the disease progresses, abscesses
enlarge and hepatic cells become necrotic. This is followed by extensive
liquefaction of abscesses and bacterial multiplication in blood-vessels in various
parts of the liver. Hepatic cells have fatty degeneration, while ulcers develop in
the body musculature adjacent to the diseased liver (Miyazaki and Egusa,
1976b).
Histopathology of E. tarda in Japanese flounders, red sea bream (Pagrus
major), Japanese eels and tilapia (Tilapia sp.; Oreochromis sp.) were compared
(Miyazaki and Kaige, 1985). The major difference from infections in eels is the
predominance of granulomatous inflammation in the Japanese flounder and red
sea bream (Fig. 13.3). In infected tilapia, the abscesses in internal organs also
progressed to granulomas (Kubota et al., 1981). Histopathology of striped bass
includes epithelial hyperplasia, necrosis associated with the lateral-line canals
and abscess formation in the anterior kidney and other internal organs (Herman
and Bullock, 1986).
At least some E. tarda isolates produce toxic extracellular products (ECP).
Ullah and Arai (1983) isolated an exotoxin from E. tarda culture media and
found no evidence of endotoxins, therefore postulating that the exotoxin was
responsible for pathogenicity. However, factors that regulate pathogenicity of
E. tarda are unclear. Suprapto et al. (1995) detected a heat-labile ECP in a
virulent strain of E. tarda belonging to serogroup A, which was lethal to
Japanese eel and Japanese flounder. The optimum incubation temperature for
ECP production was 2530C, which coincides with most reports of optimum
temperature for fish susceptibility. An intracellular component (ICC) was also
detected in bacteria, associated with cell lysis. Japanese flounder appeared to be
more susceptible to E. tarda (about 15 times higher) than the Japanese eel.
Results of these studies suggest that the toxin produced by E. tarda plays an
important role in its virulence.
The ability of E. tarda to infect warm blooded animals, humans in particular,
was shown by Janda et al. (1991), who demonstrated its ability to invade HEp-2
cell monolayers, produce cell-associated haemolysin and siderophores and
express mannose-resistant hemagglutination against guinea-pig erythrocytes.
Some strains of E. tarda were virulent to mice. Janda and Abbott (1993) showed
that strains of E. tarda, a known pathogen of warm-blooded animals, produced
3040% higher levels of cell-associated haemolytic activity (haemolysins)
than strains of E. ictaluri, known only from fish. The increased haemolytic
activity could contribute to the pathogenicity of E. tarda to humans. When
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J.A. Plumb
Fig. 13.3. Histopathology of Edwardsiella tarda infection in several different fish. (A) Abscess
(arrow) in kidney of J apanese eel (haematoxylin and eosin (H & E), 31). (B) Early E. tarda
infection in liver of tilapia. Affected hepatic cells are necrotized (N) which is followed by
macrophage infiltration (arrow) (Giemsa, 80). (C) Granuloma (arrow) formation in liver of E.
tarda-infected red sea bream (Giemsa, 200). (D) Neutrophils gorged with E. tarda (arrow) from
abscess in the kidney of eel (Giemsa, 1000). (All photographs by T. Miyazaki.)
grown under iron-restricted conditions in the presence of ethylenediamine
di(o-hydroxyphenylacetic acid), haemoglobin, haematin and haemin stimulated
bacterial growth in both liquid and agar bioassays. Haemolysin activity under
these conditions was increased three- to > 40-fold. All of these studies indicate
that, in addition to its invasive capabilities, E. tarda produces a haemolysin,
which is partially regulated by availability of iron and may also play a role in
human disease.
ENTERIC SEPTICAEMIA OF CATFISH
(EDWARDSIELLA ICTALURI)
Enteric septicaemia of catfish, caused by E. ictaluri, was first reported by Hawke
(1979), but it has become the most important infectious disease of the catfish
industry in the USA especially in the south-east (A.J. Mitchell, Fish Farming
Research Laboratory, Stuttgart, Arkansas, USA, personal communication).
Estimates of the cost of the disease to the catfish industry have been in the
US$l0s of millions annually, but a carefully calculated assessment of losses is
not available. Because of its comparatively narrow host specificity, ESC is not a
great economic problem in regions where channel catfish are not cultured.
Host range and geographical distribution
Edwardsiella ictaluri has a narrower host range than that of E. tarda; however, it
is still diverse (Table 13.4). Cultured channel catfish are most severely affected,
but less susceptible ictalurids include white catfish (Ameiurus catus), blue
catfish (Ictalurus furcatus), and, rarely, brown bullhead (Ameiurus nebulosus).
Experimental infections in blue catfish have been difficult, and variances in
susceptibility in channel catfish strains have been demonstrated (Wolters and
Johnson, 1994). Additionally, Wolters et al. (1996) showed that, following
experimental infection, channel catfish had the lowest survival (62%) and blue
catfish had the highest survival (90%), while survival of hybrids of the two
species was intermediate (74%). Natural infections in non-ictalurids include
walking catfish (Clarias batrachus) (Kasornchandra et al., 1987) and two
ornamental species, Bengal danio (Danio devario) (Waltman et al., 1985) and
green knife fish (Eigemmannia virescens) (Kent and Lyons, 1982). Experimental
infections were established in chinook salmon and rainbow trout (Oncorhyn-
chus mykiss) (Baxa et al., 1990), but Plumb and Sanchez (1983) could not
experimentally infect golden shiners (Notemigonus chrysoleucas), tilapia
(Tilapia aurea), largemouth bass or bighead carp (Aristichthys nobilis). Plumb
and Hilge (1987) demonstrated that the European catfish (sheatfish) (Silurus
glanis) was only slightly susceptible.
Edwardsiella ictaluri has been confirmed only in the USA, Thailand and
Australia (see Table 13.2); however, there is a report of its possible presence in
Taiwan (Chung and Kou, 1983) and there have been unconfirmed reports of
clinical signs typical of ESC in other parts of the world. In the USA, E. ictaluri is
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J.A. Plumb
found primarily across the south-eastern region, where channel catfish are
grown commercially. However, the bacterium has been reported to cause disease
among cultured channel catfish in other states, such as Arizona, California,
Idaho, Indiana and New Mexico. With the continual worldwide dissemination of
channel catfish for aquaculture purposes and an inadequate method of detecting
non-clinical infections, it is likely that E. ictaluri will occur in other
geographical regions. Enteric septicaemia is generally considered a disease of
cultured catfish, but Chen et al. (1994) found indications that the pathogen may
also occur in wild populations of channel catfish in California.
There is no indication that E. ictaluri poses a health threat to aquatic animals
other than a limited number of fish species. The temperature limitations under
which E. ictaluri grows essentially preclude this bacterium from being a
pathogen for humans or other warm-blooded animals (Janda et al., 1991).
The disease
Enteric septicaemia of catfish may be mild, chronic or acute, in which some
clinical signs are nearly pathognomonic. Diseased fish are listless at the surface,
with head-up, tail-down posture, and sometimes spin in circles before death.
More characteristic clinical signs are petechial haemorrhage or inflammation in
the skin under the jaw, on the operculum and belly (Fig. 13.4); haemorrhaging
often becomes so severe that the skin is bright red. Haemorrhage also occurs at
the base of fins. Small white (13 mm) depigmented areas appear on the skin and
they progress into similar-sized inflamed cutaneous ulcers. An open lesion
develops between the frontal bones of the skull posterior to, or between, the eyes
in chronically ill fish hence the common name hole-in-the-head (Fig. 13.5). It
should be noted that other bacteria (A. hydrophila, for example) can cause the
same lesion. Infected fish also have pale gills, exophthalmia and sometimes
abdominal distension with ascites. The ascites fluid is usually cloudy and/or
Table 13.4. Species of fish from which Edwardsiella ictaluri has been
isolated.
Fish species Reference
Natural inf ections
Blue catfish, Ictalurus furcatus J .A. Plumb, unpublished
Brown bullhead, Ameiurus nebulosus Plumb and Sanchez, 1983
Channel catfish, Ictalurus punctatus Hawke, 1979
Danio, Danio devario Waltman et al., 1985
Green knife fish, Eigemmannia virescens Waltman et al., 1985
J apanese eel, Anguilla japonica Chung and Kou, 1983
Puntius, Puntius conchonus Humphrey et al., 1986
Walking catfish, Clarias batrachus Kasornchandra et al., 1987
White catfish, Ameiurus catus Plumb and Sanchez, 1983
Experimental inf ections
Chinook salmon, Oncorhynchus tshawytscha Baxa et al., 1990
Rainbow trout, Oncorhynchus mykiss Baxa et al., 1990
bloody and rarely clear yellow. The kidney and spleen are hypertrophied, while
the spleen is dark red. Inflammation occurs in adipose tissue, peritoneum and
intestine, and the liver is either pale or mottled with congestion.
Enteric septicaemia of catfish is considered a seasonal disease, occurring
primarily in the late spring to early summer and again in the autumn (Fig. 13.6).
This pattern generally coincides with, but is not confined to, water temperatures
of 1828C. Francis-Floyd et al. (1987) demonstrated that the highest mortality
in experimentally infected channel catfish fingerlings was at 25C, lower at 23
and 28C and no deaths at 17, 21 or 32C. Several experiments have further
substantiated the temperature preference of ESC. Baxa-Antonio et al. (1992)
used immersion of channel catfish in a bath containing E. ictaluri to show peak
mortality at 25C (98%) and lower mortality at 20C (47%), 30C (25%), 35C
(4%) and 15C (0%). The effect of 25C on clinical ESC was further
demonstrated by Plumb and Shoemaker (1995), using a naturally infected
Fig. 13.4. Channel catfish infected with Edwardsiella ictaluri. The upper fish has haemorrhage
in the skin under the jaw and isthmus (arrow). The lower fish has white depigmented lesions on
the pigmented skin (arrowheads), red ulcerated lesions on the lower gill cover (arrow) and
abdominal distension, caused by ascitic fluid in the coelomic cavity.
496
J.A. Plumb
Fig. 13.5. Channel catfish infected with Edwardsiella ictaluri exhibiting open lesion (large
arrow) in the cranial region and inflamed nares (arrowhead) and exophthalmia typical of chronic
infection.
Fig. 13.6. Seasonal occurrence of Edwardsiella ictaluri, showing greatest incidence of disease
in May, J une, September and October, when average water temperatures are 2027C.
population of channel catfish, in which 10% were culture-positive for E. ictaluri
while being held at 15C. When these fish were elevated to 25C, 77% mortality
occurred due to E. ictaluri, which was significantly higher than that at 18C or
30C (10% and 23%, respectively).
In spite of the compelling experimental data that implicates a mid-20C
optimum temperature, the author has noted an increasing incidence of ESC in
diagnostic case work during winter and summer, indicating possible adaptation
of E. ictaluri to a broader temperature range (J.A. Plumb, unpublished). During
the early years following the discovery of ESC, relatively few outbreaks of the
disease were reported, but the number of E. ictaluri isolates soon began to climb
at an alarming rate. In 1981, there were 47 outbreaks diagnosed in the south-
eastern USA and, in 1985, there were 1420 diagnosed outbreaks, thus accounting
for 28% of all reported fish-disease cases in the region (A.J. Mitchell, Fish
Farming Experiment Station, Stuttgart, Arkansas, 1996, personal communica-
tion). In 1988, there were 1605 documented reports of ESC (30.4% of reported
fish diseases); however, the prevalence levelled off during 1990 and 1991 and
has remained constant since then.
Mortality in naturally infected channel catfish populations varies from less
than 10% to over 50%. It occurs in juvenile as well as food-sized fish, and under
all types of cultural conditions (including ponds, raceways, recirculating
systems and cages). Few fish diseases occur without some environmental
stressor preceding the infection, but E. ictaluri can probably cause disease
independent of stressors. This is not to suggest that adverse environmental
conditions do not influence the severity of infection, because Wise et al. (1993a)
showed that, when channel catfish were stressed by confinement in tanks prior to
E. ictaluri exposure, there was 97% mortality in stressed fish and 77% mortality
in non-stressed fish. To further illustrate the effects of stress, Ciembor et al.
(1995) netted and handled channel catfish and then exposed them to water-borne
E. ictaluri, leading to a mortality of 53% in stressed fish and 16% in unstressed
fish. It was also shown by Plumb et al. (1993b) that stocking density in ponds
may even affect susceptibility to E. ictaluri.
The microorganism
Edwardsiella ictaluri (ATCC 33202), described by Hawke et al. (1981), is a
typical member of the Enterobacteriaceae in most respects; it is a Gram-
negative, short, pleomorphic rod, which measures about 0.75 1.52.5 m (see
Table 13.3). It is weakly motile at 2530C, but not at higher temperatures. The
organism is catalase-positive, cytochrome oxidase-negative and glucose-
fermentative and reduces nitrate to nitrite (Shotts and Teska, 1989). It is also
lactose- and indole-negative and produces an alkaline slant and acid butt without
H
2
S on TSI agar. Although E. ictaluri ferments and oxidizes glucose while
producing gas at 2030C but not at 37C, the organism is non-reactive on most
sugars and is intolerant of NaCl higher than 1.5% in culture medium. Growth on
culture media is slow, requiring 3648 h to form punctate colonies on BHI agar
at 2830C; it grows poorly, if at all, at 37C.
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J.A. Plumb
By most accounts, E. ictaluri appears to be a rather homogeneous species
biophysically, biochemically and serologically. Waltman et al. (1986b) and Plumb
and Vinitnantharat (1989) found almost no differences in biophysical or
biochemical characteristics among many isolates of E. ictaluri from a variety of
species of fish and geographical regions. Data presented by Rogers (1981), Plumb
and Klesius (1988), Bertolini et al. (1990) and Vinitnantharat and Plumb (1993)
showed little serological diversity. In this same regard, Chen and Light (1994)
reported no cross reactivity of E. ictaluri-specific antibodies in domesticated or
wild channel catfish to nine other fish bacterial pathogens, nor did fish immunized
with these nine pathogens possess antibody titres to E. ictaluri. However, Lobb
and Rhoades (1987) reported some serological and possibly plasmid differences in
various strains of E. ictaluri. Subsequently, Lobb et al. (1993) utilized plasmid and
serological methods to show that differences existed between E. ictaluri strains,
particularly those isolated from non-ictalurid fishes.
Edwardsiella ictaluri possess at least two DNA plasmids (Lobb and
Rhoades, 1987), while Newton et al. (1988) found five E. italuri isolates that
contained one to three plasmids each, based on their molecular mass. It was
proposed by Reid and Boyle (1989) that all E. ictaluri isolates have plasmids.
The role of all plasmids in E. ictaluri is not known; however, Starliper et al.
(1993) transferred antibiotic resistance from Escherichia coli to E. ictaluri via
plasmids by cohabitation of the two bacteria. The transfer rate was at the high
frequency of 1.197 10
2
. In a companion study, it was shown by Cooper et al.
(1993) that the plasmids of E. coli and E. ictaluri are either identical or very
similar. Contrastingly, plasmids of E. ictaluri were thought to be specific enough
for Speyerer and Boyle (1987) to suggest that they could be used as a
deoxyribonucleic acid (DNA) probe to detect E. ictaluri in fish.
Edwardsiella ictaluri has peritrichous flagella and occasionally pili have
been detected in scanning electron micrographs. Newton and Triche (1993)
isolated and purified two proteins from the flagella of E. ictaluri, with apparent
molecular mass of 42 and 38 kDa, respectively. These authors also showed that
the LPS from 40 different E. ictaluri isolates were all the same when examined
by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
and that an immunoblot analysis revealed a high degree of antigenic similarity
among the isolates. However, according to Saeed and Plumb (1987), the LPS of
E. ictaluri is different from that of other Enterobacteriaceae, and Weete et al.
(1988) demonstrated a rough LPS that has no O side-chains.
The use of monoclonal antibody and colloidal gold to localize the pre-
dominant antigens of E. ictaluri showed that the organism possess major outer-
membrane antigens, with molecular masses of 60 and 36 kDa (Klesius and
Horst, 1991). These antigens had previously been demonstrated by Plumb and
Klesius (1988), as well as Newton et al. (1990).
When first described, E. ictaluri was thought to be an obligate pathogen,
unable to live for an extended period outside the host (Hawke, 1979).
However, later work indicated that the organism could survive in sterilized
pond-bottom mud for over 90 days at 25C (Fig. 13.7) (Plumb and Quinlan,
1986). The survival of E. ictaluri in the environment may be influenced by
microbial competition, because Earlix (1995) showed that, in contrast to
earlier studies, the organism does not survive well in water or mud containing
other microbes.
Diagnostic methods
Clinical signs of E. ictaluri infection are more pathognomonic than most other
infectious fish diseases and therefore are helpful in ESC diagnosis. However,
isolation of the organism and/or serological tests are essential for
confirmation. Edwardsiella ictaluri is isolated from clinically infected fish on
BHI or TSA agar, but Shotts and Waltman (1990) described EIM, which
enhances E. ictaluri isolation and aids identification. The organism forms
small, translucent, greenish colonies on EIM (Fig. 13.8), while the media
inhibits Gram-positive and most Gram-negative contaminating organisms.
Colonies of E. tarda have black centres and A. hydrophila colonies are
brownish and larger, while Pseudomonas fluorescens colonies are blackish and
punctate on EIM. Using the biochemical and biophysical characteristics in
Table 13.3, E. ictaluri can be easily separated from E. tarda, because the
former is indole-negative and does not produce H
2
S on TSI agar. Commercial
identification systems, such as Minitek and API 20E, are not as accurate for E.
ictaluri as they are for some other fish pathogens (Taylor et al., 1995).
Fig. 13.7. Survival of Edwardsiella ictaluri in pond water and mud at different temperatures.
(From Plumb and Quinlan, 1986; reprinted with permission of the American Fisheries Society.)
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J.A. Plumb
Careful observation of culture plates is essential to detect growth of
E. ictaluri, because of its slow growth and the possible presence of more rapidly
growing bacteria, such as Aeromonas spp. Definitive identification is by using
biochemical characteristics (Table 13.3) or serological identification by specific
antiserum agglutination or other serological tests. These include polyclonal and
monoclonal antibody in indirect FAT (Ainsworth et al., 1986) and ELISA
(Rogers, 1981; Hanson and Rogers, 1989; Klesius, 1993; Earlix et al., 1996).
Twenty E. ictaluri isolates were positive using FAT and ELISA, while no cross-
reactivity was detected with E. tarda, Salmonella sp. or A. hydrophila (Rogers,
1981). Ainsworth et al. (1986) also used monoclonal antibodies against
E. ictaluri in an indirect FAT application for diagnosing E. ictaluri. They
compared the FAT techniques with bacterial isolation from brain, liver, spleen,
anterior kidney and posterior kidney, and 90.3% of the culture-positive, fish
were also FAT-positive, using tissues from these organs. The greatest
discrepancy occurred in the brain samples, but tissues from the spleen were 90%
positive by FAT and 85% by culture. All of these studies emphasized the time-
saving advantage of immunoassay techniques of 2 h for results versus 48 h
required for culture results. It is also possible to diagnose E. ictaluri in the
carcasses of dead fish, using an ELISA system (Hanson and Rogers, 1989).
Apparently, upon death, the bacterium escapes from lysed macrophages and uses
the nutrients of lysed cells to proliferate, thus providing a large number of
organisms.
Detection of E. ictaluri carrier fish when there is no clinical disease may
present a problem; however, Mgolomba and Plumb (1992) and Klesius (1992)
Fig. 13.8. Edwardsiella ictaluri, E. tarda, Aeromonas hydrophila and Pseudomonas fluorescens
on Edwardsiella isolation media incubated at 25C for 48 h. (Photo by D. Earlix.)
found significant bacteria in the blood and all organs (Fig. 13.9) of fish 65 and
270 days, respectively, after initial exposure to E. ictaluri. Edwardsiella ictaluri
is also readily phagocytized by macrophages in nave fish (Miyazaki and Plumb,
1985; Klesius et al., 1991), but immunization increases the phagocytic activity
(Shoemaker, 1996). Indications are that the phagocytized bacteria are not
destroyed in these cells, which could lead to a lengthy carrier state and could
provide a site of identifying carrier fish (Miyazaki and Plumb, 1985; Klesius et
al., 1991; Klesius, 1993; Shoemaker, 1996). Application of the Falcon assay
screening test (FAST)-ELISA was utilized by Klesius (1993) to rapidly and
accurately detect E. ictaluri antibody in adult fish and he proposed that the
method could be used to identify possible E. ictaluri carrier fish. Earlix et al.
(1996) utilized an ELISA and monoclonal antibody in conjunction with tissue
homogenization, digestion of tissue with Triton X-100 and filtration on 0.45 m
nitrocellulose membrane to detect an 80% E. ictaluri carrier state in
asymptomatic channel catfish. This is compared with a 24% carrier-state
detection by conventional bacteriological isolation methods. Implementation of
these procedures could be useful in determining E. ictaluri carrier populations.
Serum agglutination, passive haemagglutination, complement-dependent
Fig. 13.9. Edwardsiella ictaluri isolated from organs and tissues 44, 51 and 65 days after initial
exposure to the pathogen. HK, head kidney; BL, blood; BR, brain; L, liver; TK, trunk kidney; SP,
spleen; G, gonads; GB, gall bladder; M, muscle. (From Mgolomba and Plumb, 1992, reprinted with
permission of Elsevier Science Publishers.)
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J.A. Plumb
passive haemolysis, indirect immunofluorescence, agar gel immunodiffusion
and agglutination with fractionated immunized fish sera were used for detecting
humoral antibody to LPS of E. ictaluri (Saeed and Plumb, 1987). All tests were
sensitive to the LPS antibody, with the complement-dependent haemolysis titres
being the highest (average titre of 1 : 2360). Waterstrat et al. (1989) found a
close relationship of antibody titres measured by optical density and
corresponding agglutination titres to E. ictaluri in channel catfish. Using the
FAST-ELISA system employing a monoclonal antibody against an
immunodominant epitope of E. ictaluri, Klesius et al. (1991) found no cross-
reactivity with sera from experimentally immunized channel catfish against
E. tarda or A. hydrophila. The ELISA system was further refined so that either
E. ictaluri antigen or antibody in fish can be detected in 30 min. Time is usually
of the essence in diagnosing clinical E. ictaluri; hence, the faster serological
techniques should be used in conjunction with bacterial isolations.
Transmission of the disease
In aquaculture, infected channel catfish are the primary source of E. ictaluri,
with natural transmission occurring primarily through the water column.
Horizontal transmission of E. ictaluri was demonstrated by Klesius (1994), in
which nave fish showed clinical infections 12 days postexposure to fish that had
died of ESC. Experimental transmission of E. ictaluri is easily achieved by
water-borne exposure, i.m. or i.p. injection, intestinal intubation or introducing
the bacterium into the nares only (Plumb and Sanchez, 1983; Shotts et al., 1986;
Newton et al., 1989; Morrison and Plumb, 1994). Acute clinical disease usually
appears at 57 days postbath exposure at 25C. Nusbaum and Morrison (1996)
showed that the pathogen invaded the gill and then migrated to other organs and
tissues. The nares are a primary site of E. ictaluri invasion, and exposure of this
organ to E. ictaluri can initiate chronic ESC (Morrison and Plumb, 1994). It was
shown by Mgolomba and Plumb (1992) and Klesius (1992) that survivors of an
epizootic still carry E. ictaluri long after clinical disease has disappeared;
therefore, they can serve as reservoirs of the pathogen. Recent research indicated
that, in a pond where fish were dying of E. ictaluri, the water in the vicinity of
dead fish had significantly higher E. ictaluri counts than waters where there
were no carcasses (Earlix, 1995). Removal of dead fish should reduce the
number of bacteria to which non-infected fish are exposed. Transmission from
adults to offspring during spawning is likely but as yet unproved.
MacMillan and Santucci (1990) were unable to isolate E. ictaluri from the
intestine of channel catfish, but Earlix (1995) isolated the organism on EIM from
intestines of approximately 50% of clinically infected channel catfish. Using
fluorescent antibody, E. ictaluri was also detected in the lower intestines of
cormorants and herons (Taylor, 1992) and these bacteria were later shown to be
viable. This reservoir could serve as a source of infection for nave channel
catfish, but actual transmission from the birds to fish has not been demonstrated.
Once E. ictaluri is introduced into a particular body of water, it probably
remains there as a source of infection, either in carrier fish or in the environment.
The way in which E. ictaluri is transferred from farm to farm is speculative, but
there is little doubt that the transfer of infected fish is the primary instrument of
transmission, as well as other means; for example, seines, nets, etc. that are not
disinfected or thoroughly air-dried between use could be sources of infections,
as could fish-eating birds and terrestrial animals moving from pond to pond or
farm to farm.
Treatment and protection
Management of cultured fish to reduce the effects of clinical disease is
important. In addition to the application of the best management practices
outlined earlier, the use of strains of channel catfish that are less susceptible to
E. ictaluri or utilizing channel catfish blue catfish hybrids shows promise of
providing possible culture animals on farms where E. ictaluri is endemic
(Wolters and Johnson, 1994; Wolters et al., 1996).
Diet may be important in altering susceptibility of channel catfish to
E. ictaluri. Channel catfish fed no zinc had 100% mortality, compared with
2530% mortality for those fish fed 1530 mg of zinc (Paripatananont and
Lovell, 1995). Other factors may affect susceptibility of channel catfish to
E. ictaluri, because the addition of 60 mg or more of vitamin E in the diet of
channel catfish increased agglutinating antibody titres and enhanced the
ability of macrophages to phagocytose virulent bacteria (Wise et al., 1993b).
Contrastingly, Tyler and Klesius (1994) showed that the injection of squalene,
an oil-based adjuvant, actually increased the susceptibility of channel catfish
to E. ictaluri. Similarly, Stanley et al. (1995) showed that a normally
immunoenhancing compound, an extract from the tunicate Ecteinascidia
turbinata, had an adverse effect on disease susceptibility when injected i.p. into
channel catfish.
Channel catfish are less susceptible to E. ictaluri, and possibly other
potential pathogens, in water with salinity of up to 4000 mg l
1
(G. Whitis,
Aquaculture Extensionist, Alabama Fish Farming Center, Greensboro, Alabama,
1995, personal communication). In view of this observation, Plumb and
Shoemaker (1995) exposed an E. ictaluri carrier population (about 10%
incidence in 15C water) of channel catfish to waters containing 03000 mg l
1
NaCl and raised the temperature to 25C. Mortality in 0 and 100 mg NaCl l
1
were 95100% and the mortality in the populations transferred to water with
over 1000 mg NaCl l
1
were 1742%. It is apparent that many factors, including
genetics, nutrition and environmental quality, affect the susceptibility of channel
catfish to E. ictaluri, the reasons for which are poorly understood.
Although there is a tendency for the aquaculturist to do something when
ESC strikes, it has been suggested that simply stopping feeding the fish every
day may be as good a management decision as applying antibiotics. It has been
shown that not feeding and feeding medicated feed (sulphadimethoxine
ormetoprim) every third day resulted in the highest survival of E. ictaluri-
infected fish, compared with daily feeding with a normal ration (D. Wise, Delta
Research Station, Stoneville, Mississippi, 1996, personal communication).
504
J.A. Plumb
Actually, skipping 1 or 2 days between feeding was also better than a daily
feeding regime. Cessation of feeding when ESC occurs has been adopted by
many channel catfish farmers in place of feeding a medicated diet, generally
with satisfying results. In an attempt to evaluate the value of winter feeding on
ESC susceptibility in the spring, Okwoche (1996) demonstrated that production-
size channel catfish held over winter without receiving feed were more resistant
to E. ictaluri in the spring than those fish that had received normal or partial
feeding during the winter months.
Two drugs, oxytetracycline and sulphadimethoxineormetoprim, are
approved by the FDA for E. ictaluri infections in the USA (Schnick et al., 1989).
Both are incorporated into manufactured feed; oxytetracycline is fed at 50
75 mg kg
1
of fish day
1
for 1214 days, followed by a 21-day withdrawal period.
Sulphadimethoxineormetoprim is also fed at 5075 mg kg
1
of fish for 5 days,
followed by a 3-day withdrawal for catfish. The importance of an early diagnosis
cannot be over emphasized in ESC, because successful therapy depends on
expedience and immediate application of medicated feed. The sulpha-
dimethoxineormetoprim may create palatability problems if the concentration
in the feed is too high, and the fish will go off feed. To prevent palatability
problems, Johnson and Smith (1994) suggested that the concentration of drug in
the feed be reduced by at least half and the pellet made smaller and fed at 3% of
body weight.
The drug sarafloxacin, a quinolone, was shown to be effective for treating E.
ictaluri infections of channel catfish at 10 mg kg
1
of fish day
1
for 5 or 10 days
under experimental conditions (Plumb and Vinitnantharat, 1990). The super-
iority of the 10-day feeding of sarafloxacin was demonstrated by Thune and
Johnson (1992), because, 15 days after cessation of treatment, the number of
carrier fish with the shorter feeding duration was nearly twice that of the 10-day
feeding. Field trials by Johnson et al. (1992, 1993) demonstrated the efficacy of
sarafloxacin on E. ictaluri-infected channel catfish held in either ponds or cages.
Although sarafloxacin has been submitted by the manufacturer to the FDA for
use on channel catfish, its approval has not been forthcoming.
Waltman and Shotts (1986b) screened 118 isolates of E. ictaluri for suscep-
tibility to 37 antimicrobials, including oxytetracycline and sulphadimethoxine
ormetoprim. They found no evidence of resistance to either antibiotic, however,
there are more recent reports of resistance to both of these drugs (Plumb et al.,
1995; P. Taylor, US Fish and Wildlife Service, Marion, Alabama, 1995, personal
communication). The increase in resistance to drugs is exacerbated by improper
use of the antibiotics by feeding medicated feed when it is not necessary, feeding
at an incorrect rate or applying the medicated feed for too long, too often or for
too short a time. The increased resistance could also be plasmid-induced, as was
suggested by Waltman et al. (1989).
Edwardsiella ictaluri is a strong immunogen, especially when injected, and
therefore is an excellent candidate for vaccine development (Vinitnantharat and
Plumb, 1992). Antibody titres against E. ictaluri are measured by agglutination
or ELISA, but the antibody titres do not necessarily correlate with protection
(Salati et al., 1983; Klesius and Sealey, 1995). However, Vinitnantharat and
Plumb (1993) showed that channel catfish which survived a natural epizootic of
E. ictaluri had varying levels of antibody and that those fish with high titres
(>1 : 1024) suffered 6.5% mortality when challenged by injection, compared
with 25% and 51% in fish with titres of 1 : 256512 (medium) and 01 : 128
(low), respectively. Most of the E. ictaluri vaccination studies discussed below
do not elicit humoral antibody titres that approach even the medium range noted
above, which, therefore, may help explain why some experimental vaccinations
have been unsuccessful in providing protection against ESC.
The kinetics of the immune response of channel catfish to i.p. injection with
either cell extracts or whole-cell preparations of E. ictaluri and held in cages at
2030C showed peak antibody titres of 1 : 2001 : 2000, 28 weeks following
injection (Vinitnantharat and Plumb, 1992). Channel catfish at 25C produced a
rapid immune response to whole-cell bacterins; however, if fish were
vaccinated, held at 25C for 4 days and then the temperature reduced to 12C,
the agglutination titre was higher and had greater longevity (Plumb et al., 1986).
These fish also showed a greater antibody titre in the memory response than in
the primary response, when given a second injection. Specific E. ictaluri
antibody following immersion in a live bacterial bath can occur as quickly as 5
days after exposure (Klesius and Sealey, 1995). Significant antibody stimulation
occurred 1421 days after exposure and began to decline at 28 days. These fish
developed a second response when re-exposed 84 days after initial contact, but
this response was not greater than the initial response. After the initial exposure,
the fish were protected against subsequent exposure to live, pathogenic
E. ictaluri.
Several studies have examined the potential of fractionating E. ictaluri,
followed by isolating and purifying an immunodominant antigen (Plumb and
Klesius, 1988; Klesius and Horst, 1991; Vinitnantharat et al., 1993). It appears
that proteins with a molecular mass of 36 kDa and 60 kDa are primary
immunodominant antigens in the cell membrane, which provide protection to
channel catfish when injected (Vinitnantharat et al., 1993). The 36 kDa protein is
not lost by cells when cultured for up to 30 passages on media. Saeed and Plumb
(1986) demonstrated that E. ictaluri LPS was an excellent antigen when injected
along with adjuvant, but immersion in LPS was not immunogenic.
Immersion-vaccinating channel catfish against ESC has been used
experimentally; however, there is some question about its protective efficacy.
Immersion in a 1 : 10 dilution of the killed bacterin is the optimum concentration
(Morsey, 1988; Vinitnantharat and Plumb, 1992) and length of immersion is
0.52 or more minutes, but the longer the exposure the better the response.
Laboratory studies indicate that E. ictaluri vaccine incorporated into the feed is
immunogenic and may serve as a booster vaccination. Plumb and Vinitnantharat
(1993) combined immersing juvenile channel catfish in 1 : 10 formalin-killed
bacterin and then feeding an encapsulated preparation in the feed for two 5-day
periods, with a 10-day non-vaccination period between. Six months later,
agglutinating antibody titres in the immersion- plus oral-vaccinated fish
averaged about 1 : 1700, compared with 1 : 931 in the non-vaccinated fish. The
immune response may have been aided by natural exposure to E. ictaluri, but no
clinical ESC was detected. When subsamples of fish were challenged with
E. ictaluri, the survivals were: non-vaccinated control 42.7%; immersion
506
J.A. Plumb
vaccinates only 56.3%; immersion + oral vaccinates 70.8%. Accordingly,
exposure of channel catfish to a formalin-killed E. ictaluri bacterin by
immersion may provide marginal protection, but immersion plus oral
application increases protection. The concentration of encapsulated ESC
vaccine in the feed should be at least 0.5%, with 1% vaccine being optimum,
although 10% vaccine was not immunosuppressive (Plumb et al., 1994).
In a field study involving 12,500 to 2.4 million juvenile channel catfish on
each of four different farms, Plumb et al. (1993a) investigated the practical
application of vaccinating against E. ictaluri. There was no experimental
laboratory challenge, but the overall harvest of non-vaccinated fish was 43.6%, a
single immersion was 56.7%, a double immersion was 64.2% and immersion
plus oral application resulted in 68.8% survival. Similar results were reported by
Thune et al. (1993), but they found one immersion/oral vaccination regime to
have resulted in very high survival (95%), compared with immersion vaccinates
(23.3%) and non-vaccinates (38.3%). In a study of channel catfish vaccinated by
immersion followed by the 5105-day oral booster regime, and non-vaccinated
fish and stocked at 6000, 12,000 and 24,000 0.04 ha
1
, vaccinated fish at the two
lower densities experienced 20% higher survival (P<0.05) than non-vaccinated
fish (Plumb et al., 1993b). Vaccination induced no benefit at the higher stocking
density.
All immersion and/or oral vaccination studies with E. ictaluri bacterins and
encapsulated products have not been successful. One possible reason for these
failures is that the fish may not absorb the killed antigen and therefore it does not
reach immunogenic tissues (Nusbaum and Morrison, 1996). Also, humoral
agglutinating antibody may not be produced and, even if present, may not be
protective (Klesius and Sealey, 1995).
Until recently, vaccines have been either formalin-killed whole-cell
bacterins, sonicated cell preparations or cell extracts, such as LPS or
immunodominant antigens, but attenuated preparations of E. ictaluri are now
receiving some attention. Recently, Shoemaker (1996) strongly suggested that
an effective E. ictaluri immersion vaccine needs to be a live bacterium and that
the cell-mediated immune response must be activated, which killed preparations
do not do. He demonstrated that channel catfish which survived exposure to a
live, moderately virulent E. ictaluri isolate were 100% protected against
subsequent exposure to virulent E. ictaluri, but channel catfish vaccinated by
immersion or orally with killed bacterins had 68% and 50% survival,
respectively. In support of the efficacy of the exposure to live bacteria, the in
vitro bactericidal activity by peritoneal macrophages from live-cell vaccinates
was also significantly greater (P<0.05) than bactericidal activity of
macrophages from the other groups of vaccinates. The protection afforded by
initial exposure was correlated to cell-mediated immunity rather than humoral
antibody. An irreversible attenuated E. ictaluri would be required for live-
vaccine preparation. Cooper et al. (1996) describe an E. ictaluri isolate that is
chondroitinase-negative, and preliminary studies show that, when this mutant is
injected into channel catfish, they are protected when the fish are subsequently
exposed to pathogenic E. ictaluri.
Pathogenesis
The pathogenic mechanism of E. ictaluri infection in channel catfish is not fully
understood. Janda et al. (1991) demonstrated that the bacteria did not invade
HEp-2 cell monolayers at 35C, nor did they produce cell-associated haemolysin
or siderophores, as does E. tarda. In examining ECP being associated with the
pathogenesis of E. ictaluri, Stanley et al. (1994) found a fibrillar network
connecting virulent cells that could aid in attachment. Virulent isolates had
greater amounts of capsular material and surface proteins, and they demonstrat-
ed a greater ability to degrade chondroitin than did avirulent cells. These authors
reported no clear correlation between haemolytic activity and virulence.
Edwardsiella ictaluri infects fish by several routes. Water-borne bacteria
can invade the olfactory organ via the nasal opening and migrate into the
olfactory nerve, then into the brain meninges and finally to the skull and skin
(Miyazaki and Plumb, 1985; Shotts et al., 1986; Morrison and Plumb, 1994).
Injury to the nares includes loss of sensory cilia and microvilli from the olfactory
mucosal surface (Morrison and Plumb, 1994) within 1 h of exposure to
E. ictaluri. By 24 h, the olfactory receptors and supporting cells were
degenerating; electron microscopy confirmed the presence of E. ictaluri on the
mucosal surface and within the epithelium (Fig. 13.10). Host leucocytes
migrated through the olfactory epithelium into the lamellar lumen and
phagocytized the bacterium. With regard to the attachment mechanism of E.
ictaluri, Wolf (1996) showed that bacterial lectins were instrumental in this
attachment by utilizing specific sugar residues, specifically D-mannose,
N-acetylneuraminic acid and L-fucose, in the nasal mucosa.
Edwardsiella ictaluri apparently colonizes capillaries in the dermis and
causes necrosis and depigmentation of the skin. In the intestine, E. ictaluri enters
the blood through the intestinal wall and is engulfed by bacteriophages, resulting
in septicaemia (Shotts et al., 1986; Newton et al., 1989). Channel catfish
exposed to E. ictaluri via oral infection developed enteritis, hepatitis, interstitial
nephritis and myositis within 2 weeks of infection. Francis-Floyd et al. (1987)
described gastrointestinal lesions, including petechia or ecchymoses in the
mucosa of the gastrointestinal tract and intestinal distension associated with gas
production. The gill is also a primary site of E. ictaluri invasion. Using
radiolabelled E. ictaluri, Nusbaum and Morrison (1996) demonstrated that,
during immersion, the organism colonizes the gill epithelium in large numbers in
272 h. Bacterial numbers then increased rapidly in the liver and less rapidly in
the trunk kidney, gut and brain. It is worth noting that formalin-killed radio-
labelled bacteria did not appear to cross the gill epithelium membrane.
Histopathologically, the trunk kidney and spleen are the most severely
affected organs in channel catfish, both of which are necrotic, while the liver is
oedematous and necrotic (Fig. 13.11) (Areechon and Plumb, 1983).
Proliferation occurs in interlamellar tissue in gills (Jarboe et al., 1984; Miyazaki
and Plumb, 1985; Shotts et al., 1986). Also, a mild focal infiltration, necrosis and
granulomatous inflammation take place in the underlying musculature in areas
where the epidermis is missing. Intact E. ictaluri cells are also seen in
macrophages, similarly to E. tarda (see Fig. 13.3).
508
J.A. Plumb
The most complete pathological study of E. ictaluri was that of Newton
et al. (1989). Following experimental exposure of channel catfish to 5 10
8
cfu
ml
1
, 93% of the affected fish developed acute ESC and 7% developed chronic
infection. Acute disease was characterized grossly by haemorrhage and
ulceration, and microscopically by enteritis and by olfactory sacculitis at 2 days
postexposure, followed by hepatitis and dermatitis. Chronic ESC, seen at 34
weeks postexposure, was characterized by dorsocranial swelling and ulceration,
granulomatous inflammation and meningoencephalitis of the olfactory bulbs,
olfactory tracts and olfactory lobes of the brain. The granulomatous
inflammation in chronic E. ictaluri infection is a key histopathological
characteristic of ESC (Fig. 13.11). Skeletal muscle becomes necrotic, with
infiltration of macrophages, while internal organs, especially the liver, have
normal tissue displaced by macrophages.
TOPICS FOR FURTHER STUDY
Edwardsiella tarda and E. ictaluri both cause economically important diseases
in aquaculture, but their epidemiological implications and manifestation need
elucidation. Since neither bacterium is a strict obligate pathogen, we need to
learn how and where they survive in fish and the environment. Knowledge of
how cultural practices enhance their spread, particularly E. ictaluri throughout
the catfish industry, would be most helpful. The role and effect of weather, water
quality and environmental conditions, fish genetics, nutrition and other
management practices on ES and ESC should be further investigated and would
aid in better management of these diseases. Why these bacteria are more
pathogenic at certain temperatures and the nature of their primary pathogenesis
are other areas worthy of study. To understand the pathogenesis of these
organisms and their mechanism of attachment and invasiveness to the host and
the identification of pathogenic components could enhance the utilization of
preventive measures.
Chemotherapeutic control of E. tarda and E. ictaluri is a problem, because
they have transmissible R plasmids, which enhance antibiotic resistance.
Educating and encouraging aquaculturists to practise health maintenance and to
apply drugs properly and only when necessary will slow the evolution of
resistance to commonly used drugs. New therapeutics must be developed for
aquaculture to avoid overdependence on one specific drug.
Fig. 13.10. (Opposite) Electron micrographs of Edwardsiella ictaluri in olfactory organ of
channel catfish. (A) Scanning electron micrograph of the olfactory mucosal surface following
direct experimental E. ictaluri infection with a cluster of bacteria attached to the epithelial surface.
Note the fine filamentous processes extending from E. ictaluri (arrows) ( 13,500). (B)
Transmission electron micrograph of the olfactory mucosal surface 1 h following direct
experimental E. ictaluri infection. The bacteria (large arrows) are close to the epithelial surface
(arrowhead), which has no cellular cilia but has increased secretory vesicles of host mucosal cells
(small arrows) ( 2565). (C) Transmission electron micrograph showing several catfish leucocytes
in the interlamellar space of the olfactory rosette. A number of E. ictaluri can be seen within
cellular phagosomes (arrows) ( 4590). (Photographs courtesy of E.E. Morrison and K.G. Wolfe.)
510
J.A. Plumb
Fig. 13.11. Paraffin sections of tissue from a channel catfish infected with Edwardsiella ictaluri.
(A) Necrotic skeletal muscle (N) with infiltrating macrophages (arrow) typical of granulomatous
inflammation. A giant cell (GC) is present within the accumulation of macrophages. (H & E, 450.)
(B) Focal accumulation of macrophages (M) in the liver. Within this lesion, the macrophages have
displaced most of the liver tissue, but scattered hepatocytes (arrow) are still present. (H & E,
1000.) (Photograph by A. Goodwin.)
A concerted effort must continue on developing vaccines. Although a
simple, killed bacterin may be the least expensive to use, it may not be the most
effective. Basic research in extraction and purification of the immunodominant
antigen, aided by genetic engineering, should continue. The development of an
irreversible attenuated strain of E. ictaluri for vaccination and an effective
delivery system would be very beneficial to the catfish industry.
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Fish 13

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479

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

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