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

16Other Bacterial Pathogens

J.G. Daly

Division of Natural Sciences, Purchase College, State University of New York,Purchase, New York, New York 10577, USA.

INTRODUCTION

Some bacteria are well-recognized as pathogens of fish and have received asubstantial amount of attention by scientists, as is evident from previouschapters. For other bacterial pathogens of fish, there is less informationavailable, either because the disease is rarely encountered or because it is anew and emerging problem that is only beginning to be studied. In othercases, it is not clear if the organism should be recognized as a pathogen offish or only an unusual concatenation of organism, host and environmentalstresses.

This chapter focuses on four disparate groups of bacteria, which can berecognized as fish pathogens, but about which there is not enough informationto warrant separate chapters. These groups are: Photobacterium damselasubsp. piscicida (formerly Pasteurella piscicida), the pseudomonads, members ofthe family Enterobacteriaceae other than Yersinia and Edwardsiella, andthe atypical lactobacilli, including Carnobacterium piscicola and Vagococcussalmoninarum. Various other Gram-positive and negative bacteria that haverecently been described as causing pathological conditions of fish aresummarized briefly in Table 16.1. A short review on newly identified bacterialfish pathogens has been published by Austin and McIntosh (1991).

A bias exists towards recognition of pathogens that affect those fish speciesreared in intensive aquaculture. Thus, the bacterial pathogens receivingindividual chapter attention are pathogens of salmonid fish (aeromonads,vibrios, Renibacterium), channel catfish (Edwardsiella and Aeromonas) andyellowtail (Enterococcus). Increased contact between fish and opportunity fortransmission are factors in this prejudice. Stress of numbers and cultureconditions will also increase effects attributed to the pathogen. Not only arepathogens more readily recognized in intensive aquaculture, but this isparticularly the case in regions where there is an infrastructure of diagnostic andpathology laboratories and fish health regulations and where the costs of such

578 J.G. Daly

infrastructure can be met. Warm-water aquaculture is probably underrepresentedin lists of significant pathogens.

Factors that may result in a changed status for a bacterium could includenew host species being introduced, more intensive and stressful conditions in

Table 16.1. Fish bacterial pathogens for which there is limited information.

Bacterial species Disease condition

Rhodococcus sp.Speare et al. (1991) Granulomatous lesions in Atlantic salmonBackman et al. (1990) Panophthalmitis in chinook salmonPlanococcusAustin et al. (1988); Gastroenteritis; slight swelling of the kidneyAustin and Stobie (1992a)Janthinobacterium lividumAustin et al. (1992) Mortalities in rainbow troutAcidovorax delafieldiiAznar et al. (1992) Eel, Anguilla anguilla pathogen?Unspeciated Gram-negativeFerguson et al. (1989) Visceral necrosis of goldfish, Carassius

auratusAcinetobacter sp.Roald and Hastein (1980) Infection in Atlantic salmonMoraxella sp.Baya et al. (1990a) Mortalities in striped bassMycoplasma mobile Gill isolate on tench, Tinca tinca L., with redKirchhoff and Rosengarten disease (1984)(1984); Kirchhoff et al. (1987)Bacillus mycoidesGoodwin et al. (1994) Mortalities in channel catfish, Ictalurus

punctatus

Criteria for ‘real pathogens’:

1. Disease situation in fish, with significant mortalities (acute or persistent)or substantial damage or effect (e.g. growth rate, appearance, behaviour).2. Problem recurs at different times and/or in different places (i.e. not a one-time event related to special stresses or environmental conditions. (A caveathere is that increasing awareness of the potential pathogenicity of abacterium often leads to increased numbers of diagnoses and an apparent‘explosion’ in occurrences of the disease.)3. A recognizable agent can be isolated or detected from disease situations.Consistency of isolates? Virulent and avirulent strains?4. Importance of predisposing stress and/or environmental factors forproduction of the disease.5. Predominant isolate (or sole isolate) from organs, blood and/or lesions. Asubclinical disease or a carrier state may be present when the same isolateoccurs in many fish in a tested group. (Tissue samples taken aseptically fromthe organs of healthy fish show relatively low numbers of culturableorganisms, even with enrichment steps.)6. Reinfection produces the disease (fulfilling Koch’s postulates). Someconcern with this criterion, as stress factors, route of infection (N.B. hole inthe head syndrome/Edwardsiella) and an appropriate level of exposure allneed to be taken into account. Loss of virulence with culture may be aconcern.


aquaculture, exposure to new sources of potential pathogens, and successfulprevention of infection by a previously important species or strain of pathogen.In dealing with rare or emerging bacterial pathogens of well-studied systems andhosts or in assessing the significance of pathogens in other hosts or aquacultures,it is important to designate the criteria that are used to recognize when abacterium should be designated as a ‘fish-pathogenic bacterium’, rather than anisolation incidental to disease. How often are ‘pathogens’ isolated as a result ofhigh loads of saprophytic bacteria (e.g. from sewage) meeting an unusuallysusceptible host or a host under unusually stressful conditions (e.g. Proteus)?What criteria should be used to determine the ‘status’ of a bacterium as a fishpathogen?

PHOTOBACTERIUM DAMSELA SUBSPECIES PISCICIDA(FORMERLY PASTEURELLA PISCICIDA)

Photobacterium damsela subsp. piscicida was known until recently asPasteurella piscicida (Gauthier et al., 1995). It is a Gram-negative rod whichcauses a disease in fish known either as pseudotuberculosis or fish pasteurel-losis. This serious problem in Japanese yellowtail culture can result in losses onindividual farms of up to 50%. The bacterium’s taxonomic position as a bonafidePasteurella has been questioned for a number of years and, based on small-subunit ribosomal ribonucleic acid (rRNA) sequences, whole deoxyribonucleicacid (DNA) relatedness and biochemical characterization, Gauthier et al. (1995)have reassigned the bacterium to a subspecies of P. damsela. In this review, Ihave adopted the newer, taxonomically valid name for the bacterium. Forhistorical reasons, the disease is still referred to as pasteurellosis.

The microorganism

Photobacterium damsela subsp. piscicida is a non-motile, Gram-negative rodthat exhibits bipolar staining and is oxidase- and catalase-positive, fermentative,sensitive to the vibriostatic agent O/129 (150 µg disc–1), does not produce gasfrom glucose and is halophilic (Janssen and Surgalla, 1968; Magarinos et al.,1992a). It differs from Pasteurella species because it does not grow at 37°C, it ishalophilic and it does not reduce nitrate. Magarinos et al. (1992a) provide auseful table for differentiating Photobacterium damsela subsp. piscicida fromthe common fish pathogens Vibrio anguillarum and Aeromonas salmonicidasubsp. salmonicida, as well as Pasteurella spp. Further biochemicalcharacteristics of this species are shown in Table 16.2.

Magarinos et al. (1992a) have found that isolates from several Europeancountries, Japan and the USA are biochemically and antigenically similar, withhomogeneous lipopolysacharide (LPS) electrophoretic patterns and membraneprotein profiles. Romalde et al. (1995) also reported that the fatty acid profilesare very similar. Sakai et al. (1993) found that all of their six Japanese isolateshad hydrophobic surfaces and agglutinated sheep red blood cells.

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Species of fish affected and geographical distribution

Photobacterium damsela subsp. piscicida has been isolated from diseased fish inthe USA (Janssen and Surgalla, 1968), Japan (Kimura and Kitao, 1971), Taiwan(Tung et al., 1985) and, most recently, Spain (Toranzo et al.,1991), France and

Table 16.2. Characteristics of isolates of Photobacterium damsela subsp.piscicida (from Janssen and Surgalla, 1968; Yasunaga et al., 1983, 1984;Toranzo et al., 1991).

Gram stain – D-Tartrate –Bipolar staining + Gelatinase –Cell morphology Short rods Caseinase –Motility – Lipase (Tween 80) +Growth on nutrient agar + Phospholipase +

In nutrient broth + Amylase –In peptone water + Haemolysis:On heart-infusion agar + Sheep erythrocytes –On BHI agar + Salmon erythrocytes –On SS agar – Acid production from:On MacConkey agar – Glucose +On Endo agar – Mannose +

Growth at 5°C – Galactose +10°C – Fructose +15°C + Maltose –25°C + Sucrose –30°C + Rhamnose –37°C – Arabinose –

Growth in 0% NaCl – Amygdaline –Growth in 0.5% NaCl + Melibiose –Growth in 3% NaCl + Mannitol –Growth in 5% NaCl – Inositol –Cytochrome oxidase + Sorbitol –Catalase + Glycerol –Methyl red + Xylose –Voges–Proskaur + Lactose –Indole production – Trehalose –Nitrate production – Raffinose –Ammonium production – Cellobiose –Citrate production – Dextrin –H2S – Inulin –O/F F Glycogen –Gas from glucose – Adonitol –Arginine dihydrolase + Inositol –Lysine decarboxylase – Dulcitol –Ornithine decarboxylase – Erythritol –Tryptophan deaminase – Salicin –β-Galactosidase (ONPG) – Aesculin –Urease –Phenylalanine deamination –Gluconate utilization –

H2S, hydrogen sulphide; OPNG, o-nitrophenyl β-D-galactopyranoside.


Greece (Baudin Laurencin et al., 1991; Bakopoulos et al., 1995), Italy (Ceschiaet al., 1991; Doimi et al., 1991), Portugal (Baptista et al., 1996) and Norway(Speilberg et al., 1991). The bacterium has been isolated from numerous speciesin both wild and farmed fish, as summarized in Table 16.3.

The disease

Photobacterium damsela subsp. piscicida causes a disease in fish known eitheras pseudotuberculosis or fish pasteurellosis. It is characterized by the presenceof numerous, white bacterial colonies throughout the internal viscera, parti-cularly the spleen and kidney (Fukuda and Kusuda, 1981). This disease is veryserious in Japanese yellowtail culture, causing losses in individual farms of up to50%. In Japan, the disease seems to occur in the early summer, when watertemperatures are 20–25°C. Similarly, in a recent outbreak in Spain watertemperatures were 25°C and the outbreak was in the middle of the summer(Toranzo et al., 1991).

This is one of the few fish pathogens that can cause massive mortalities inwild fish. Severe epizootics in white perch and striped bass populations occurredon the eastern seaboard of the USA in 1963 and 1977. Landings of white perch inthe year after the 1963 epizootic were only 42% of those of any of the previous 3years. The only significant cause for this decrease in the fishery appears to have

Table 16.3. Isolations of Photobacterium damsela subsp. piscicida from fish.

Fish source Citation

White perch Roccus americanus Snieszko et al. (1964)Striped bass Morone saxatilis Snieszko et al. (1964)Menhaden Brevoortia tyrannus Lewis et al. (1970)Striped mullet Mugil cephalus Lewis et al. (1970)Yellowtail Seriola quinqueradiata Kimura and Kitao (1971)Ayu Plecoglossus altivelis Kusuda and Miura (1972)Black sea bream Mylio macrocephalus Ohnishi et al. (1982)Red sea bream Acanthopagrus schlegeli Yasunaga et al. (1983,

1984)Oval file fish Navodan modestus Yasunaga et al. (1983,

1984)Snakehead fish Channa maculata Tung et al. (1985)Red grouper Epinephelus okaara Ueki et al. (1990)Gilthead sea bream Sparus aurata Toranzo et al. (1991)Turbot Scophthalmus maximus Toranzo et al. (1991)Sole Solea solea Baudin Laurencin et al.

(1991)Mullet Mugil cephalus Baudin Laurencin et al.

(1991)Sea bass Dicentrarchus labrax Baudin Laurencin et al.

(1991)Atherine Atherina boyeri Ceschia et al. (1991)Sea bream Pagrus pagrus Ceschia et al. (1991)Atlantic salmon Salmo salar Speilberg et al. (1991)

582 J.G. Daly

been the Pasteurella epizootic in 1963 (Sindermann, 1990).Little is known about the transmission of the disease; however, the

bacterium does not appear to survive in sea water for periods of greater than 3–5days (Janssen and Surgalla, 1968; Toranzo et al., 1982). This suggests that directfish-to-fish contact is required for maintaining the infection in a population offish. Magarinos et al. (1994a) have recently reported that the bacterium maysurvive in sea water in a ‘viable but not culturable’ state. Although these starvedcells were not culturable by traditional methods, they were still virulent forturbot.

Diagnostic methods

Photobacterium damsela subsp. piscicida can grow on most bacteriologicalmedia, provided that there is added sodium chloride (NaCl) to a finalconcentration of 0.5%. The identification of the bacterium is typically based onits biochemical and morphological characteristics. Table 16.2 shows thecharacteristics of P. damsela subsp. piscicida strains isolated from varioussources. Kent (1982) reported on the usefulness of the API 20E identificationsystem. Photobacterium damsela subsp. piscicida gives positive reactions forarginine dihydrolase and glucose, galactose, mannose and fructose, while allother tests are negative. The usefulness of the fluorescent antibody technique fordetecting the bacterium in diseased tissues has been reported by Kitao andKimura (1974) and Kawahara et al., (1986). A DNA hybridization probe usefulboth for bacterial colony identification and for directly detecting the pathogen infish has been described (Zhao and Aoki, 1989). The probe was able to detect 3.9ng of purified DNA and 105 cells of P. damsela subsp. piscicida. The authorswere successful in detecting the pathogen in spleen and kidney tissues that hadbeen smeared on to nitrocellulose filters but did not report on the sensitivity ofthis method. Zhao and Aoki (1992) have also reported on the use of a 5.1 kbplasmid probe for rapid detection and identification of the bacterium.

Control and treatment

ChemotherapyAoki and Kitao (1985) described the antibiotic susceptibility of Japanese strainsof the bacterium to 14 chemotherapeutic agents. All strains were susceptible toampicillin and several strains were resistant to several antibiotics. Of 60 strainstested, 21 were resistant to furazolidone and five were resistant to chlorampheni-col, tetracycline, kanamycin, furazolidone and sulphamonomethoxine.Transferable R plasmids were detected in these latter five strains, with markersfor chloramphenicol, tetracycline, kanamycin and sulphamonomethoxine.Takashima et al. (1985) reported on drug-resistant strains from various districtsof Japan. Of 281 strains, 262 were resistant to combinations of chloramphenicol,tetracycline, kanamycin, ampicillin, nalidixic acid, furazolidone and/or sulpha-monomethoxine and R plasmids were detected in 168 strains. In contrast to


previous results (Aoki and Kitao, 1985), 41 ampicillin resistant strains wereisolated and a transferable R plasmid was also detected.

Yasunaga and Yasumoto (1988) reported that florfenicol was effective intreating experimentally induced pseudotuberculosis when administered in feedor given as a single oral dose. Oxolinic acid, ampicillin and sodium nifur-styrenate were not effective. Toranzo et al. (1991) have recently reported the oraladministration of chloramphenicol and oxytetracycline for the control of anepizootic of gilthead sea bream in Spain.

ImmunotherapyFukuda and Kusuda (1985) used three different vaccine preparations againstpseudotuberculosis in yellowtail: a formalin-killed whole cell bacterin, an LPSvaccine and a culture supernatant fluid containing components of the bacteriathat had undergone autolysis. The fish were challenged by gastral adminis-tration. The vaccines were administered by either immersion or by spray. Thebest protection was observed with the LPS vaccine, followed by the precipitatedculture fluid vaccines. There was 87% protection in the LPS vaccinates versus40% in the controls (immersion method) and 80% protection in the LPSvaccinates and 30% in the controls (spray method). The authors found thatvaccinated fish had higher specific antibody titres in serum and mucus, butunfortunately did not perform passive transfers of antibody to show thatantibodies were able to protect fish against pseudotuberculosis.

Kusuda and Hamaguchi (1988) compared the efficacy of three bacterins inpreventing pseudotuberculosis. These were composed of: (i) a formalin-killedbacterin; (ii) bacteria heated to 75°C for 1 h; and (iii) an attenuated live bacterin.All were delivered by the immersion method. The best protection was with theattenuated bacterin (25% mortality) and the formalin-killed bacterin (57%mortality), while controls showed a 81% mortality. No significant protectionwas achieved with the heated bacterin. Both the attenuated live bacterin and theformalin-killed bacterin increased the phagocytic activity of the fish, with theformer giving the highest phagocytic activity.

Kusuda et al. (1988) reported that yellowtail could be effectively protectedagainst an immersion challenge of the bacterium after immunization with P.damsela subsp. piscicida ribosomal antigen P or S. The protection achieved wasgreater than that obtained in animals that were immunized with bacterial LPS.

Using the fluorescent antibody technique, Kawahara and Kusuda (1988)histologically examined the tissue localization of antigens (formalin-killedbacteria, sonicated cells and LPS) given by immersion. After the fish wereimmersed in antigen for 5 min, the antigens were detected in the skin from 0 to60 min, skin and stomach for 0–12 h, in the intestine from 30 min to 3 days, inthe heart from 60 min to 6 h and in the liver, spleen and kidney from 6 to 24 h.Their results suggested that the main antigen uptake was via the gills.

Magarinos et al. (1994b) compared the efficacy of two vaccine preparations,a whole-cell bacterin and a toxoid-enriched whole-cell vaccine. No protectionwas demonstrated with the whole-cell bacterin and limited (41% relative percent survival) protection was demonstrated in gilthead sea bream with thetoxoid-enriched preparation.

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Pathogenesis and immunity

Little is known about the virulence mechanisms of the bacterium. The fact that itproduces granulomatous lesions during chronic infections (Toranzo et al., 1991)suggests that it is able to reside within phagocytic cells of the host. Magarinos etal. (1992b) have shown that P. damsela subsp. piscicida extracellular products(ECP) possess phospholipase activity and haemolytic activity for sheep, salmonand turbot, but not trout, erythrocytes. Gelatinase, caseinase and elastaseactivities differed among the strains. Skarmeta et al. (1995) found that bothvirulent and avirulent strains of the bacterium were killed by rainbow trout, seabass and gilthead sea bream macrophages, in an in vitro assay. Bonet et al.(1994) have recently demonstrated that a capsule is produced when thebacterium is grown under glucose-rich conditions.

Currently, this bacterium has become a problem in warm-water Europeanmariculture. Control of the disease via vaccination is required, as the bacteriumreadily becomes resistant to antibiotics. Where the bacterium resides in thenatural environment is also of concern, as well as determining the importance ofcarrier fish. The possibility of viable but non-culturable forms must also beexamined.

PSEUDOMONAS SPECIES

Taxonomy of pseudomonads

Pseudomonads exist throughout the aquatic environment and are associated withboth healthy (Evelyn and McDermott, 1961; Bullock and Sniesko, 1969) anddiseased fish. It is generally believed that these bacteria can be opportunisticpathogens or produce damaging secondary infections. For example, when carpare infected with A. salmonicida ssp. nova, the causative agent of carperythrodermatitis, both pseudomonads and motile aeromonads are readilyisolated from the internal viscera as the disease progresses (Evenberg et al., 1988).

The term ‘pseudomonads’ and the genus name Pseudomonas have beenused rather broadly for aerobic Gram-negative rods. The taxonomic relation-ships of Pseudomonas anguilliseptica are not yet defined, while [Pseudomonas]putrefaciens has been reassigned to the genus Shewanella (MacDonell andColwell, 1985).

Pseudomonas fluorescens caused white nodules in the spleen and abscessesin the swim-bladder of tilapia (Sarotherodon niloticus) (Miyashita, 1984).Natural mortalities were highest when water temperatures were 15–20°C.Intramuscular injections of the bacteria caused mortalities and pathology similarto those observed in naturally infected fish. Granulomas were found in thespleen, liver and kidney (Miyashita et al., 1984). The same bacterium has beenreported to cause mortalities in 2-week-old tilapia fry (Duremdez and Lio-po,1985). This bacterial genus has also been reported to cause disease in a numberof other species, including goldfish (Carassius auratus) (Bullock, 1965), tench(Tinca tinca) (Ahne et al.,1982) and rainbow trout (Li and Fleming, 1967; Li and


Traxler, 1971). Bullock et al. (1965) reported on the biochemical character-ization of a number of isolates from diseased fish in the USA.

Pseudomonas (Alteromonas) putrefaciens was identified as the causativeagent of a disease outbreak in cultured rabbit fish (Siganus rivulatus) in the RedSea (Saeed et al., 1987). Moribund fish were discoloured, had haemorrhagicnecrosis on the body and mouth and exhibited exophthalmia and frayed fins. Theauthors were able to reinfect and kill fish when 109 bacteria were injectedintraperitoneally into healthy rabbit fish (Saeed et al., 1987, 1990).

Pseudomonas chlororaphis has been reported to cause high mortalities inAmago trout (Oncorhynchus rhodurus) in a single hatchery in Japan (Hatai etal., 1975), where fish exhibited large amounts of ascitic fluid and haemorrhagingin various parts of the body.

Pseudomonas pseudoalcaligenes was isolated from skin lesions on rainbowtrout infected with Yersinia ruckeri type I (Austin and Stobie, 1992b).Experimental injection of 105 cells of P. pseudoalcaligenes, either intra-peritoneally (i.p.) or intramuscularly (i.m.), resulted in total mortailities within 7days. Intramuscular injection resulted in some muscle liquefaction around theinjection site.

Pseudomonas anguilliseptica

Species of fish affected and geographical distributionOf all the pseudomonads that have been isolated from fish, the most informationis available about P. anguilliseptica. This bacterium was first described as theaetiological agent of ‘Sekiten-byo’, or red-spot disease of Japanese eels(Anguilla japonica), by Wakabayashi and Egusa (1972). Since that time it hasbeen isolated from European eel (Anguilla anguilla) (Stewart et al., 1983), blacksea bream (Acanthopagrus schlegeli) (Nakajima et al., 1983), ayu (Plecoglossusaltivelis) (Nakai et al., 1985c), Atlantic salmon (Salmo salar), sea trout (Salmotrutta), rainbow trout (Oncorhynchus mykiss), whitefish (Coregonus sp.) andBaltic herring (Clupea harengus membras) (Wiklund and Bylund, 1990; Lonn-strom et al., 1994; Wiklund and Lonnstrom, 1994), gilthead sea bream (Sparusaurata), sea bass (Dicentrarchus labrax), turbot (Scophthalmus maximus)(Berthe et al., 1995) and possibly the giant sea perch (Lates calcarifer) and theestuarine grouper Epinephelus tauvina) (Nash et al., 1987). Experimentalchallenges have caused mortalities in bluegill sunfish (Lepomis macrochirus),goldfish (Carassius auratus), carp (Cyprinus carpio) and loach (Misgurnusanguillicaudatus) (Muroga et al., 1975). The disease has been found in Japan(Wakabayashi and Egusa, 1972), Scotland (Stewart et al., 1983), Taiwan (Nakaiet al., 1985b) and, most recently, in Finland (Wiklund and Bylund, 1990) andFrance (Berthe et al., 1995).

The diseaseThe disease was first known as ‘Sekiten-byo’, or red-spot disease of Japaneseeel, where it can cause great losses of cultured fish. Clinical signs of the diseaseappear to be the same in all species affected, namely petechial haemorrhages of

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the skin, peritoneum and liver. The kidneys can be severely affected, with someliquefactive necrosis (Wakabashi and Egusa, 1972; Ellis et al., 1983; Wiklundand Bylund, 1990).

The microorganismPseudomonas anguilliseptica is a Gram-negative motile rod, cytochromeoxidase-positive, producing no acid from glucose (negative in Hugh Liefson’sO/F medium), is negative for indole, arginine dihydrolase, lysine decarboxylase,and ornithine decarboxylase, but utilizes citrate and usually produces gelatinase.The Finnish isolates, unlike others described to date, do not hydrolyse Tween 80.The bacteria grow slowly on tryptone soya agar (TSA), forming round, greyish,convex colonies of 1 mm diameter after 3–4 days of incubation at 25°C. Thebacteria will grow at temperatures ranging from 4 to 30°C (no growth at 37°C)and at salinities of 0–4%. Stewart et al. (1983) have estimated the mol % guanineplus cytosine (G + C) content of the type strain NCMB 1949 to be 57.4% andtheir Scottish isolate to be 56.5 mol % G + C. (Accepted ranges are 55–64% forPseudomonas vs. 38–40% for pseudomonads reclassified as Alteromonas.) Thewhole-cell fatty acid analysis of 79 strains demonstrated that all were related tobut distinct from other pseudomonads (Nakajima et al.,1983).

Three different serotypes of P. anguilliseptica have been identified, basedon the presence or absence of a heat-labile K antigen. There are a K+ and a K–

serotype isolated from Japanese eels (Nakai et al., 1981) and a separate K+

serotype from ayu (Nakai et al., 1985b). The Finnish isolates are similar to theserotype isolated from ayu (Wiklund and Bylund, 1990). Outer-membraneprotein patterns appear to be useful for showing the similarity among strains(Nakajima et al.,1983).

Diagnostic methodsThe bacterium will grow on basic laboratory media, such as brain–heart infusion(BHI) agar, TSA and blood agar, but would not grow on Pseudomonas isolationagar (Difco laboratories) (Wiklund and Bylund, 1990). The bacterium can bereadily identified by slide agglutination with specific rabbit serum, but it isimportant to dilute the serum 1:10 to prevent cross-reactions with otherpseudomonads or Vibrio anguillarum (Nakai et al., 1981; Toranzo et al., 1987).

Control and treatmentThe disease can be effectively eliminated in eels by raising the watertemperature to 26–27°C (Muroga et al., 1975; Stewart et al., 1983). This methodof control cannot be applied to rainbow trout or other fish species that cannottolerate these high temperatures. It is postulated that this treatment is effectivebecause the bacterium cannot grow at these temperatures and that the bacteria aremore quickly lysed by normal eel serum at 28°C than at 20°C (Nakai et al., 1985a).

Wiklund and Bylund (1990) attempted to treat Atlantic salmon withoxytetracycline (75 mg kg–1 for 8 days), but this had only a limited effect. Theseauthors found that the most effective drugs in vitro were ampicillin andtrimethoprim/sulphamethoxazole.


Pathogenesis and immunityNakai and Muroga (1979) studied the immune response of Japanese eels toformalin-killed P. anguilliseptica bacterin, with or without Freund’s completeadjuvant. The fish were twice injected i.m., with a 1 week interval, at doses of 1mg 100 g–1 body weight. Higher serum agglutinating titres were obtained in thefish receiving the bacterin with adjuvant. Fish vaccinated, held in fresh water at19–25°C and then placed in outdoor tanks for the winter were refractory to anartificial challenge 5 months later. Since this disease is most prevalent in thespring, vaccination seems to be a good method for preventing the disease.

ENTEROBACTERIAL PATHOGENS

The most important members of the family Enterobacteriaceae that arepathogenic to fish are the enteric redmouth disease agent, Y. ruckeri (see Chapter12) and two species of Edwardsiella, E. tarda and E. ictaluri (Chapter 13). Thereare only a few reports suggesting that other enterics may be pathogenic for fish.However, recent reports suggest that they may represent an emerging problem.Serratia, Proteus and Citrobacter have all been implicated as potential fishpathogens. These bacteria share some common characteristics with othermembers of the Enterobacteriaceae: they are small Gram-negative rods,catalase-positive and oxidase-negative and produce acid from glucose byoxidative and fermentative metabolism. Motile species have peritrichousflagella and most reduce nitrate to nitrite. Further biochemical characterizationcan be carried out using the various commercial test strips that are available.

Proteus (Providencia)

Proteus rettgeri (reclassified as Providencia rettgeri in the 1984 Bergey’sManual, Penner, 1984) was isolated consistently from the internal organs andlesions of silver carp (Hypophthalmichthys molitrix) in Israel (Bejerano et al.,1979). The disease appeared as an acute bacterial septicaemia, with severemortalities occurring within 3 days when pond water temperatures were 20°C orhigher, compared with 8 days at lower temperatures. Some ulceration andmuscle degeneration were observed and there were large, red and often deepulcerative lesions on the external surface. Intramuscular injection of the isolatesof P. rettgeri or exposure of scarified fish to bacteria produced mortalities andlesions, although with a lower level of mortality and less severe lesions than innatural infections.

Serratia

Two species of Serratia, S. liquefaciens and S. plymuthica, have been associatedwith bacterial septicaemia and mortalities in salmonid fish. Serratia lique-faciens was the predominant isolate recovered from dead and dying Atlantic

588 J.G. Daly

salmon in Scottish marine cages (McIntosh and Austin, 1990). The internalorgans were affected, particularly the kidney, spleen and liver, but there wereexternal signs of infection. Serratea liquefaciens was also a common isolatefrom the internal organs of lake trout (Salvelinus namaycush) and brook trout(Salvelinus fontinalis) from an Ontario, Canada, hatchery with a low butinconsistent level of mortalities (S. Lord, 1991, unpublished observations). Anaxenic culture of the bacterium has also been isolated from an ulcerative skinlesion on a wild rock bass (Ambloplites rupestris) (J. Daly, 1985, unpublishedobservations). In all these cases, the results of biochemical tests on the isolatesclosely resembled those reported for S. liquefaciens (Grimont and Grimont,1984). However, the Atlantic salmon isolate was difficult to identify initially,because its anomalous oxidase reaction and arrangement of flagella suggestedthat it was an aeromonad (McIntosh and Austin, 1990). When injected intoAtlantic salmon, an isolate from Atlantic salmon caused pronounced damage tothe musculature at the site of injection and rapid mortalities (72 h for an infectivedose of 103 bacteria). Rainbow trout also showed muscle and internal organnecrosis when injected with 107 bacteria, but mortalities did not occur (McIntoshand Austin, 1990). Extensive, spreading muscle necrosis can appear when S.liquefaciens is injected subcutaneously into rainbow trout (S. Lord, 1991,unpublished observation), perhaps due to the action of the many exoenzymesproduced by this bacterium.

Serratia plymuthica, a red pigmented species, was isolated repeatedly frommoribund rainbow trout fingerlings in a Spanish hatchery (Nieto et al., 1990).There were no external signs of disease and the organism was detected afterbacteriological culture of the organs. A mean lethal dose (LD50) of 105 cells wasreported for i.p. injections into rainbow trout and again there were signs of tissuedamage from the challenge. Austin and Stobie (1992b) isolated S. plymuthicafrom skin lesions on rainbow trout which were otherwise infected with Y. ruckeritype I. Experimental injection of 104 cells of S. plymuthica per fish, either i.m. ori.p., resulted in total mortality within 7 days. The bacterium caused severeerosion of the skin and musculature.

Baya et al. (1992) have recently shown that Serratia marcescens could alsopotentially be a fish pathogen. These authors isolated the bacterium from thekidneys of healthy white perch (Morone americanus). The bacterium has anLD50 of 5 × 103 for rainbow trout and 1 × 105 for striped bass, regardless ofwhether the bacteria were given i.p. or i.m. However, the fish died sooner whenthe i.m. route was used. The LD50 of the ECP was 0.8 µg protein g–1 fish forstriped bass and 0.4 µg protein g–1 fish for rainbow trout.

Other enteric bacteria

Based on the above reports, some species of Serratia and Proteus appear to beopportunistic pathogens of fish. Not only have they been found as thepredominant isolate from diseased fish but they also produce disease in fishexperimentally exposed to them. Other species of enteric bacteria, notablyCitrobacter freundii (Sato et al., 1982; Baya et al., 1990b; Sanz, 1991;


Karunasagar et al., 1992) and Enterobacter agglomerans (Hansen et al., 1990),have been suggested as possible fish pathogens, on the basis of isolations frominfections and mortalities. However, further evidence is needed to substantiatethese species as more than incidental pathogens. Toranzo et al. (1994) found thatnine fish isolates were of low virulence (LD50 of greater than 5 × 107) but couldestablish a carrier state within the fish. As enterobacteria are ubiquitous in theenvironment, it is not surprising that some should be opportunistic pathogens offish. They would present a particular risk in warm-water ponds that arefrequently exposed to faecal material, such as silver carp ponds regularlyfertilized with poultry manure (Bejerano et al., 1979) or other polluted waters. Acase in point is the recent report of a C. freundii infection of carp fingerlings.This may have resulted from a contaminated water-supply after the onset ofthe monsoon (Karunasagar et al., 1992). Baya et al. (1992) have reportedthat fish living in river water that receives city sewage carry a significantlyhigher number of bacterial species in comparison with fish from less pollutedwaters.

ATYPICAL LACTIC ACID BACTERIA

This section discusses atypical lactic acid bacteria that have been implicated inpathological conditions of fish. Lactic acid bacteria have been shown to mediatedisease in fish that have undergone some kind of stress and are usuallyassociated with postspawning mortalities. Ross and Toth (1974) first reportedthe condition of pseudokidney disease associated with a Lactobacillus sp. Hiu etal. (1984) classified some isolates into a new species, Lactobacillus piscicola,and, later, Collins et al. (1987) reclassified L. piscicola into a new genus,Carnobacterium piscicola. Most recently, Wallbanks et al. (1990) haveclassified some fish lactic acid bacteria into the new genus and species,Vagococcus salmoninarum.

Species of fish affected and geographical distribution

Bacteria belonging to the genus Lactobacillus or Carnobacterium have beenisolated from the following species of fish with disease conditions: rainbow trout(O. mykiss) (Cone, 1982), lake trout S. namaycush (Daly and Stevenson, 1986),cutthroat trout (Salmo clarki) and chinook salmon (Oncorhynchus tshawytscha)(Hiu et al., 1984), brown trout (S. trutta) and carp (C. carpio) (Michel et al.,1986), striped bass (Morone saxatilis) and channel catfish (Ictalurus punctatus)(Baya et al., 1991). The bacterium has also been isolated from brown bullheads(Ictalurus nebulosus) that showed no signs of disease (Baya et al., 1991). Thebacterium is widely distributed, having been isolated in Canada, the USA,Australia, Belgium and France (Cone, 1982; Hiu et al., 1984; Michel et al.,1986; Humphrey et al., 1987).

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The disease

A septicaemic condition of fish, known as pseudokidney disease, was firstreported by Ross and Toth (1974) as caused by Lactobacillus sp. These authorsand Cone (1982) reported the disease only in female fish. Cone (1982) reportedthe disease in ‘overripe’ fish that had not lost all their eggs after spawning. Thebacterium was isolated from kidney and ascites. Gram staining revealed largenumbers of Gram-positive bacteria both in ascites and within eggs. Herman et al.(1985) reported that the disease was in both sexes of fish. The fish hadhaemorrhaging at the base of the fins, and some males had hyperaemic skinlesions. Internally, many fish had ascites, pale livers and haemorrhages of theintestine and gonads and some organs had grey membranes.

The microorganisms

Lactic acid bacteria have been reported from fish since first being described byEvelyn and McDermott (1961). Hiu et al. (1984) classified some isolates into anew species L. piscicola, and, later, Collins et al. (1987) reclassified L. piscicolaand Lactobacillus carnis (which is isolated from meat) into a new genusCarnobacterium as C. piscicola. The bacteria are Gram-positive, non-spore-forming rods, occurring singly or in chains. They do not produce catalase orcytochrome oxidase, do not reduce nitrate and do not produce indole, urease,gelatinase, lysine decarboxylase, ornithine decarboxylase, but they possessarginine dihydrolase. All strains characteristically do not grow on acetatemedium. Strains also differ in carbohydrate fermentation. The reader is referredto the articles by Hiu et al. (1984), Michel et al. (1986), Baya et al. (1991),Starliper et al. (1992) and Toranzo et al. (1993) for further description ofbiochemical profiles.

Members of the genus Carnobacterium differ from true lactobacilli only intheir ability to grow on acetate media and their synthesis of oleic acid rather thancis-vaccenic acid (Collins et al., 1987). Most recently, Wallbanks et al. (1990)have classified some fish lactic acid bacteria into the new genus and species,Vagococcus salmoninarum. Schmidtke and Carson (1994) examined 13 isolates ofV. salmoninarum from Australia and Norway and compared them with the isolatesdescribed by Wallbanks et al. (1990). They amended and extended the descriptionof the bacterium and suggested that V. salmoninarum could be phenotypicallyseparated from Lactococcus piscium and C. piscicola, using tests for hydrogensulphide (H2S) production and α-haemolysis of sheep red blood cells, and byexamining for the production of acid from a battery of carbohydrates. Toranzoet al. (1993) have suggested a series of tests to help differentiate Lactobacillussp., C. piscicola, Carnobacterium divergens, V. salmoninarum and L. piscium.These include growth on acetate agar, production of arginine dihydrolase, acidproduction from mannitol and inulin, H2S production and starch hydrolysis.

Other lactic acid bacteria, such as those described by Starliper et al. (1992)and Austin and van Poucke (1993), are different from the known, describedspecies of fish-pathogenic lactic acid bacteria and have yet to be speciated.


Diagnostic methods

These bacteria can be readily isolated on TSA or BHI agar (Hiu et al. 1984;Herman et al., 1985; Baya et al., 1991). Fermentation patterns can be quicklydetermined with the API 50 CHI test strips. Michel et al. (1986) and Baya etal. (1991) found that weak reactions with the API 50 CHI were difficult toread. Carnobacterium piscicola can be separated from the relatedC. divergens by the fermentation patterns for inulin and mannitol.Carnobacterium piscicola ferments both of these, while C. divergens doesnot (Montel et al., 1991). Baya et al. (1991) showed that a limited number ofCarnobacterium strains isolated from fish were serologically heterogeneous.These workers also showed that there were differences in outer-membraneprotein profiles of their isolates.

Williams and Collins (1992) have designed synthetic oligonucleotideprobes specific for the genus Vagococcus. Brooks et al. (1992) have reported onthe use of the polymerase chain reaction (PCR) and oligonucleotide probes,designed, from 16S rRNA sequence data, for the detection and identification ofCarnobacterium from meat. One of their probes is specific for C. piscicola/Carnobacterium gallinarum. Specific probes, based on their 16S rRNAsequences, could not be designed to differentiate these two species, because ofthe 99% 16S rRNA sequence relatedness of the two species (Brooks et al.,1992). To overcome this, alternative methods need to be developed.

A note of caution is warranted when diseased animals are examinedhistologically using the Brown and Brenn Gram stain. One should be aware thatthe very important pathogen Renibacterium salmoninarum is also Gram-positive. This latter bacterium, however, is smaller in length.

Control and treatment

There is no information available on transmission of these atypical lactobacilli.Baya et al. (1991) found that their Carnobacterium isolates were resistant toantibiotics in common use in aquaculture, such as oxytetracycline, quinolones,nitrofurans and potentiated sulphonamides. Michel et al. (1986), Baya et al.(1991) and Toranzo et al. (1993) have reported that their isolates were sensitiveto erythromycin in vitro.

Pathogenesis and immunity

Ross and Toth (1974), Cone (1982), Herman et al. (1985), Michel et al. (1986)and Baya et al. (1991) have all reported difficulty in transmitting the disease byi.p. injection of the bacterium. Either another challenge route is necessary or thebacterium only causes disease when the animals are stressed or at a certain age.Baya et al. (1991) reported that the LD50 for the reference strain, ATCC 35586,given i.p. to rainbow trout was 2.0 × 106 cells, whereas striped bass wereresistant to the bacterium at doses of 8.0 × 107. Every strain tested created a

592 J.G. Daly

carrier state in the kidney of fish that survived the i.p. challenge. This was truefor both striped bass and rainbow trout. Toranzo et al. (1993) reported a strainfrom rainbow trout that was unusually virulent. This isolate had an LD50

<5 × 106 after either an injection or a water-borne challenge.

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