魚 病 研 究 Fish Pathology,33(4),229-238,1998.10

Cytophaga, Flavobacterium, Flexibacter and Chryseobacterium Infections in Cultured Marine Fish

J.-F.Bernardet

Unite de Virologieet ImmunologieMoleculaires, Centre de Recherches INRA,78352 Jouy-en-Josascedex, France

(Received February 2,1998)

Many cases of fin or tail rot, skin ulcer, and jaw erosion were reported over the years in cultured marine fish. The aetiological role of various bacteria belonging to the Flavobacterium-Cytophaga group was frequently suspected, but only 3 bacterial species pathogenic for marine fish were fully described until now. Flexibacter maritimus was first identified in Japan in the late seventies from several fish species reared in sea water. Subsequently, the disease was recognized in very different geographic areas such as France, Scotland, Spain, Malta, Tasmania and California. The pathogenicity of the bacterium was demonstrated experimentally, phenotypic and genomic characteristics as well as virulence mechanisms were investigated, and treatment methods were proposed. Significant losses due to Flexibacter maritimus may occur locally. In Norway, Flexibacter ovolyticus was isolated from the adherent bacterial epiflora of Atlantic halibut eggs and was shown to be an opportunistic pathogen for halibut eggs and larvae. Chryseobacterium scophthalmum (first described under the epithet Flavobacterium scophthalmum) was isolated 10 years ago in Scotland from turbot suffering from gill hyperplasia and haemorrhagic septicaemia. A large collection of strains was studied, and investigations of the disease included experimental infection, histopa thology and immunization trials. Until now, Flexibacter ovolyticus and Chryseobacterium scophthalmum have not been recognized in fish species or regions other than those from which they were initially isolated.

Key words : Cytophaga, Flexibacter, Flavobacterium,Chryseobacterium, marine fish

Over the years, many authors have reported losses attempts for isolating these organisms were frequently among feral or cultured marine fish involving bacteria unsuccessfUl. Until now, only 3 bacterial species patho- belonging to the Flavobacterium-Cytophaga group. genic fbr marine fish have been isolated, fully described These conditions are worldwide and may be economi and named: Flexibacter maritimus (Wakabayashi et al., cally significant, locally. They have been described in 1986),Flexibacter ovolytic〃5(Hansen et al.,1992), and many different fish species, anadromous salmonids m scophthalmum(Mudarris et al., Chryseobacteriu raised in sea water as well as strictly marine species, cul 1994). tured in net-pens or concrete tanks (Devesa et al., 1989). In this review, recent taxonomic modifications con- Diseased fish usually exhibit severe necrotic lesions, ceming these bacteria will first be presented. Some such as fin and tail rot, skin ulcers, stomatitis or jaw examples of diseases in marine fish related to unidenti erosion, but septicaemiamay also occur. In many cases, fied bacteria belonging to this group will then be given, the aetiological role of Cytophaga-, Flexibacter-, or as well as more detailed informations about the 3 named Flavobacterium-like organisms was suspected because bacterial species. Lastly, the pathogenicity of some numerous long, slender, Gramnegative rods exhibiting Cytophaga-and Flexibacter-like organisms fbr molluscs gliding motility were observed in the lesions. However, and crustacea will be considered.

Address for correspondence:Jean-FrangoisBernardet, Unit6 de Virologieet lmmunologie Mol6culaires, Centre de RecherchesINRA, Taxonomic background 78352 Jouy-en-Josascedex, France. Facsimilenumber:33134652591. E-mail address:jfb@biotec. Following extensivephylogenetic investigations jouy.inra.fr based on the study of rRNA sequence,menaquinone 230 J. F. Bernardet content, as well as fatty acid and whole-cell protein pro genus (i.e. S. myxococcoides) is a cellulolytic soil files (Nakagawa and Yamasato, 1993; Bernardet et al., organism (Reichenbach, 1989) phylogenetically related 1996), the taxonomy of the Flavobacterium-Bacteroides to the genus Cytophaga as recently emended (Bernardet phylum (also called rRNA superfamily V), to which the et al., 1996). Flavobacterium-Cytophaga group belongs, has gone through considerable modifications. Among other "Flavobacterium "-like organisms taxa, these modifications particularly concerned the Several bacteria phenotypically similar to members family Flavobacteriaceae (Bernardet et al., 1996) and of the genus Flavobacterium (as it was defined before related genera such as Cytophaga and Flexibacter. As the recent emendation restricted it to soil and freshwater a consequence of the very isolated position occupied by organisms) have been recognized from diseased marine the type species of these 2 genera within the superfam fish. From a yellowish slime on halibut skin, Harrison ily, the genus Cytophaga was restricted to 2 cellulolytic (1929) isolated Chryseobacterium (Flavobacterium) soil organisms (Nakagawa and Yamasato, 1996) and F. balustinum, but this bacterium has later been considered flexilis was considered the type and only species within a fish spoilage agent rather than a pathogen (Austin and the genus Flexibacter (Nakagawa and Yamasato, 1993; Austin, 1987). However, a very similar organism was Bernardet et al., 1996). Hence, all other bacterial later isolated from the heart blood of a dace (Leuciscus species previously included in these genera had to be leuciscus) exhibiting signs of haemorrhagic septicaemia reclassified. For instance, the freshwater fish patho in a French river (Brisou et al., 1959). As no strain gens Flexibacter (Cytophaga) columnaris, Flexibacter from halibut was preserved, the dace isolate became the (Cytophaga) psychrophilus, and Cytophaga johnsonae type strain of C. balustinum. All Chryseobacterium were allocated to the emended genus Flavobacterium, species being highly tolerant to salt (Bernardet, unpub together with Flavobacterium branchiophilum (Bernardet lished data), it is possible C. balustinum was isolated et al., 1996). The turbot pathogen Flavobacterium from both a marine and a freshwater fish. Very scophthalmum (Mudarris et al., 1994) was transferred recently, this bacterial species was also found in the to the new genus Chryseobacterium, together with intestines of healthy chum salmon (Oncorhynchus keta) several other nongliding organisms previously consid (Morita et al., 1997). ered Flavobacterium, such as F. balustinum (Vandamme During mass mortalities associated with granuloma et al., 1994). Flexibacter maritimus and F. ovolyticus tous lesions in various marine fish species from Florida should also be reclassified in a new genus, but their in 1951, an organism was isolated from sea water and generic epithet was maintained pending further taxo named "Flavobacterium piscicida" (Bein, 1954). Sub nomic investigations (Bernardet et al., 1996). Conse sequent investigations led to the reclassification of the quently, all fish pathogenic bacterial species within bacterium as "Pseudomonas piscicida" (Buck et al., rRNA superfamily V are now included in the family 1963), but it was then listed among the incertae sedis of Flavobacteriaceae, but only some of them exhibit a the genus Alteromonas to which it phenotypically gliding motility. resembles (Baumann et al., 1984). A phylogenetic study finally resulted in the allocation of this species to the new genus Pseudoalteromonas (Gauthier et al., Unidentified or doubtful pathogenic organisms 1995). Mortality could have resulted from a simulta "Sporocytophaga" sp. neous phytoplankton bloom ("red tide") rather than the Bacteria exhibiting microcyst-like structures have bacterium (B. Austin, personal communication), but a occasionally been observed in diseased marine fish, but certain toxicity of liquid cultures and their filtrates was identification of the pathogen is doubtful as no strain indeed demonstrated for several fish and crab species was ever isolated and very scant information is avail (Hansen et al., 1965). able (Pacha and Ordal, 1970; Wood, 1974; Austin and The most recent description of a "Flavobacterium" Austin, 1987). The most recent case report concerned like organism pathogenic for marine fish deals with Atlantic salmon smolt suffering from severe jaw ero a bacterium isolated during the summer of 1978 sion within 6 to 8 weeks of introduction into the ocean from coho salmon reared in sea water in Galicia, Spain (Hicks, 1989). Affiliation of the bacteria observed in (ACUIGROUP, 1980). The outbreak caused 20-25% fish lesions to the genus Sporocytophaga is most un mortality with signs of haemorrhagic septicaemia, and likely as the type and only valid species within this the bacterium was isolated from kidney and liver. Cytophaga/Flexibacter in marine fish 231

Clinical signs and 100% mortality resulted from intra rium involved in the Washington outbreak was not iso venous injection of a bacterial suspension, but the au lated (Frelier et al., 1994), and the organism causing the thors surmised that the virulence of the bacterium was disease in British Columbia was erroneously identified probably revealed by several factors such as high water as "Sporocytophaga" based on microscopic observation temperature, high density of fish population, and severe only (Hicks, 1989) (see above). A yellow organism fouling of cages with algae and mussel larvae. Generic growing on media containing seawater or at least 1% identification of the isolate was based on a rather scant NaCl was recovered from the lesions of diseased coho phenotypic study, and motility alone was enough to salmon (Sawyer, 1976), whereas the bacterium isolated prevent its assimilation to a "Flavobacterium", even from Atlantic cod was also able to grow on a freshwater according to the then definition of the genus. medium (Hilger et al., 1991). It is thus likely the bac teria involved in these cases were not identical, in spite "Cytophaga/Flexibacter" -like organisms of the similarity of gross lesions. Numerous reports of filamentous gliding bacteria in The other kind of clinical cases was also characterized diseased marine fish have been published, but their study by ulcerative lesions, but mainly localized to the skin was insufficient to result in a definite identification, even and fins. They occurred in penreared Atlantic salmon when they were successfully isolated (Austin and Aus smolts on the coast of Washington State, USA (Kent et tin, 1987). Only a selection of the most recent and al., 1988) and in cultured turbot (Scophthalmus maxi detailed reports is discussed below. On the basis of mus) in the North West of Spain (Devesa et al., 1989). predominant gross lesions, these cases may be divided White foci and patches appeared on the caudal peduncle into 2 different groups. and posterior region of the flanks in salmon and on the In most cases, the lesions consisted of severe and flanks, the operculum, and the base of dorsal and caudal extensive erosion and ulceration of the nose, mouth and fins in turbot. These lesions increased in size and pro jaws (Sawyer, 1976; Hicks, 1989; Hilger et al., 1991; gressively transformed into severe skin ulcers exposing Frelier et al., 1994). Destruction usually involved skin, the underlying muscle, sometimes concerning extensive integument, and cartilage, but bones, especially of the areas on moribund fish (Kent et al., 1988; Devesa et al., jaws, could also be damaged and teeth were often lost 1989). Masses of filamentous bacteria exhibiting glid (Hilger et al., 1991; Frelier et al., 1994). Infected tissue ing motility were revealed by microscopic examination was frequently haemorrhagic (Hilger et al., 1991) and of infected samples. In the case of diseased turbot, some haemorrhage was also occasionally noticed at the these bacteria were only observed in the intermediate base of the fins and along the intestine, demonstrating zone of the lesions, whereas the external and central that the infection was septicaemic (Hicks, 1989). Yel zones mainly yielded a ciliate protozoa and a halophilic low layers of bacterial growth were observed on ulcer Vibrio sp., respectively. In both cases, high mortality ated areas (Sawyer, 1976; Frelier et al., 1994), which occurred shortly after introduction into sea water, and were occasionally characterized by distinct yellow the outbreak in turbot paralleled a sudden increase in nodules consisting of necrotic areas surrounded by col water temperature (Devesa et al., 1989). The causal lagenous fibres (Hilger et al., 1991). These outbreaks agent of turbot mortality was not isolated. Lesions in of ulcerative lesions limited to the head and causing Atlantic salmon yielded yellow rhizoid colonies of a sea high mortality were mostly observed on salmon reared water-dependent gliding bacterium which shared many in net-pens in North America, namely coho salmon characteristics with Flexibacter maritimus but proved (Oncorhynchus kisutch) in a Maine estuary, USA (Saw serologically distinct. Lesions similar to those observed yer, 1976), and Atlantic salmon (Salmo salar) smolt in during the natural disease were induced by inoculating British Columbia, Canada (Hicks, 1989) and in Wash lightly abraded skin zones with a pure culture of the bac ington State, USA (Frelier et al., 1994). In these 3 terium (Kent et al., 1988). cases, epizootics typically occurred within a few weeks Another yellow gliding bacterium resembling F. following introduction to sea water, and in one case maritimus was involved in high mortality in Japanese losses were particularly severe after water temperature flounder (Paralichthys olivaceus) fry cultured in Korea suddenly increased (Sawyer, 1976). A similar disease but, contrary to this bacterial species, the Korean isolate was also observed in young Atlantic cod (Gadus was able to grow in a medium devoid of sea water; NaCl morhua) caught during a survey on fish diseases in the alone was even not necessary to growth (Kim and Lee, German Wadden Sea (Hilger et al., 1991). The bacte 1995). No mortality was obtained by immersion of 232 J. F. Bernardet flounder fry in a bacterial suspension, whereas intra culture of Dover sole (Solea solea) in Scotland was hin muscular injection resulted in mortality up to 100%. dered by outbreaks of "black patch necrosis", an infec tious disease causing skin and fin necrosis (McVicar and Flavobacterium columnare White, 1979 and 1982). The F. columnare-like organ Flavobacterium columnare (previously known as ism isolated from diseased Dover sole (Campbell and Cytophaga columnaris or Flexibacter columnaris) is the Buswell, 1981 and 1982) was later identified as F. agent of columnaris disease in many freshwater fish maritimus by DNA homology studies (Bernardet et al., worldwide (Austin and Austin, 1987). It has also long 1990). The same bacterium was subsequently recog been suspected to be responsible for mortality in marine nized in diseased sea bass (Dicentrarchus labrax) fish suffering from similar conditions (Nigrelli and farmed along the Mediterranean coast in France (Pepin Hutner, 1945; Borg, 1948; Bullock et al., 1971). Sev and Emery, 1993; Bernardet et al., 1994), Malta eral authors have later studied the effects of sodium and (Tabone, 1996), and Greece (A. Le Breton, personal other ions on the survival of F. columnare (Kingsbury, communication). At about the same time, disease 1964; Fijan and Voorhees, 1969; Chowdhury and problems attributable to this pathogen threatened the Wakabayashi, 1988). They all demonstrated that most culture of turbot as well as Atlantic and coho salmon in strains are unable to tolerate more than 0.5% NaCl, and Galicia, a province located on the Atlantic coast of Spain thus cannot survive in sea water. (Alsina and Blanch, 1993; Pazos et al., 1993 and 1996). In Tasmania, Australia, F. maritimus was reported from several cultured fish including Atlantic Flexibacter maritimus salmon, rainbow trout (Oncorhynchus mykiss), green After outbreaks of a disease caused by a gliding back flounder (Rhombosolea tapirina), and striped trum bacterium in red (Pagrus major) and black sea bream peter (Latris lineata) (Soltani and Burke, 1994; (Acanthopagrus schlegeli) cultured in Japan were Handlinger et al., 1997). F. maritimus was also identi reported (Masumura and Wakabayashi, 1977), the fied along the southern coast of California from chinook bacterium was isolated and the authors announced their salmon (Oncorhynchus tschawytscha) reared in net-pens intention to make a separate formal proposal of the name as well as from captured white seabass (Atractoscion "Flexibacter marinus" (Hikida et al., 1979). However, nobilis), Pacific sardine (Sardinops sagax), and north this organism was eventually formally published under ern anchovy (Engraulis mordax) (Chen et al., 1995). the epithet Flexibacter maritimus, the strain NCIMB In Chile, F. maritimus is suspected to be responsible of 2154 being designated as the type strain (Wakabayashi disease problems in Atlantic salmon and turbot (P. et al., 1986; Holmes, 1992). The organism was listed Bustos and R. Enriquez, personal communications), but as Cytophaga marina in the Bergey's Manual of Sys the isolates have not been fully identified and no confir tematic Bacteriology with strain NCIMB 2153 as the mation has been published yet. This review of pub type strain (Reichenbach, 1989), but the priority of the lished case reports clearly shows that F. maritimus is name Flexibacter maritimus and of the type strain able to infect many species of anadromous and marine NCIMB 2154 was later recognized (Holmes, 1992). fish worldwide. Phenotypical characteristics were extensively studied Several predisposing factors influencing the outbreaks (Wakabayashi et al., 1984; Baxa et al., 1986) and DNA and the severity of the losses were identified, such as investigations further demonstrated the homogeneity of high water temperature (Wakabayashi et al., 1984; this taxon (Baxa et al., 1987a; .Bernardet and Grimont, Handlinger et al., 1997), recent transfer of fish from 1989; Bernardet et al., 1990 and 1994). tanks to net-pens (Wakabayashi et al., 1984; Pepin and For several years, the disease seemed restricted to Emery, 1993), absence of a layer of sand in the tanks Japan. F. maritimus was isolated from the body sur (McVicar and White, 1982), existing gill pathology face, skin lesions, or kidney of several fish species other related to poor feeding management and water quality than sea breams, such as Japanese flounder, leather (Handlinger et al., 1997), and physical trauma due to jacket (Aluterus monoceros), rock bream (Oplegnathus net abrasion, cannibalism, or skin parasites (Chen et al., fasciatus), plaice (Cleisthenes pinetorum herzensteini), 1995). puffer (Takifugu rubripes), and yellowtail (Seriola Gross lesions exhibited by diseased fish have been quinqueradiata) (Baxa et al., 1986; Wakabayashi et al., extensively described (e.g. McVicar and White, 1979; 1986; Baxa, 1988). During the early eighties, the Campbell and Buswell, 1982; Wakabayashi et al., 1984; Cytophaga/Flexibacter in marine fish 233

Baxa et al., 1986; Baxa, 1988; Alsina and Blanch, 1993; Wakabayashi et al., 1984; Baxa et al., 1986; Bernardet Pazos et al., 1993; Chen et al., 1995; Handlinger et al., and Grimont, 1989; Bernardet et al., 1990 and 1994; 1997). An overall similarity was noticed in the pathol Pazos et al., 1993; Alsina and Blanch, 1993; Soltani and ogy in salmonid and nonsalmonid species (Handlinger Burke, 1994; Chen et al., 1995). The Gramnegative et al., 1997). However, some variations in the nature, rods, usually filamentous in external lesions and much siting, and extend of lesions were found with regard to shorter in the kidney as well as in liquid and solid cul the species (Chen et al., 1995) and age of fish (Baxa, tures, display an active gliding motility on wet surfaces. 1988). Usually, lesions begin as white or yellowish Round degenerative forms called spheroplasts appear patches or small blisters involving the head, mouth, fins, within a few days. Colonies are flat, rhizoid, yellowish, and flanks. Local scale loss results in haemorrhagic mucoid and rather adherent to the agar; they adsorb dermal erosion, frequently covered with a layer of bac Congo red but no change in colour occurs when they are teria. In severe cases, eroded zones subsequently flooded with 20% KOH. Different types of colonies, evolve into deep skin ulcers, jaw erosion, or fin rot. varying in their rhizoid aspect and their adherence, may Necrotic gill lesions are not common, but were reported coexist on the same agar plate (Sorogon et al., 1991; in chinook salmon (Chen et al., 1995) as well as Atlan Bernardet, unpublished observations). A strong pro tic salmon and rainbow trout (Handlinger et al., 1997). teolytic activity is exhibited but no simple or complex Eyes are occasionally affected (Handlinger et al., 1997). polysaccharide is known to be degraded. Histopathology findings include many filamentous Median lethal doses (LD50) of extracellular enzymes, bacteria adhering to the eroded surface and extending exotoxin and endotoxin were determined following deeply into the connective tissue, as well as congestion, intraperitoneal injection in red and black sea bream fry, petechial haemorrhages, and necrosis. A considerable and in vitro activity of each toxin was also measured consistency in histopathology was noticed, as well as a (Baxa et al., 1988a). Injection of precipitated extracellular remarkable lack of inflammatory response in mature product (ECP) or haemolysin caused high lesions (Baxa, 1988; Chen et al., 1995; Handlinger et mortality and severe pathological signs, whereas no al., 1997). mortality occurred in crude or pure lipopolysaccharide Routine subculture of F. maritimus is relatively easy, (LPS) or proteaseinjected fish. However, simulta whereas isolation of the organism from diseased fish is neous injection of protease and crude LPS resulted in not always successful although many bacterial cells are 40% mortality. A definite toxic factor could not be commonly observed in smears (Handlinger et al., 1997; identified in this study but ECP and haemolysin are prob Bernardet, unpublished observations). Moreover, F. ably responsible for the pathogenicity of F. maritimus. maritimus is usually mixed with several other bacterial Moreover, all strains tested were shown to adhere species within the lesions and most of them produce strongly to the skin mucus of 3 fish species and to resist substances that inhibit its growth (Pazos et al., 1996). its bactericidal properties (Magarinos et al., 1995). The Although external lesions are most common, a septicae skin is thus a possible portal of entry for the bacterium. mia may occur in severely affected fish and the bacte Experimental infection was reported by many authors rium may then be isolated from the kidney (Alsina and but comparison of their results is uneasy because differ Blanch, 1993). Among the 75 strains included in one ent fish species, routes of inoculation, and concentra study, 13% were recovered from the kidney and 87% tions of bacteria wereused. On the whole, intramuscular from superficial lesions (Baxa, personal communica and intraperitoneal injections are not considered effica tion). F. maritimus is a strictly marine organism; ad cious (Wakabayashi et al., 1984; Alsina and Blanch, dition of NaCl only to the medium does not allow its 1993; Pepin and Emery, 1993). Similarly, bath chal growth as it requires at least 30% sea water (Wakabayashi lenge is not a reliable method of inducing the disease et al., 1986). Several media were compared for the (Wakabayashi et al., 1984; Baxa et al., 1987b), unless growth of F. maritimus; while some media proved ap the skin was previously scarified or abraded (Tabone, propriate for the routine culture, a new medium inhibi 1996) or the bacterial strain was first passaged twice in tory for most heterotrophic marine bacteria and most fish (Handlinger et al., 1997). On the contrary, serious effective for the recovery of F. maritimus from diseased lesions and high mortality result from subcutaneous fish was devised (Pazos et al., 1996). injection (Campbell and Buswell, 1982; Baxa et al., Phenotypical characteristics of F. maritimus have b; Pepin and Emery, 1993) and from topical appli1987 been detailed in many publications (Hikida et al., 1979; cation of F. maritimus cultures (Wakabayashi et al., 234 J. F. Bernardet

1984; Tabone, 1996). drugs were recently banned from use. Efficacy of sev Because the isolation of F. maritimus is fastidious, eral antibiotics for F. maritimus was recently tested in diagnostic of the diseases caused by this organism is vitro and in vivo (Soltani et al., 1995). The best corre frequently limited to the observation of filamentous glid lation between the 2 methods occurred for amoxycillin ing bacteria within the lesions. However, definite iden and trimethoprim which both produce adequate serum tification of the bacterium is only possible after it has levels when given as a bath or orally to Atlantic salmon been isolated on agar so that at least a limited number of and rainbow trout. Amoxycillin is clinically effica morphological and biochemical characteristics may be cious against the disease, but trimethoprim is even more evidenced. Polyclonal antisera have been raised against protective. F. maritimus, and all strains tested were shown to share a common antigen which may thus provide a rapid iden Flexibacter ovolyticus tification of the bacterium (Wakabayashi et al., 1984). This was later confirmed on a larger number of strains Because of its restricted distribution, Flexibacter and 6 serotypes were established by agglutination and ovolyticus has received much less scientific attention. adsorption tests using antisera raised against 2 different However, commercial success of Atlantic halibut strains (Baxa, 1988). However, Pazos et al. (1993) (Hippoglossus hippoglossus) farming in Norway has found that whole-cell protein and lipopolysaccharide long been hampered by serious losses during the early analyses did not support the existence of antigenic di stages of life (Hansen et al., 1992). In the early nine versity among F. maritimus isolates. Recently, a pair ties, these mortalities were shown to be correlated with of specific primers was designed by comparison of the the dominant presence of Flexibacter sp. among the 16S rRNA sequence of F. maritimus and 23 related bac adherent egg epiflora, resulting in ulcerations colonized terial species. Polymerase chain reaction (PCR) using by large numbers of bacteria. Within most of these these primers resulted in the amplification of the 16S lesions, the chorion is dissolved and the zona radiata is rDNA of all F. maritimus strains tested whereas no other damaged (Bergh et al., 1992). Experimental infection bacterial species was amplified (Toyama et al., 1996). of eggs using Flexibacter sp. further demonstrated its When adapted to infected biological samples, this role in the process (Bergh et al., 1992). After the eggs method will provide a very sensitive detection of the were exposed to the bacterium, halibut yolk sac larvae bacterium. show reduced activity and increased mortality, as well It seems that immunization of fish against F. maritimus as increased density (Skiftesvik and Bergh, 1993). has not been investigated. Protection could be difficult These responses are not found when turbot is similarly to achieve since, at least in the case of Dover sole infec exposed. Extensive phenotypic and genomic studies tion, "in any subsequent outbreak, survivors appeared of 35 strains isolated from halibut eggs and larvae to be equally susceptible" (McVicar and White, 1979). resulted in the description of a new bacterial species, F. Because the host response is prevented by exotoxins, it ovolyticus (Hansen et al., 1992). Many characteristics was the opinion of Handlinger et al. (1997) that "any resemble that of F. maritimus, but DNA studies show efficacious vaccine would need to include this toxin". that halibut isolates form a tight genomic species with Routine formalin treatments are effective in control low DNA relatedness with F. maritimus (Hansen et ling "black patch necrosis" in Dover sole (McVicar and al., 1992; Bernardet, unpublished data). The rather White, 1979), and flush treatments with potassium fastidious Gramnegative rods display gliding motility permanganate are used to control disease outbreaks in and produce pale yellow colonies on marine media. F. penreared sea bass in Malta (Le Breton, personal com ovolyticus is considered an opportunistic pathogen, caus munication). Pharmaceuticals that contain hydrogen ing losses in halibut eggs and larvae when becoming peroxide are presently used for controlling F. maritimus dominant among their microflora or when their resis infections in cultured turbot in France. The minimal tance is weakened by unknown factors (Hansen et al., inhibitory concentration of a range of antibiotics was 1992). determined in vitro, revealing the high sensitivity of F. maritimus to furans, penicillin, ampicillin, and erythro Chryseobacterium scophthalmum mycin; its sensitivity to oxytetracycline was lower (Baxa et al., 1988b). Bath treatments with furans controlled The first members of a new bacterial species were the disease (Baxa et al., 1988b; Tabone, 1996), but these recovered in Scotland during investigations of the Cytophaga/Flexibacter in marine fish 235 microflora of wild turbot and coastal water samples, and C. scophthalmum has not been recovered yet from similar organisms were subsequently isolated in 1987 other fish species and other location than those from from diseased turbot during an outbreak in a local farm which it was initially isolated. (Mudarris and Austin, 1989; Mudarris, 1989). The bacterium caused gill hyperplasia and haemorrhagic Cytophaga and Flexibacter infections in marine septicaemia. Diseased turbot exhibited haemorrhages molluscs and crustacea in the eyes, skin, and jaw, as well as several internal organs. Interestingly, bacteria similar to those cited above Histopathology of the disease was studied (Mudarris have also been shown to be pathogenic for cultured shell and Austin, 1992) and extensive phenotypic investigations fish (Elston, 1989;Lavilla-Pitogo, 1995). Filamentous were conducted on a large group of strains isolated in Gram-negative bacteria attributed to these genera may pure culture from the gills and internal organs (Mudarris heavily colonize gills and body surface of diseased and Austin, 1989). The organism was first considered penaeid shrimps (Lightner, 1988; Lavilla-Pitogo, 1995 a Cytophaga after the presence of a gliding motility was and references therein), but no strain was isolated. suspected on fresh isolates, but this character was not Serious mortalities and hinge ligament erosion were confirmed after storage. The Gram-negative rods, described in a variety of cultured juvenile bivalve mol characterized by a thick cell wall, produce round, raised, luscs (Elston, 1989). Electron microscopy investiga orange colonies on agar. Growth occurs on marine tions of eroded ligament revealed the presence of a dense media and also on media devoid of NaCl or sea water, population of bacteria which were subsequently shown demonstrating that the bacterium is halotolerant and not to possess enzymes degrading the ligament. Based obligately marine. This trait, together with all other upon phenotypical and genomic characteristics, the phenotypic and genomic characteristics, led to the clas isolates were considered Cytophaga spp (Dungan et al., sification of the new species in the genus Flavobacte 1989). rium (Mudarris et al., 1994). Following phylogenetic investigations, it was subsequently transferred to the new This review has shown that the culture of many ma genus Chryseobacterium (see above). rine fish and shellfish worldwide is seriously hampered Experimental infection was performed injuvenile tur by various Chryseobacterium, "Flexibacter" and bot, immersion and intraperitoneal injection both result "Cytophaga" species . Few bacterial species have been ing in high mortalitieswith typical lesions. Interestingly, extensively studied and identified to date, and it is likely young rainbow trout kept in fresh water were also suc several new species should be described in the future. cessfully infected by the same method (Mudarris and Austin, 1989). Control of the disease was possible by References injection or bathing with furazolidone, but sulphafurazole ACUIGRUP(Laboratorio de IctiopathologiaAcuicultura proved much less efficacious. As C. scophthalmum is Bioter,Madrid, Spain)(1980):Flavobacteriosisin coho also consistently recovered from the gills of healthy tur salmon(Oncorhynchus kisutch),In"Fish diseases"(ed.by bot, it is considered that it could actually belong to its W.Ahne). SpringerVerlag, Berlin,212-217. normal microflora and that unknown factors could Al-Harbi,A. H. and B. Austin(1992a):Theimmune response weaken the host and/or enhance the virulence of the bac of turbot,Scophthalmus maximus(L.),tolipopolysaccharide terium (Mudarris and Austin, 1989). from a fishpathogenic Cytophaga-like bacterium.J. Fish Dis.,15,449-452. Immunizationof turbotwith LPS from C. scophthalmum Al-Harbi,A. H. and B. Austin(1992b):Influenceof skinand was shown to induce high haemagglutination activities gut mucus from turbot(Scophthalmus maximusL.)on the in the serum and skin and gut mucus when fish were survivalof a fishpathogenic Cytophaga-likebacterium. intraperitoneally injected, whereas administration of Bull.Eur.Ass. Fish Pathol.,12,80-82. LPS by the oral route or by immersion is generally Al-Harbi,A. H. and B. Austin(1992c):Distributionof formal unsuccessful (Al-Harbi and Austin, 1992a). Skin and izedcells and lipopolysaccharide(LPS)ofa fish pathogenic Cytophaga-likebacterium in tissuesof turbot(Scophthalmus gut mucus of LPS-immunized turbot are also inhibitory maximus L.)followinguptake by injection,immersion and to C. scophthalmum (Al-Harbi and Austin, 1992b). the oralroute. Bull.Eur.Ass. Fish Pathol.,12,100-103. Immunohistochemical techniques were used to study the Alsina, M. and A. R. Blanch(1993):First isolationof distribution of the bacterium and LPS following differ Flexibactermaritimus from cultivatedturbot(Scophthalmus ent routes of inoculation (Al-Harbi and Austin, 1992c). maximus). Bull.Eur.Ass. Fish Pathol.,13,157-160. 236 J.F. Bernardet

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