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). 483 Edwardsiella Septicaemias 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). 485 Edwardsiella Septicaemias 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. 487 Edwardsiella Septicaemias 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, 489 Edwardsiella Septicaemias 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 491 Edwardsiella Septicaemias 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 492 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.) 493 Edwardsiella Septicaemias 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 494 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 495 Edwardsiella Septicaemias 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. 497 Edwardsiella Septicaemias 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. 498 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 499 Edwardsiella Septicaemias 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.) 500 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.) 501 Edwardsiella Septicaemias 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.) 502 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. 503 Edwardsiella Septicaemias 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 505 Edwardsiella Septicaemias 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. 507 Edwardsiella Septicaemias 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 509 Edwardsiella Septicaemias 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.) 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