427

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

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
11
Motile Aeromonads
(Aeromonas hydrophila)
T. Aoki
Laboratory of Genetics and Biochemistry, Department of Aquatic Biosciences,
Tokyo University of Fisheries, Konan 4-5-7, Minato-ku, Tokyo 108, Japan.
INTRODUCTION
Motile aeromonads of the Aeromonas hydrophila complex cause a haemorrhagic
septicaemia in fish (Bullock et al., 1971; Egusa, 1978; Schperclaus et al.,
1992). This bacterium has been observed in numerous species of freshwater fish
and occasionally in marine fish and in amphibians, reptiles, cattle and humans
throughout the world (Bullock et al., 1971; Khardori and Fainstein, 1988).
However, the most significant diseases occur in cultured freshwater fish. The
bacterium is distributed widely in fresh water and bottom sediments containing
organic material, as well as in the intestinal tract of fish (Aoki, 1974; Egusa,
1978; Hazen et al., 1978; Seidler et al., 1980; Kaper et al., 1981; Van der Kooij
and Hijnen, 1988; Sugita et al., 1994, Dumontet et al., 1996).
Infectious abdominal dropsy in common carp has been attributed to the
A. hydrophila group (Aeromonas punctata) and was first described by
Schperclaus (1930), who reported on this condition in cultured, wild and
stocked carp in central and eastern Europe. The causative agent has since been
shown to be rhabdovirus carpio (spring viraemia of carp) (Fijan, 1972; Wolf,
1988). During the 1960s, outbreaks of red fin disease, caused by A. hydrophila,
occurred frequently in cultured eels in Japan (Hoshina, 1962; Egusa, 1978)
(Fig. 11.1), with concurrent infections by Saprolegnia parasitica (Egusa, 1978).
Currently, only sporadic outbreaks of A. hydrophila occur in cultured eels.
Aeromonas hydrophila is typically recognized as an opportunistic pathogen or
secondary invader (Austin and Austin, 1987).
Conversely, there have been reports of A. hydrophila acting as a primary
pathogen in fish. Isolates differ greatly in their pathogenicity with some strains
being highly virulent and others non-virulent. Eddy (1960) and Kou (1972a)
reported that non-virulent or weakly pathogenic strains did not produce gas and
acetone from glucose. Wakabayashi et al. (1981) recognized common and
identical biochemical characteristics in virulent strains, in particular the
production of elastase and enzymes involved in lysis of Staphylococcus. These
428
T. Aoki
characteristics were identical to the A. hydrophila biovar. hydrophila, following
classification by Popoff and Vron (1976). Economically, the disease attributed
to these bacteria is of greatest importance in cultured freshwater fish.
Recent advances in biochemistry, molecular biology and virulence factors
associated with A. hydrophila have led to new understanding of this bacterial
group. These are reviewed in this chapter.
THE DISEASE AND AGENT
Species of fish affected and geographical distribution of
Aeromonas hydrophila
Most cultured and wild freshwater fish are susceptible to infection by
A. hydrophila, but particularly cold-water fish, such as brown trout (Salmo
trutta), rainbow trout (Oncorhynchus mykiss), chinook salmon (Oncorhynchus
tshawytscha), ayu (Plecoglossus altivelis), carp (Cyprinus carpio), channel
catfish (Ictalurus punctatus), clariid catfish (Clarias batrachus) (Fig. 11.2),
Japanese eel (Anguilla japonica), American eel (Anguilla rostrata), gizzard
shad (Dorosoma cepedianum), goldfish (Carassius auratus), golden shiner
(Notemigonus crysoleucas), snakehead fish (Ophicephalus striatus) and tilapia
(Tilapia nilotica) (Bullock et al., 1971; Egusa, 1978; Saitanu, 1986).
Fig. 11.1. Eel (Anguilla japonica) with red-fin disease (haemorrhagic septicaemia) caused by
Aeromonas hydrophila (courtesy of Dr Teruo Miyazaki).
429 Motile Aeromonads
The disease
Diseased fish usually display cutaneous haemorrhage of the fins and trunk, and
the condition is often refered to as red fin disease (Hoshina, 1962) (Fig. 11.1).
The bacteria multiply inside the intestine, causing a haemorrhagic mucous
desquamative catarrh. Toxic metabolites of A. hydrophila are absorbed from the
intestine and induce a toxaemia. Capillary haemorrhage occurs in the dermis of
fins and trunk and in the submucosa of the stomach. Hepatic cells and epithelia
of renal tubules show degeneration. Glomeruli are destroyed and the tissue
becomes haemorrhagic, with exudates of serum and fibrin (Miyazaki and Jo,
1985; Miyazaki and Kaige, 1985) (Figs 11.311.5).
European carp infected with A. hydrophila show severe tail and fin rot and
visible haemorrhage and ulceration of the body surface. Widespread
proliferation of bacteria is usually observed in the intestine. In some reports
(Fijan, 1972; Wolf, 1988), the histopathological phenomena associated with the
rhabdovirus infection haemorrhagic septicaemia of carp have been erroneously
attributed to motile aeromonads (Bullock et al., 1971). Aeromonas hydrophila is
widely distributed in the intestinal tract of cultured fish and the water and
sediments of freshwater ponds which are rich in organic materials. Virulent
strains of A. hydrophila in these environments are possible sources of infection.
Outbreaks of disease are usually associated with a change in environmental
conditions. Stressors, including overcrowding, high temperature, a sudden
change of temperature, rough handling, transfer of fish, low dissolved oxygen,
Fig. 11.2. Clariid catfish (Clarias batrachus) with ulcerative form of haemorrhagic septicaemia
caused by Aeromonas hydrophila (courtesy of Dr Kriengsag Saitanu).
430
T. Aoki
Fig. 11.3. Aeromonas hydrophila infection in J apanese eel. Intestine shows bacterial
multiplication in the contents and mucousdesquamative catarrh (haematoxylin and eosin stain)
(courtesy of Dr Teruo Miyazaki).
Fig. 11.4. Aeromonas hydrophila infection in J apanese eel. Skin shows capillary haemorrhage
in the dermal loose connective tissue and the thinned epithelium (haematoxylin and eosin stain)
(courtesy of Dr Teruo Miyazaki).
431 Motile Aeromonads
poor nutritional status and fungal or parasitic infection, contribute to physiological
changes and heighten susceptibility to infection. This applies to fish of all ages.
Causative bacterium
There are three mesophilic motile aeromonad species in Bergeys Manual of
Systematic Bacteriology: A. hydrophila, Aeromonas caviae and Aeromonas
sobria (Popoff et al., 1981; Popoff, 1984). Currently, the following additional
Aeromonas species have been recognized: A. allosaccharophila (Martinez-
Murcia et al., 1992a; Esteve et al., 1995a), A. encheleia (Esteve et al., 1995b),
A. eteropelogenes (Collins et al., 1993), A. eurenophila (Martinez-Murcia et al.,
1992b), A. ichthiosmia (Collins et al., 1993), A. jandaei (Carnahan et al., 1991a),
A. media (Allen et al., 1983), A. schuberti (Hickman-Brenner et al., 1988),
A. trota (Carnahan et al., 1991b) and A. veronii (Hickman-Brenner et al., 1987).
The similarity of the above-mentioned ten species of motile aeromonads was
determined as a dendogram, using numerical taxonomy (Carnahan and Joseph,
1993). Millership (1996) reviewed characteristics for the differentiation of the
common phenotypes of these species in aeromonads. The genus Aeromonas has
been classified into the family Vibrionaceae; however, Colwell et al. (1986)
proposed that these aeromonads constituted a separate family, the Aero-
monadaceae.
Fig. 11.5. Aeromonas hydrophila infection in J apanese eel. Kidney shows destroyed glomeruli
accompanying exudation of serum and fibrin. Epithelia of renal tubules show necrosis and
atrophy (haematoxylin and eosin stain) (courtesy of Dr Teruo Miyazaki).
432
T. Aoki
Kou (1972a, 1973) and Wakabayashi et al. (1981) recognized that almost all
pathogenic strains of motile aeromonads relevant to aquaculture were
encompassed within A. hydrophila biovar. hydrophila, proposed by Popoff and
Vron (1976).
Aeromonas hydrophila is a Gram-negative rod-shaped bacterium and is
motile, due to a monotrichous polar flagellum. The bacterium measures 0.31.0
m in diameter and 1.03.5 m in length. It has no spore stage or capsule. The
optimum growth temperature is 28C, but growth can occur at 37C. Colonies
on nutrient agar are white to pale pink, round and convex, with entire margins.
The biochemical characteristics are shown in Table 11.1. Cytochrome oxidase
Table 11.1. Biological characteristics of Aeromonas hydrophila.
Characteristics Response
Indole production in 1% peptone water +
Aesculin hydrolysis +
Growth in potassium cyanide (KCN) broth +
L-Histidine and L-arginine utilization +
L-Arabinose utilization +
Acetoin from glucose (Voges-Proskauer (VP) reaction) +
H
2
S from cysteine +
Oxidase +
Cytochrome oxidase +
Catalase +
Methyl red (MR) experiment d
Acetylmethylcarbinol production +
2,3-Butanediol production +
2,3-Butanediol dehydrogenase +
-Galactosidase production +
Phosphatase +
Nitrate reduction +
Urease
Malonate
Gelatin liquefaction +
Casein digestion +
Loeffler serum digestion +
Starch hydrolysis +
Lipase +
Lecithinase +
Glucuronate utilization +
Ornithine decarboxylase
DNAse +
RNAse +
Haemolysis +
Carbohydrate decomposition
Adonitol
Aesculin d
Arabinose d
Cellobiose d
Dextrin +
Dulcitol
Fructose +
Galactose +
433 Motile Aeromonads
and catalase reactions are positive. It is a facultative anaerobe, fermenting
carbohydrates to acid, or acid and gas. Aeromonas hydrophila is resistant to the
vibriostatic agent O/129 (phosphate: 2,4-diamino-6,7-diisopropylpteridine
phosphate) 150 g, reduces nitrates to nitrite, is unable to grow in media
containing 6.5% sodium chloride (NaCl) and is generally resistant to ampicillin
and carbenicillin. The guanine plus cytosine (G + C) content of the deoxy-
ribonucleic acid (DNA) is 5763% (MacInnes et al., 1979).
Aeromonas hydrophila contains thermostable O, thermolabile K and
flagellar H antigens. Serologically, the O antigen of A. hydrophila is hetero-
geneous (Sakazaki and Shimada, 1984; Janda et al., 1994, 1996). Different
serotypes have been observed from various sources of fish, isolated in different
years and places (Eddy, 1960; Bullock, 1966). Interestingly, a common antigen
has been found among virulent strains (Kou, 1972b; Leblanc et al., 1981;
Merino et al., 1996). Protein fingerprints do not correlate with biochemical
characteristics. Both phenotype and protein fingerprints show clustering of
epizootiologically related isolates (Millership and Want, 1993).
The outer membrane component protein of A. hydrophila isolates is variable
(Aoki and Holland, 1985), but Maruvada et al. (1992) detected species-specific
polypeptides of the outer membrane from A. hydrophila, and Wilcox et al.
(1992) suggested that outer membrane protein profiles were useful for
confirming the identity of A. hydrophila. Howard et al. (1993) cloned eight exe
genes of extracellular protein secretion and membrane assembly from
A. hydrophila and found them to have a high similarity with the extracellular
secretion operons of a number of different Gram-negative bacteria. Shaw and
Hodder (1978) showed that O-polysaccharides were remarkably similar
Glucose +
Glycerol +
Glycogen +
Inositol
Inulin
Lactose d
Maltose +
Mannitol +
Mannose +
Melezitose
Raffinose d
Rhamnose d
Salicin d
Sorbitol d
Sorbose
Starch +
Sucrose d
Trehalose +
Xylose
+, Typically positive; , typically negative; d, differs among strains; H
2
S,
hydrogen sulphide; DNAse, deoxyribonuclease; RNAse, ribonuclease.
Table 11.1. Continued
Characteristics Response
434
T. Aoki
structures in motile Aeromonas species, including A. hydrophila.
MacInnes et al. (1979) investigated the DNA homology of 17 strains of
A. hydrophila, which had been collected from various sources, using
A. hydrophila ATCC7966 as a reference strain. The percentage homology DNA
ranged from 39 to 100%, with a mean value of 64.7%. Aeromonas hydrophila
does not seem to show any significant divergence among the 17 strains
investigated. The 16S ribosomal DNA (rDNA) from ten species of Aeromonas
was sequenced to analyse relatedness (Martinez-Murcia et al., 1992b).
Homology for 1052 nucleotides of 16S rDNAs from ten species of Aeromonas
exhibited very high levels, ranging from 98 to 100%. The nucleotide sequences
of 16S rDNA of A. hydrophila are therefore useful in species identification. East
and Collins (1993) showed that the region encoding 23S ribonucleic acid (RNA)
from A. hydrophila was identical to that of the gamma division of Proteo-
bacteria, Escherichia coli and Plesiomonas shigelloides. Small-subunit ribo-
somal RNA (rRNA) sequences of Aeromonas were examined for a phylogenetic
analysis (Ruimy et al., 1994).
Aeromonas hydrophila strains possessing a common plasmid, with a
molecular size of 2.528 MDa, were isolated by Toranzo et al. (1983). Small
plasmids from 2.6 to 6 MDa were found in A. hydrophila isolated in Malaysia
(Ansary et al., 1992). Cryptic plasmids were also detected from A. hydrophila
strains from freshwater fish and fresh water (Noterdaeme et al., 1991). No
relationship between their virulence or biochemical characteristics and the
presence of plasmids was found (Brown et al., 1997).
DIAGNOSTIC METHODS
The gross signs of disease are haemorrhagic septicaemia and fin rot. The
aetiological agent can be grown on brainheart infusion medium, tryptosoy agar
(tryptone soya agar), nutrient agar and MacConkey agar with incubation at
2025C for 2448 h. Numerous selective media have been developed for the
isolation and presumptive identification of A. hydrophila or motile aeromonads
(Moyer, 1996), including RimlerShotts medium (Shotts and Rimler, 1973),
modified peptone beef-extract glycogen agar (McCoy and Seidler, 1973),
RippeyCabelli (membrane filter method (mA)) agar (Rippey and Cabelli,
1979), MacConkeys agar supplemented with trehalose (Kaper et al., 1981) and
starchampicillin agar (Palumbo et al., 1985). Davis and Sizemore (1981)
reported that Rimler and Shotts medium and RippeyCabelli agar were not
suitable for A. hydrophila. Arcos et al. (1988) compared six media for selective
isolation of A. hydrophila and showed that mA agar gave the best recovery rate
and also an acceptable specificity, but its selectivity was low.
An API-20E test strip is used widely for identification of the
Enterobacteriaceae (Kaper et al., 1979). Toranzo et al. (1986) indicated that the
importance of biochemical characteristics must be backed up by standardized
testing.
For diagnosis, the aetiological agent is isolated on nutrient agar at 30C
from kidney of a fish with haemorrhagic septicaemia. Isolates are motile
435 Motile Aeromonads
Gram-negative bacilli. Wakabongo et al. (1992) found that only four tests
aesculin hydrolysis, acetoin production, lysine decarboxylation and gas from
glucose were sufficient to distinguish A. hydrophila, A. caviae and A. sobria.
A number of A. hydrophila-lytic bacteriopahges were isolated from river
water, river mud, sewage and human clinical origin (e.g. stool, urine) and phage
typing was attempted (Chow and Rouf, 1983; Demarta and Peduzzi, 1984;
Altwegg et al., 1988; Merino et al., 1990; Fukuyama et al., 1991, 1992). A
comprehensive phage-typing system for A. hydrophila will provide a useful tool
for epizootiological and ecological studies.
Aeromonas hydrophila has been identified by the gel-diffusion technique
(Bullock, 1966), direct fluorescent antibody technique (Lewis and Allison,
1971), indirect fluorescent antibody technique (Lewis and Savage, 1972),
immunoblotted sodium dodecylsulphate (SDS)-polyacrylamide gel electro-
phoresis (PAGE) (Mulla and Millership, 1993) and enzyme-linked
immunosorbent assay (Merino et al., 1993). These methods are of limited value,
because many different serological types of A. hydrophila are distributed in fish
farms (Eddy, 1960; Bullock, 1966).
Lucchini and Altwegg (1992) differentiated ribotyping of restriction
genomic DNAs of aeromonads, using different fragments of a 16S rDNA gene of
E. coli as a probe and achieved identification of most Aeromonas strains to
species level. This method is easier than DNADNA hybridization, because only
minimal amounts of genomic DNA are needed and several strains can be
analysed on a single gel. Pulsed-field gel electrophoresis is a rapid and
discriminatory technique for the typing of A. hydrophila where a common origin
of infection is suspected (Talon et al., 1996).
Deoxyribonucleic acid probe hybridization technology is now becoming
available for the direct detection and identification of microorganisms. The
colony hybridization technique (Grunstein and Hogness, 1975) can be applied
easily to identify and count the causative organism. Fish-pathogenic bacteria,
such as Vibrio anguillarum (Hirono et al., 1996) and Pasteurella piscicida (Zhao
and Aoki, 1992a), can be detected by probe hybridization with their specific
DNA nucleotide sequence or cryptic plasmid. However, there are many common
DNA fragments between A. hydrophila and Aeromonas salmonicida (Miyata et
al., 1995), which makes it unlikely that this probe technique will be successful
for this species.
The sensitivity obtained using hybridization with non-radiolabelled probes
is lower than with radiolabelled probes (Zhao and Aoki, 1992a). However,
radiolabelled probes are generally unacceptable for use in diagnosis and further
development of detection procedures based on non-radiolabelling is still
required.
The polymerase chain reaction (PCR) (Mullis and Faloona, 1987) is useful
for detecting fish pathogens from diseased fish and their environment. This
technique is available for A. salmonicida (Miyata et al., 1996), Edwardsiella
tarda (Aoki and Hirono, 1995), P. piscicida (Aoki et al., 1997) and
V. anguillarum (Hirono et al., 1996). However, recently, Cascn et al. (1996)
found a specific PCR primer set for the detection of A. hydrophila hybridization
group 1.
436
T. Aoki
The DNA fingerprinting method, AFLP (amplified fragment length
polymorphism), is a valuable high-resolution genotype tool for classification of
Aeromonas species (Huys et al., 1996).
CONTROL AND TREATMENT
Transmission of the disease
The aetiological agent is transmitted horizontally but not vertically. It is
distributed widely in water and sediments of ponds and can be transmitted by
discharge from the intestinal tract and external lesions on the skin (Aoki, 1974;
Egusa, 1978; Hazen et al., 1978; Seidler et al., 1980; Kaper et al., 1981; Van der
Kooij and Hijnen, 1988; Sugita et al., 1994; Dumontet et al., 1996). Parasite
damage and fungal infection of the epidemics may allow entry and spread of
bacterial infection (Egusa, 1978).
Aeromonas hydrophila has been recognized as a pathogen not only of
amphibians, reptiles and snakes but also cattle and humans (Eddy, 1960;
Khardori and Fainstein, 1988). It has been implicated as the causative agent of
clinical infections in humans, including septicaemia and peritonitis (Janda and
Abbott, 1996). Recently, its role as a psychrotrophic spoiler of meat, seafood and
vegetables has been recognized (Palumbo, 1996). No confirmed cases of
A. hydrophila food poisoning have been reported, but its association with
gastrointestinal illness suggests that it plays a role in food-borne disease. The
epidemiological relationship among A. hydrophila isolated from fish, human and
environmental resources is particularly difficult to assess.
Aeromonas hydrophila is cosmopolitan in distribution. Infection has been
seen in many freshwater fish, e.g. ayu, carp, channel catfish, eel, gizzard shad,
salmon, snakehead fish and trout (Bullock et al., 1971; Egusa, 1978; Saitanu,
1986).
CONTROL OF DISEASE
In general, bacterial infection occurs when fish are physiologically stressed or
sanitation is poor. Good hygiene, periodic drying and disinfection of ponds are
important in prevention. Avoidance of overcrowding, low oxygen and rough
handling are the best methods of prevention.
Chemotherapy
Chemotherapeutic agents are used for the treatment of A. hydrophila in fish
farms (Aoki, 1992). Isolates of A. hydrophila have been found to be sensitive to
chloramphenicol, florfenicol, tetracycline, sulphonamide, nitrofuran derivatives
and pyridonecarboxylic acids (Aoki and Egusa, 1971; Endo et al., 1973; Katae
et al., 1979; Fukui et al., 1987). Fluorinated analogue, tetracycline derivatives,
437 Motile Aeromonads
nitrofuran derivatives, sulphonamide and pyridonecarboxylic acids are effective
in oral treatments (Austin and Austin, 1987). A review by the Fisheries Agency
of the Japanese government on chemotherapeutic use against bacterial infections
in cultured fish has been carried out (Okamoto, 1992). The use of chlor-
amphenicol and almost all nitrofuran derivatives in fish have been restricted in
Japan since 1983.
The extensive use of antibacterial agents has led to an increase in resistant
strains of A. hydrophila. Drug-resistant strains carrying transferable R plasmids
appeared in 1971 in cultured eel and multiple resistant strains carrying R
plasmids are now distributed widely in cultured amago, ayu, carp and eel in
Japan (Aoki et al., 1971, 1972; Aoki and Watanabe, 1973; Aoki, 1974, 1975).
Almost all strains have intrinsic resistance to ampicillin, and drug-resistant
strains carried transferable R plasmid, which encoded resistance to sulpho-
namide and tetracycline and to chloramphenicol, sulphonamide and
streptomycin (Akashi and Aoki, 1986). Drug-resistant strains have been isolated
from the water, as well as from the intestinal tracts of cultured fish (Aoki, 1974,
1975) in France, Ireland (Hedges et al., 1985), Malaysia (Ansary et al., 1992;
Son et al., 1997), Taiwan (Kou and Chung, 1980) and the USA (Shotts et al.,
1976). Recently, drug-resistant strains have been isolated from cultured
snakehead (O. striatus) in Thailand (Aoki et al., 1990). The DNA of transferable
R plasmids was classified into incompatibility (Inc) (Couturier et al., 1988)
groups AC, which had been detected in different areas, and resistance to
sulphonamide and tetracycline and showed high homology with each other
(Akashi and Aoki, 1986). R plasmids classified into Inc group U, with resistance
to chloramphenicol, sulphonamide and streptomycin, showed homology within
a specific species and with R plasmids from A. salmonicida. Aoki (1988)
demonstrated that A. hydrophila strains carrying R plasmids having identical
DNA structures are widely distributed in cultured freshwater fish in various
areas.
Tetracycline-resistance (tet) genes on the R plasmids of Gram-negative
bacteria have been classified on the basis of their DNA structure into six classes;
Tet A, B, C, D, E, F and G (Levy, 1988; Zhao and Aoki, 1992b). The tet gene of
the R plasmids detected from A. hydrophila, which were isolated in Japan, was
classified into Tet D class (Aoki and Takahashi, 1987). Class D and E
tetracycline-resistance determinants were found in resistant strains of
A. hydrophila isolated in catfish ponds in the USA (DePaola and Roberts, 1995).
A class D tetracycline-resistance determinant of RA1 from A. hydrophila
contained the resistance (tet A) and repressor (tet R) genes. The tet A gene
encoded 394 amino acids, with a calculated molecular size of 41.1 kDa (Varela
and Griffith, 1993), and the tet R encoded 218 amino acids, with a calculated
molecular size of 24.4 kDa (Unger et al., 1984).
The 40 MDa plasmid encoded with resistance to carbenicillin and
novobiocin also regulated adherence and production of a potent haemolysin
(Hanes and Chandler, 1993).
438
T. Aoki
PATHOGENESIS AND IMMUNITY
A variety of possible virulence factors of A. hydrophila have been suggested,
including lipopolysaccharides (endotoxins), extracellular products (ECP),
siderophores, the ability of attachment to host cells and surface proteins. The
ECP include a cytotoxin, enterotoxin, haemolysins, protease, haemagglutinin
and acetyl cholinesterase (Cahill, 1990; Gosling, 1996; Howard et al., 1996).
Aeromonas hydrophila enters through the epithelium of the intestinal tract of
fish. Enterotoxins of A. hydrophila cause fluid to accumulate in ligated rabbit
ileal loops. Enterotoxins are divided into two types, cytotonic and cytotoxic.
Boulanger et al. (1977) isolated two different haemolysins, an - and a
-haemolysin, both of which have been implicated in the pathogenesis of
infection. Allan and Stevenson (1981) investigated the production of protease
and haemolysin in the ECP of A. hydrophila strains and showed a close
correlation between the quantity of haemolysin and toxicity to fish. Two
biologically similar but immunologically distinct haemolysins were purified by
Asao et al. (1986) and Kozaki et al. (1987, 1989). These haemolysins (cytotoxic
enterotoxin) had enterotoxic activity and caused fluid accumulation in infant
mice, in mouse intestine and in rabbit ligated ileal loops. Purified haemolysin
also displayed cytotoxicity to Vero cells and lethal toxicity to mice. The
molecular weight of the haemolysin was estimated at 48,00050,000 kDa and
biological activity was inactivated with heating for 5 min at 65C (Asao et al.,
1984).
Cross-reaction of A. hydrophila haemolysin and cholera toxin has been
reported, and a specific synthetic oligonucleotide probe for regions of A + B
subunits of cholera toxin was found to hybridize with chromosomal DNA from
some strains of A. hydrophila. The enterotoxin purified by Rose et al. (1989) had
a molecular size of 52,000, but only 25 residues of the N-terminal sequences
were found to be identical to the haemolysin of A. hydrophila (aerolysin)
(Howard et al., 1987). The amino acid residues differed significantly between
enterotoxin and aerolysin over the vast majority of the protein. The structure of
proaerolysin was determined by X-ray crystallography at 2.8 (Parker et al.,
1994). The aerolysin secretion system indicates that it is a haemolysin which is
directed outside the cell through periplasm (Wong and Buckley, 1993).
Five haemolysin genes (AHH1, AHH2, AHH3, AHH4 and AHH5) were
cloned from A. hydrophila into a E. coli vector (Aoki and Hirono, 1991; Hirono
and Aoki, 1991; Hirono et al., 1992). Each was classified into three groups,
depending on their nucleotide sequences. AHH1 belonged to one group (group
1), which contained part of a homologous sequence of the haemolysin genes of
Vibrio cholerae El Tor and Vibrio vulnificus (Rader and Murphy, 1988). There
were two highly conservative regions, and the location of the cysteine residue
was conserved. These regions may be important in the control of haemolytic
activity (Fig. 11.6). The other group (group 2) included AHH3, AHH4 and
AHH5, the previously reported aerolysin gene from A. hydrophila and A. trota
(Howard et al., 1987; Husslein et al., 1988) (Fig. 11.7). Chopra et al. (1993)
cloned a cytolytic enterotoxin gene from A. hydrophila. The gene was also
classified into group 2 by the nucleotide sequence. The remaining gene, AHH2,
439 Motile Aeromonads
is placed in group 3. From colony hybridization analysis, using the cloned
haemolysin genes, it was found that AHH1 (group 1) and AHH5 (group 2) were
widely distributed among aeromonads (Table 11.2). It is interesting that all
tested strains of A. salmonicida have AHH1 and AHH5 genes. One strain of A.
hydrophila has two or three haemolysin genes (Hirono and Aoki, 1991; Hirono
et al., 1992).
A significant qualitative as well as quantitative difference in the protease
components of ECP was produced by A. hydrophila and A. sobria, which were
pathogenic for fish (Nieto and Ellis, 1991). The role of protease in the virulence
of A. hydrophila is currently controversial. Wakabayashi et al. (1981) described
most of the virulent strains of A. hydrophila biovar. hydrophila as having a high
proteolytic activity. A single protease purified from A. hydrophila was lethal to
carp and was dermonecrotic to guinea-pigs. Thune et al. (1982) and Lallier et al.
(1984) found A. hydrophila protease to be lethal. Lallier et al. (1984) also
showed that both virulent and weakly virulent strains were dermonecrotic in the
guinea-pig, and a dermonecrotic factor was observed in sonicated cells of an
A. hydrophila strain isolated from eel (Shimizu, 1968). Chabot and Thune
Fig. 11.6. Comparison of deduced amino acid sequences among the AHH1, ASH4 and Vibrio
cholerae El Tor haemolysin (Rader and Murphy, 1988). The same amino acid residues are
indicated as on the aligned sequences. VCH, V. cholerae El Tor haemolysin.
440
T. Aoki
(1991) characterized three proteases and found no correlation between either
qualitative or quantitative protease production and virulence in age-0 channel
catfish. A metalloprotease with a molecular size of 38 kDa and a serine
proteinase with a molecular size of 22 kDa was purified from A. hydrophila
strain B52. These were stable at 56C for 10 min and had a lethal effect in fish,
with a median lethal dose (LD
50
) of 150 ng g
1
. Only the serine protease
possessed cytotoxic activity (Rodriguez et al., 1992). Leung and Stevenson
(1988) found two distinct types of extracellular protease: thermostable
Fig. 11.7. Comparison of deduced amino acid sequences among the AHH3, AHH4, AHH5,
ASH3, ASA1, Aeromonas hydrophila aerolysin (Howard et al., 1987) and A. trota aerolysin
(Husslein et al., 1988). The same amino acid residues are indicated as on the aligned sequences.
AHAER, A. hydrophila aerolysin; ATAER, A. trota aerolysin.
441 Motile Aeromonads
metalloprotease and thermolabile serine protease. The relationship between
these and the two purified by Nieto and Ellis (1986) is not known, but they were
also lethal to fish. Protease from A. hydrophila enhanced haemolysin activity
(Howard and Buckley, 1985), but Lallier et al. (1984) found that purified
haemolysin from A. hydrophila was not lethal to fish. A novel zinc-proteinase
was purified and characterized from A. hydrophila (Loewy et al., 1993), and
Rivero et al. (1990) cloned an extracellular protease gene from A. hydrophila.
Aeromonas hydrophila strains, which had haemolytic activity, enterotoxin
productivity and cytotoxic ability, were not virulent for rainbow trout (Santos et
al., 1988).
Yadav et al. (1992) noted that the fish cell lines, especially the BB (brown
bullhead, Ictalurus nebulosus) cells were sensitive targets for A. hydrophila
cytotoxins, even when assayed in conditions vastly different from those of
mammalian cells. Cytotoxin-producing strains were frequently associated with
epizootic ulcerative syndrome (EUS)-infected fish, compared with healthy fish.
Cytotoxic A. hydrophila strains have a role in the pathogenicity and progression
of EUS (Yadav et al., 1992).

Table 11.2. Colony hybridization analysis of Aeromonas species A.

hydrophila, A. sobria, A. caviae, A. veronii and A. salmonicida, using AHH1,
AHH5, ASH1 and ASA1 DNA probes.
Sources DNA probes
(no. of tested
Strains strains) AHH1 AHH5 ASH1 ASA1
Aeromonas Canned milk (1) 1* (1) 1 (1) 0 (0) 0 (0)
hydrophila Human (14) 9 (9) 4 (11) 0 (0) 8 (8)
Fish (31) 15 (16) 11 (23) 3 (3) 21 (26)
Environment (5) 5 (5) 4 (5) 0 (0) 5 (5)
Turtle (1) 1 (1) 1 (1) 0 (0) 1 (1)
Total (52) 31 (32) 21 (41) 3 (3) 35 (40)
Aeromonas sobria Human (18) 2 (3) 3 (7) 0 (0) 18 (18)
Fish (4) 0 (1) 1 (3) 0 (0) 4 (4)
Environment (2) 1 (1) 0 (2) 0 (0) 2 (2)
Bovine (1) 0 (0) 0 (0) 0 (0) 1 (1)
Frog (1) 0 (0) 0 (1) 0 (0) 1 (1)
Total (26) 3 (5) 4 (12) 0 (0) 26 (26)
Aeromonas caviae Fish (6) 2 (2) 1 (4) 0 (0) 6 (6)
Environment (4) 2 (2) 1 (2) 0 (0) 3 (3)
Pig (1) 1 (1) 1 (1) 0 (0) 1 (1)
Total (11) 5 (5) 3 (7) 0 (0) 10 (10)
Aeromonas veronii Human (1) 0 (0) 0 (0) 0 (0) 1 (1)
Aeromonas Salmonid 104 (104) 104 (104) 1 (1) 104 (104)
salmonicida fish (104)
*Hybridized number using high-stringency condition.

Hybridized number using low-stringency condition.

442
T. Aoki
Aoki and Holland (1985) observed that iron-binding proteins with a
molecular size of 6870 kDa in A. hydrophila were induced under iron-limiting
conditions. Enterobactin, the catecholate siderophore produced by a strain of
A. hydrophila (Andrus and Payne, 1983), and amonabactin, a novel phenolate
siderophore in A. hydrophila 495A2, were identified (Barghouthi et al., 1989,
1991). Esteve and Amaro (1991) found the hydroxamate-type siderophores
produced by A. hydrophila. The iron-uptake system was similar in A. hydrophila
and A. sobria isolated from European eels (Anguilla anguilla) and in
A. salmonicida. These authors suggested that siderophore produced by
A. hydrophila could be important and play a role in virulence for acquisition of
iron from the host. Recently, the amonabactins were synthesized and their
spectroscopic properties were elucidated (Telford et al., 1994).
The receptor cell surface of A. hydrophila can bind to iron-containing
proteins lactoferrin, transferrin, ferritin, cytochrome C and haemin of the
host (Kishore et al., 1991; Ascencio et al., 1992), demonstrating a relationship
between lactoferrin binding and siderophore production by the bacteria. A
carbohydrate-reactive outer-membrane protein, which may contribute as an
adhesive mechanism, was isolated from A. hydrophila strain A6 (Quinn et al.,
1993, 1994). This protein is related to the colonization strategies of aeromonads
associated with human enteric disease. However, the protein has not been proved
to relate to pathogenicity in fish.
Two cytotonic enterotoxin genes were cloned from A. hydrophila; one of the
enterotoxins was heat-labile at 56C, while the other was heat-stable (Chopra
et al., 1994).
The role of haemolytic activity and cytotoxic activity in contributing to
pathogenicity to fish still needs to be elucidated.
The attachment of a pathogen to the epithelial tissue is the first step in the
host disease process. The W pili of A. hydrophila strain Ae6 is the colonization
factor for the intestine, as well as a haemagglutinin (Hokama and Nakasone,
1990; Hokama et al., 1990). Haemagglutinating activity was also detected in
A. hydrophila (Atkinson and Trust, 1980; Corral et al., 1990). Aeromonas
hydrophila exhibited aggregative adherence to HEp-2 cells (Neves et al., 1994),
fish tissue culture and mucus-coated glass slides (Krovacek et al., 1987).
Adherence and haemagglutination were not significantly correlated with
virulence in fish (Corral et al., 1990).
De Meuron and Peduzzi (1979) have suggested that the K-antigen is a
pathogenicity factor. Virulent strains of A. hydrophila possessed an S-layer on
the cell surface (Dooley et al., 1986; Dooley and Trust, 1988; Ford and Thune,
1991, 1992).
Recently, Rodriquez et al. (1993) purified acetylcholinesterase from ECP
of A. hydrophila and showed it to be lethal to fish. Glycerophospholipid
: cholesterol acyltransferase (GCAT) and lipopolysaccharide complex enhanced
the lethal exotoxicity and cytolysin of A. salmonicida (Lee and Ellis, 1990). A
gene encoding GCAT was cloned from A. hydrophila (Thornton et al., 1988).
Experimental vaccination for prophylaxis against infection of A. hydrophila
has been examined (Stevenson, 1988). Fish immunized either intramuscularly or
intraperitoneally with vaccine showed protection against challenge. The
443 Motile Aeromonads
agglutinating antibody titre increased in the serum of immunized fish (Song
et al., 1976; Ruangpan et al., 1986; Karunasagar et al., 1991).
Immersion vaccination of channel catfish using polyvalent sonicated
antigens of A. hydrophila provides protection (Thune and Plumb, 1982). Lamers
et al. (1985) noted that agglutinating antibody was recognized in the serum of
carp immunized with A. hydrophila bacterin, following a second immersion with
this vaccine. However, fish vaccinated by immersion or orally showed
questionable protection.
Catfish immunized intraperitoneally by injection with the acid extract of the
S-layer protein of A. hydrophila were protected from the homologous, virulent
strain (Ford and Thune, 1992). Serological types of A. hydrophila are
heterogeneous and a polyvalent vaccine is thought to be necessary for
prevention of the infection.
TOPICS FOR FURTHER STUDY
There is significant interest in the pathogenicity of A. hydrophila to fish. Several
virulence factors, including the production of endotoxin, ECP, siderophores and
surface proteins, and the ability of attachment to host cells, are under study, but
their relative importance has not yet been elucidated. Cahill (1990) considers
that proteases are most important in fish pathogenicity, but further study on
adhesins, siderophores and surface proteins is needed to understand their role in
the pathogenesis of disease caused by A. hydrophila.
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