fish 11

427© CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)

11Motile 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 haemorrhagicsepticaemia in fish (Bullock et al., 1971; Egusa, 1978; Schäperclaus et al.,1992). This bacterium has been observed in numerous species of freshwater fishand occasionally in marine fish and in amphibians, reptiles, cattle and humansthroughout the world (Bullock et al., 1971; Khardori and Fainstein, 1988).However, the most significant diseases occur in cultured freshwater fish. Thebacterium is distributed widely in fresh water and bottom sediments containingorganic 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 Kooijand Hijnen, 1988; Sugita et al., 1994, Dumontet et al., 1996).

Infectious abdominal dropsy in common carp has been attributed to theA. hydrophila group (Aeromonas punctata) and was first described bySchäperclaus (1930), who reported on this condition in cultured, wild andstocked carp in central and eastern Europe. The causative agent has since beenshown 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 orsecondary invader (Austin and Austin, 1987).

Conversely, there have been reports of A. hydrophila acting as a primarypathogen in fish. Isolates differ greatly in their pathogenicity with some strainsbeing highly virulent and others non-virulent. Eddy (1960) and Kou (1972a)reported that non-virulent or weakly pathogenic strains did not produce gas andacetone from glucose. Wakabayashi et al. (1981) recognized common andidentical biochemical characteristics in virulent strains, in particular theproduction of elastase and enzymes involved in lysis of Staphylococcus. These

428 T. Aoki

characteristics were identical to the A. hydrophila biovar. hydrophila, followingclassification by Popoff and Véron (1976). Economically, the disease attributedto these bacteria is of greatest importance in cultured freshwater fish.

Recent advances in biochemistry, molecular biology and virulence factorsassociated with A. hydrophila have led to new understanding of this bacterialgroup. These are reviewed in this chapter.

THE DISEASE AND AGENT

Species of fish affected and geographical distribution ofAeromonas hydrophila

Most cultured and wild freshwater fish are susceptible to infection byA. hydrophila, but particularly cold-water fish, such as brown trout (Salmotrutta), rainbow trout (Oncorhynchus mykiss), chinook salmon (Oncorhynchustshawytscha), ayu (Plecoglossus altivelis), carp (Cyprinus carpio), channelcatfish (Ictalurus punctatus), clariid catfish (Clarias batrachus) (Fig. 11.2),Japanese eel (Anguilla japonica), American eel (Anguilla rostrata), gizzardshad (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 byAeromonas hydrophila (courtesy of Dr Teruo Miyazaki).

429Motile Aeromonads

The disease

Diseased fish usually display cutaneous haemorrhage of the fins and trunk, andthe 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 theintestine and induce a toxaemia. Capillary haemorrhage occurs in the dermis offins and trunk and in the submucosa of the stomach. Hepatic cells and epitheliaof renal tubules show degeneration. Glomeruli are destroyed and the tissuebecomes haemorrhagic, with exudates of serum and fibrin (Miyazaki and Jo,1985; Miyazaki and Kaige, 1985) (Figs 11.3–11.5).

European carp infected with A. hydrophila show severe tail and fin rot andvisible haemorrhage and ulceration of the body surface. Widespreadproliferation of bacteria is usually observed in the intestine. In some reports(Fijan, 1972; Wolf, 1988), the histopathological phenomena associated with therhabdovirus infection haemorrhagic septicaemia of carp have been erroneouslyattributed to motile aeromonads (Bullock et al., 1971). Aeromonas hydrophila iswidely distributed in the intestinal tract of cultured fish and the water andsediments of freshwater ponds which are rich in organic materials. Virulentstrains of A. hydrophila in these environments are possible sources of infection.Outbreaks of disease are usually associated with a change in environmentalconditions. Stressors, including overcrowding, high temperature, a suddenchange of temperature, rough handling, transfer of fish, low dissolved oxygen,

Fig. 11.2. Clariid catfish (Clarias batrachus) with ulcerative form of haemorrhagic septicaemiacaused by Aeromonas hydrophila (courtesy of Dr Kriengsag Saitanu).

430 T. Aoki

Fig. 11.3. Aeromonas hydrophila infection in Japanese eel. Intestine shows bacterialmultiplication in the contents and mucous–desquamative catarrh (haematoxylin and eosin stain)(courtesy of Dr Teruo Miyazaki).

Fig. 11.4. Aeromonas hydrophila infection in Japanese eel. Skin shows capillary haemorrhagein the dermal loose connective tissue and the thinned epithelium (haematoxylin and eosin stain)(courtesy of Dr Teruo Miyazaki).


poor nutritional status and fungal or parasitic infection, contribute to physiologicalchanges and heighten susceptibility to infection. This applies to fish of all ages.

Causative bacterium

There are three mesophilic motile aeromonad species in Bergey’s Manual ofSystematic Bacteriology: A. hydrophila, Aeromonas caviae and Aeromonassobria (Popoff et al., 1981; Popoff, 1984). Currently, the following additionalAeromonas 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 wasdetermined as a dendogram, using numerical taxonomy (Carnahan and Joseph,1993). Millership (1996) reviewed characteristics for the differentiation of thecommon phenotypes of these species in aeromonads. The genus Aeromonas hasbeen 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 Japanese eel. Kidney shows destroyed glomeruliaccompanying exudation of serum and fibrin. Epithelia of renal tubules show necrosis andatrophy (haematoxylin and eosin stain) (courtesy of Dr Teruo Miyazaki).

432 T. Aoki

Kou (1972a, 1973) and Wakabayashi et al. (1981) recognized that almost allpathogenic strains of motile aeromonads relevant to aquaculture wereencompassed within A. hydrophila biovar. hydrophila, proposed by Popoff andVéron (1976).

Aeromonas hydrophila is a Gram-negative rod-shaped bacterium and ismotile, due to a monotrichous polar flagellum. The bacterium measures 0.3–1.0µm in diameter and 1.0–3.5 µm in length. It has no spore stage or capsule. Theoptimum growth temperature is 28°C, but growth can occur at 37°C. Colonieson 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) +H2S from cysteine +Oxidase +Cytochrome oxidase +Catalase +Methyl red (MR) experiment dAcetylmethylcarbinol production +2,3-Butanediol production +2,3-Butanediol dehydrogenase +β-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 dArabinose dCellobiose dDextrin +Dulcitol –Fructose +Galactose +


and catalase reactions are positive. It is a facultative anaerobe, fermentingcarbohydrates to acid, or acid and gas. Aeromonas hydrophila is resistant to thevibriostatic agent O/129 (phosphate: 2,4-diamino-6,7-diisopropylpteridinephosphate) 150 µg, reduces nitrates to nitrite, is unable to grow in mediacontaining 6.5% sodium chloride (NaCl) and is generally resistant to ampicillinand carbenicillin. The guanine plus cytosine (G + C) content of the deoxy-ribonucleic acid (DNA) is 57–63% (MacInnes et al., 1979).

Aeromonas hydrophila contains thermostable O, thermolabile K andflagellar H antigens. Serologically, the O antigen of A. hydrophila is hetero-geneous (Sakazaki and Shimada, 1984; Janda et al., 1994, 1996). Differentserotypes have been observed from various sources of fish, isolated in differentyears and places (Eddy, 1960; Bullock, 1966). Interestingly, a common antigenhas been found among virulent strains (Kou, 1972b; Leblanc et al., 1981;Merino et al., 1996). Protein fingerprints do not correlate with biochemicalcharacteristics. Both phenotype and protein fingerprints show clustering ofepizootiologically 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-specificpolypeptides of the outer membrane from A. hydrophila, and Wilcox et al.(1992) suggested that outer membrane protein profiles were useful forconfirming the identity of A. hydrophila. Howard et al. (1993) cloned eight exegenes of extracellular protein secretion and membrane assembly fromA. hydrophila and found them to have a high similarity with the extracellularsecretion operons of a number of different Gram-negative bacteria. Shaw andHodder (1978) showed that O-polysaccharides were remarkably similar

Glucose +Glycerol +Glycogen +Inositol –Inulin –Lactose dMaltose +Mannitol +Mannose +Melezitose –Raffinose dRhamnose dSalicin dSorbitol dSorbose –Starch +Sucrose dTrehalose +Xylose –

+, Typically positive; –, typically negative; d, differs among strains; H2S,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, usingA. hydrophila ATCC7966 as a reference strain. The percentage homology DNAranged from 39 to 100%, with a mean value of 64.7%. Aeromonas hydrophiladoes not seem to show any significant divergence among the 17 strainsinvestigated. The 16S ribosomal DNA (rDNA) from ten species of Aeromonaswas sequenced to analyse relatedness (Martinez-Murcia et al., 1992b).Homology for 1052 nucleotides of 16S rDNAs from ten species of Aeromonasexhibited very high levels, ranging from 98 to 100%. The nucleotide sequencesof 16S rDNA of A. hydrophila are therefore useful in species identification. Eastand 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 phylogeneticanalysis (Ruimy et al., 1994).

Aeromonas hydrophila strains possessing a common plasmid, with amolecular size of 2.5–28 MDa, were isolated by Toranzo et al. (1983). Smallplasmids 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. hydrophilastrains from freshwater fish and fresh water (Noterdaeme et al., 1991). Norelationship between their virulence or biochemical characteristics and thepresence of plasmids was found (Brown et al., 1997).

DIAGNOSTIC METHODS

The gross signs of disease are haemorrhagic septicaemia and fin rot. Theaetiological agent can be grown on brain–heart infusion medium, tryptosoy agar(tryptone soya agar), nutrient agar and MacConkey agar with incubation at20–25°C for 24–48 h. Numerous selective media have been developed for theisolation and presumptive identification of A. hydrophila or motile aeromonads(Moyer, 1996), including Rimler–Shotts medium (Shotts and Rimler, 1973),modified peptone beef-extract glycogen agar (McCoy and Seidler, 1973),Rippey–Cabelli (membrane filter method (mA)) agar (Rippey and Cabelli,1979), MacConkey’s agar supplemented with trehalose (Kaper et al., 1981) andstarch–ampicillin agar (Palumbo et al., 1985). Davis and Sizemore (1981)reported that Rimler and Shotts medium and Rippey–Cabelli agar were notsuitable for A. hydrophila. Arcos et al. (1988) compared six media for selectiveisolation of A. hydrophila and showed that mA agar gave the best recovery rateand also an acceptable specificity, but its selectivity was low.

An API-20E test strip is used widely for identification of theEnterobacteriaceae (Kaper et al., 1979). Toranzo et al. (1986) indicated that theimportance of biochemical characteristics must be backed up by standardizedtesting.

For diagnosis, the aetiological agent is isolated on nutrient agar at 30°Cfrom kidney of a fish with haemorrhagic septicaemia. Isolates are motile


Gram-negative bacilli. Wakabongo et al. (1992) found that only four tests –aesculin hydrolysis, acetoin production, lysine decarboxylation and gas fromglucose – were sufficient to distinguish A. hydrophila, A. caviae and A. sobria.

A number of A. hydrophila-lytic bacteriopahges were isolated from riverwater, river mud, sewage and human clinical origin (e.g. stool, urine) and phagetyping was attempted (Chow and Rouf, 1983; Demarta and Peduzzi, 1984;Altwegg et al., 1988; Merino et al., 1990; Fukuyama et al., 1991, 1992). Acomprehensive phage-typing system for A. hydrophila will provide a useful toolfor 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-linkedimmunosorbent assay (Merino et al., 1993). These methods are of limited value,because many different serological types of A. hydrophila are distributed in fishfarms (Eddy, 1960; Bullock, 1966).

Lucchini and Altwegg (1992) differentiated ribotyping of restrictiongenomic DNAs of aeromonads, using different fragments of a 16S rDNA gene ofE. coli as a probe and achieved identification of most Aeromonas strains tospecies level. This method is easier than DNA–DNA hybridization, because onlyminimal amounts of genomic DNA are needed and several strains can beanalysed on a single gel. Pulsed-field gel electrophoresis is a rapid anddiscriminatory technique for the typing of A. hydrophila where a common originof infection is suspected (Talon et al., 1996).

Deoxyribonucleic acid probe hybridization technology is now becomingavailable for the direct detection and identification of microorganisms. Thecolony hybridization technique (Grunstein and Hogness, 1975) can be appliedeasily to identify and count the causative organism. Fish-pathogenic bacteria,such as Vibrio anguillarum (Hirono et al., 1996) and Pasteurella piscicida (Zhaoand Aoki, 1992a), can be detected by probe hybridization with their specificDNA nucleotide sequence or cryptic plasmid. However, there are many commonDNA fragments between A. hydrophila and Aeromonas salmonicida (Miyata etal., 1995), which makes it unlikely that this probe technique will be successfulfor this species.

The sensitivity obtained using hybridization with non-radiolabelled probesis lower than with radiolabelled probes (Zhao and Aoki, 1992a). However,radiolabelled probes are generally unacceptable for use in diagnosis and furtherdevelopment of detection procedures based on non-radiolabelling is stillrequired.

The polymerase chain reaction (PCR) (Mullis and Faloona, 1987) is usefulfor detecting fish pathogens from diseased fish and their environment. Thistechnique is available for A. salmonicida (Miyata et al., 1996), Edwardsiellatarda (Aoki and Hirono, 1995), P. piscicida (Aoki et al., 1997) andV. anguillarum (Hirono et al., 1996). However, recently, Cascón et al. (1996)found a specific PCR primer set for the detection of A. hydrophila hybridizationgroup 1.

436 T. Aoki

The DNA fingerprinting method, AFLP (amplified fragment lengthpolymorphism), is a valuable high-resolution genotype tool for classification ofAeromonas species (Huys et al., 1996).

CONTROL AND TREATMENT

Transmission of the disease

The aetiological agent is transmitted horizontally but not vertically. It isdistributed widely in water and sediments of ponds and can be transmitted bydischarge 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 derKooij and Hijnen, 1988; Sugita et al., 1994; Dumontet et al., 1996). Parasitedamage and fungal infection of the epidemics may allow entry and spread ofbacterial infection (Egusa, 1978).

Aeromonas hydrophila has been recognized as a pathogen not only ofamphibians, reptiles and snakes but also cattle and humans (Eddy, 1960;Khardori and Fainstein, 1988). It has been implicated as the causative agent ofclinical infections in humans, including septicaemia and peritonitis (Janda andAbbott, 1996). Recently, its role as a psychrotrophic spoiler of meat, seafood andvegetables has been recognized (Palumbo, 1996). No confirmed cases ofA. hydrophila food poisoning have been reported, but its association withgastrointestinal illness suggests that it plays a role in food-borne disease. Theepidemiological relationship among A. hydrophila isolated from fish, human andenvironmental resources is particularly difficult to assess.

Aeromonas hydrophila is cosmopolitan in distribution. Infection has beenseen 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 orsanitation is poor. Good hygiene, periodic drying and disinfection of ponds areimportant in prevention. Avoidance of overcrowding, low oxygen and roughhandling are the best methods of prevention.

Chemotherapy

Chemotherapeutic agents are used for the treatment of A. hydrophila in fishfarms (Aoki, 1992). Isolates of A. hydrophila have been found to be sensitive tochloramphenicol, florfenicol, tetracycline, sulphonamide, nitrofuran derivativesand pyridonecarboxylic acids (Aoki and Egusa, 1971; Endo et al., 1973; Kataeet al., 1979; Fukui et al., 1987). Fluorinated analogue, tetracycline derivatives,


nitrofuran derivatives, sulphonamide and pyridonecarboxylic acids are effectivein oral treatments (Austin and Austin, 1987). A review by the Fisheries Agencyof the Japanese government on chemotherapeutic use against bacterial infectionsin cultured fish has been carried out (Okamoto, 1992). The use of chlor-amphenicol and almost all nitrofuran derivatives in fish have been restricted inJapan since 1983.

The extensive use of antibacterial agents has led to an increase in resistantstrains of A. hydrophila. Drug-resistant strains carrying transferable R plasmidsappeared in 1971 in cultured eel and multiple resistant strains carrying Rplasmids are now distributed widely in cultured amago, ayu, carp and eel inJapan (Aoki et al., 1971, 1972; Aoki and Watanabe, 1973; Aoki, 1974, 1975).Almost all strains have intrinsic resistance to ampicillin, and drug-resistantstrains carried transferable R plasmid, which encoded resistance to sulpho-namide and tetracycline and to chloramphenicol, sulphonamide andstreptomycin (Akashi and Aoki, 1986). Drug-resistant strains have been isolatedfrom 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 culturedsnakehead (O. striatus) in Thailand (Aoki et al., 1990). The DNA of transferableR plasmids was classified into incompatibility (Inc) (Couturier et al., 1988)groups A–C, which had been detected in different areas, and resistance tosulphonamide and tetracycline and showed high homology with each other(Akashi and Aoki, 1986). R plasmids classified into Inc group U, with resistanceto chloramphenicol, sulphonamide and streptomycin, showed homology withina specific species and with R plasmids from A. salmonicida. Aoki (1988)demonstrated that A. hydrophila strains carrying R plasmids having identicalDNA structures are widely distributed in cultured freshwater fish in variousareas.

Tetracycline-resistance (tet) genes on the R plasmids of Gram-negativebacteria 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 ofthe R plasmids detected from A. hydrophila, which were isolated in Japan, wasclassified into Tet D class (Aoki and Takahashi, 1987). Class D and Etetracycline-resistance determinants were found in resistant strains ofA. hydrophila isolated in catfish ponds in the USA (DePaola and Roberts, 1995).A class D tetracycline-resistance determinant of RA1 from A. hydrophilacontained the resistance (tet A) and repressor (tet R) genes. The tet A geneencoded 394 amino acids, with a calculated molecular size of 41.1 kDa (Varelaand Griffith, 1993), and the tet R encoded 218 amino acids, with a calculatedmolecular size of 24.4 kDa (Unger et al., 1984).

The 40 MDa plasmid encoded with resistance to carbenicillin andnovobiocin 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. TheECP include a cytotoxin, enterotoxin, haemolysins, protease, haemagglutininand acetyl cholinesterase (Cahill, 1990; Gosling, 1996; Howard et al., 1996).Aeromonas hydrophila enters through the epithelium of the intestinal tract offish. Enterotoxins of A. hydrophila cause fluid to accumulate in ligated rabbitileal loops. Enterotoxins are divided into two types, cytotonic and cytotoxic.

Boulanger et al. (1977) isolated two different haemolysins, an α- and aβ-haemolysin, both of which have been implicated in the pathogenesis ofinfection. Allan and Stevenson (1981) investigated the production of proteaseand haemolysin in the ECP of A. hydrophila strains and showed a closecorrelation between the quantity of haemolysin and toxicity to fish. Twobiologically similar but immunologically distinct haemolysins were purified byAsao et al. (1986) and Kozaki et al. (1987, 1989). These haemolysins (cytotoxicenterotoxin) had enterotoxic activity and caused fluid accumulation in infantmice, in mouse intestine and in rabbit ligated ileal loops. Purified haemolysinalso displayed cytotoxicity to Vero cells and lethal toxicity to mice. Themolecular weight of the haemolysin was estimated at 48,000–50,000 kDa andbiological activity was inactivated with heating for 5 min at 65°C (Asao et al.,1984).

Cross-reaction of A. hydrophila haemolysin and cholera toxin has beenreported, and a specific synthetic oligonucleotide probe for regions of A + Bsubunits of cholera toxin was found to hybridize with chromosomal DNA fromsome strains of A. hydrophila. The enterotoxin purified by Rose et al. (1989) hada molecular size of 52,000, but only 25 residues of the N-terminal sequenceswere found to be identical to the haemolysin of A. hydrophila (aerolysin)(Howard et al., 1987). The amino acid residues differed significantly betweenenterotoxin and aerolysin over the vast majority of the protein. The structure ofproaerolysin was determined by X-ray crystallography at 2.8 Å (Parker et al.,1994). The aerolysin secretion system indicates that it is a haemolysin which isdirected outside the cell through periplasm (Wong and Buckley, 1993).

Five haemolysin genes (AHH1, AHH2, AHH3, AHH4 and AHH5) werecloned from A. hydrophila into a E. coli vector (Aoki and Hirono, 1991; Hironoand Aoki, 1991; Hirono et al., 1992). Each was classified into three groups,depending on their nucleotide sequences. AHH1 belonged to one group (group1), which contained part of a homologous sequence of the haemolysin genes ofVibrio cholerae El Tor and Vibrio vulnificus (Rader and Murphy, 1988). Therewere two highly conservative regions, and the location of the cysteine residuewas conserved. These regions may be important in the control of haemolyticactivity (Fig. 11.6). The other group (group 2) included AHH3, AHH4 andAHH5, 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 alsoclassified into group 2 by the nucleotide sequence. The remaining gene, AHH2,


is placed in group 3. From colony hybridization analysis, using the clonedhaemolysin genes, it was found that AHH1 (group 1) and AHH5 (group 2) werewidely distributed among aeromonads (Table 11.2). It is interesting that alltested strains of A. salmonicida have AHH1 and AHH5 genes. One strain of A.hydrophilahas two or three haemolysin genes (Hirono and Aoki, 1991; Hironoet al., 1992).

A significant qualitative as well as quantitative difference in the proteasecomponents of ECP was produced by A. hydrophila and A. sobria, which werepathogenic for fish (Nieto and Ellis, 1991). The role of protease in the virulenceof A. hydrophila is currently controversial. Wakabayashi et al. (1981) describedmost of the virulent strains of A. hydrophila biovar. hydrophila as having a highproteolytic activity. A single protease purified from A. hydrophila was lethal tocarp 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) alsoshowed that both virulent and weakly virulent strains were dermonecrotic in theguinea-pig, and a dermonecrotic factor was observed in sonicated cells of anA. hydrophila strain isolated from eel (Shimizu, 1968). Chabot and Thune

Fig. 11.6. Comparison of deduced amino acid sequences among the AHH1, ASH4 and Vibriocholerae El Tor haemolysin (Rader and Murphy, 1988). The same amino acid residues areindicated as • on the aligned sequences. VCH, V. cholerae El Tor haemolysin.

440 T. Aoki

(1991) characterized three proteases and found no correlation between eitherqualitative or quantitative protease production and virulence in age-0 channelcatfish. A metalloprotease with a molecular size of 38 kDa and a serineproteinase with a molecular size of 22 kDa was purified from A. hydrophilastrain B52. These were stable at 56°C for 10 min and had a lethal effect in fish,with a median lethal dose (LD50) of 150 ng g–1. Only the serine proteasepossessed 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.


metalloprotease and thermolabile serine protease. The relationship betweenthese and the two purified by Nieto and Ellis (1986) is not known, but they werealso lethal to fish. Protease from A. hydrophila enhanced haemolysin activity(Howard and Buckley, 1985), but Lallier et al. (1984) found that purifiedhaemolysin from A. hydrophila was not lethal to fish. A novel zinc-proteinasewas purified and characterized from A. hydrophila (Loewy et al., 1993), andRivero et al. (1990) cloned an extracellular protease gene from A. hydrophila.Aeromonas hydrophila strains, which had haemolytic activity, enterotoxinproductivity and cytotoxic ability, were not virulent for rainbow trout (Santos etal., 1988).

Yadav et al. (1992) noted that the fish cell lines, especially the BB (brownbullhead, Ictalurus nebulosus) cells were sensitive targets for A. hydrophilacytotoxins, even when assayed in conditions vastly different from those ofmammalian cells. Cytotoxin-producing strains were frequently associated withepizootic ulcerative syndrome (EUS)-infected fish, compared with healthy fish.Cytotoxic A. hydrophila strains have a role in the pathogenicity and progressionof 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 amolecular size of 68–70 kDa in A. hydrophila were induced under iron-limitingconditions. Enterobactin, the catecholate siderophore produced by a strain ofA. hydrophila (Andrus and Payne, 1983), and amonabactin, a novel phenolatesiderophore in A. hydrophila 495A2, were identified (Barghouthi et al., 1989,1991). Esteve and Amaro (1991) found the hydroxamate-type siderophoresproduced by A. hydrophila. The iron-uptake system was similar in A. hydrophilaand A. sobria isolated from European eels (Anguilla anguilla) and inA. salmonicida. These authors suggested that siderophore produced byA. hydrophila could be important and play a role in virulence for acquisition ofiron from the host. Recently, the amonabactins were synthesized and theirspectroscopic properties were elucidated (Telford et al., 1994).

The receptor cell surface of A. hydrophila can bind to iron-containingproteins – lactoferrin, transferrin, ferritin, cytochrome C and haemin – of thehost (Kishore et al., 1991; Ascencio et al., 1992), demonstrating a relationshipbetween lactoferrin binding and siderophore production by the bacteria. Acarbohydrate-reactive outer-membrane protein, which may contribute as anadhesive mechanism, was isolated from A. hydrophila strain A6 (Quinn et al.,1993, 1994). This protein is related to the colonization strategies of aeromonadsassociated with human enteric disease. However, the protein has not been provedto relate to pathogenicity in fish.

Two cytotonic enterotoxin genes were cloned from A. hydrophila; one of theenterotoxins was heat-labile at 56°C, while the other was heat-stable (Chopraet al., 1994).

The role of haemolytic activity and cytotoxic activity in contributing topathogenicity to fish still needs to be elucidated.

The attachment of a pathogen to the epithelial tissue is the first step in thehost disease process. The W pili of A. hydrophila strain Ae6 is the colonizationfactor for the intestine, as well as a haemagglutinin (Hokama and Nakasone,1990; Hokama et al., 1990). Haemagglutinating activity was also detected inA. hydrophila (Atkinson and Trust, 1980; Corral et al., 1990). Aeromonashydrophila 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 withvirulence in fish (Corral et al., 1990).

De Meuron and Peduzzi (1979) have suggested that the K-antigen is apathogenicity factor. Virulent strains of A. hydrophila possessed an S-layer onthe cell surface (Dooley et al., 1986; Dooley and Trust, 1988; Ford and Thune,1991, 1992).

Recently, Rodriquez et al. (1993) purified acetylcholinesterase from ECPof A. hydrophila and showed it to be lethal to fish. Glycerophospholipid: cholesterol acyltransferase (GCAT) and lipopolysaccharide complex enhancedthe lethal exotoxicity and cytolysin of A. salmonicida (Lee and Ellis, 1990). Agene encoding GCAT was cloned from A. hydrophila (Thornton et al., 1988).

Experimental vaccination for prophylaxis against infection of A. hydrophilahas been examined (Stevenson, 1988). Fish immunized either intramuscularly orintraperitoneally with vaccine showed protection against challenge. The


agglutinating antibody titre increased in the serum of immunized fish (Songet al., 1976; Ruangpan et al., 1986; Karunasagar et al., 1991).

Immersion vaccination of channel catfish using polyvalent sonicatedantigens of A. hydrophila provides protection (Thune and Plumb, 1982). Lamerset al. (1985) noted that agglutinating antibody was recognized in the serum ofcarp immunized with A. hydrophila bacterin, following a second immersion withthis vaccine. However, fish vaccinated by immersion or orally showedquestionable protection.

Catfish immunized intraperitoneally by injection with the acid extract of theS-layer protein of A. hydrophila were protected from the homologous, virulentstrain (Ford and Thune, 1992). Serological types of A. hydrophila areheterogeneous and a polyvalent vaccine is thought to be necessary forprevention of the infection.

TOPICS FOR FURTHER STUDY

There is significant interest in the pathogenicity of A. hydrophila to fish. Severalvirulence factors, including the production of endotoxin, ECP, siderophores andsurface proteins, and the ability of attachment to host cells, are under study, buttheir relative importance has not yet been elucidated. Cahill (1990) considersthat proteases are most important in fish pathogenicity, but further study onadhesins, siderophores and surface proteins is needed to understand their role inthe pathogenesis of disease caused by A. hydrophila.

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