DISEASES OF AQUATIC ORGANISMS Vol. 29: 13-20, 1997 Published April 24 Dis Aquat Org

Experimental infection of Scophthalmus maxim us and halibut Hippoglossus hippoglossus yolk sac larvae with Aeromonas salmonicida subsp. salmonicida

Oivind Berghl,*, Brit ~jeltnes~,Anne Berit Skiftesvikl

'Institute of Marine Research, Austevoll Aquaculture Research Station, N-5392 Storebe, Norway '~nstituteof Marine Research, Department of Aquaculture, PO Box 1870 Nordnes, N-5024 Bergen, Norway

ABSTRACT: The suscept~bilityof early l~festages of turbot Scophthalmus maximus and halibut Hippo- glossus h~ppoglossusto Atvomonas salmoniclda subsp. salrnonicida was studied in challenge experi- ments. Eggs of both specles (and the larvae hatching from the eggs) were exposed to the bac- tenum. Larvae of both species experienced mortality during the yolk sac stage, apparently as a result of the challenge. Examination of histolog~cal sect~onsrevealed degenerative changes in the skeletal muscle of both species of larvae. The bactenum could not be recovered from the larvae by culture, but it was shown to be present in the intestinal lumen of some of the turbot larvae examined using an imn~unohistochemical techn~que known to be spec~ficfor the bactenum. The results indicate that manne fish larvae exposed to A salmonicida subsp salmonicida may become ~nfectedand that the bacter~ummay persist In the larvae. Turbot larvae seemed to be more susceptible than hahbut larvae

KEYWORDS: Aeromonas salmonicida subsp salrnonlcida . Fish larvae . Turbot . Halibut

INTRODUCTION Marine fish larvae are susceptible to infection by opportunistic pathogenic bacteria (Bergh et al. 1992, During outbreaks of typical furunculosis in Atlantic Skiftesvik & Bergh 1993). No information is available Salmo salar, Aeromonas salmonicida subsp. concerning the susceptibility of these larvae residing salrnonicida may be released in large numbers by in the vicinity of net pens containing infected salmon infected fish (Enger et al. 1992). The bacterium can be releasing Aeromonas sahonicida subsp. salrnonicida detected in surrounding seawater and in the sediment to the environment. The bacterium has been isolated beneath the net pens containing fish (Enger & Thorsen from marine zooplankton (Nese & Enger 1993), indi- 1992). Although A. salmonicida subsp. salrnonicida oc- cating that it can become associated with planktonic casionally causes disease in marine species such as organisms, perhaps including yolk sac larvae of fish. Gadus morhua and halibut Hippoglossus hippoglossus The purpose of this study was to investigate the extent and members of the wrasse family (Labridae), adult to which A. salrnonicida subsp, salrnonicida is able to individuals of these species appear to be more resistant become associated with, or even to infect and cause to this disease than salmonids (Hjeltnes et al. 1995). mortality of yolk sac larvae of 2 commercially impor- However, isolation of the bacterium from turbot Scoph- tant marine fish species, turbot and halibut. thalmus maximus experiencing significant mortality has been reported (Nougayrede et al. 1990, Toranzo & Barja 1992, Pedersen & Larsen 1996). MATERIALS AND METHODS

Source, cultivation, and preparation of the bacterium. The strain of Aeromonas salmonicida subsp. salmo-

O Inter-Research 1997 Resale of full article not permitted Dis Aquat Org 29: 13-20, 1997

nicida used in this study was isolated from an outbreak respectively). The last group of eggs served as a con- of typical furunculosis in a stock of Atlantic salmon at trol group and nothing was added to these wells. the Institute of Marine Research, Matre Aquaculture From each group, 3 multiwell dishes (72 individual Research Station, Matredal, Norway, in 1990. The eggs) were randomly selected. These eggsAarvae were strain is included in the culture collection of the inspected for mortality daily until termination of the Institute of Marine Research as strain number As 55 experiment at Day 5 after hatching. The 96 individual Matre. All incubation of the bacterium for the experi- eggs/larvae in the remaining 4 multiwell dishes were ment with turbot was performed at 16°C in the climate- sequentially sampled for fixation for histological ex- controlled room in which the experiment with turbot aminations, or attempts to re-isolate the pathogen. larvae was performed. For the experiment with halibut, Infection of halibut. One female of the halibut brood- all cultivation of bacteria was performed at 10°C. stock of the Austevoll Aquaculture Research Station The different suspensions of the bacterium that were was stripped and the resulting eggs were fertilized used in the expenments were obtained from exponen- with sperm from 1 male. The eggs were reared in tially growing cultures of As 55 Matre in Difco Marine 250-L upwelling incubators as described by Pittman et Broth (Difco, Detroit, USA). The concentration of bac- al. (1990) at approximately 6°C for 7 d. teria in the cultures was measured by plating on Tryp- From this stage onwards, the entire experiment was tone Soya Broth (Oxoid, Basingstoke, England), 1.5% carried out in climate-controlled rooms at 6°C. The agar (Difco), and 25%0seawater. After 1 wk of incuba- eggs were transferred to polystyrene multidishes tion, the concentration of bacteria was measured in (Nunc, Roskilde, Denmark) according to Bergh et al. terms of colony-forming units (CFU) per ml. Suspen- (1992). Each of these multiwell dishes had 6 separate sions of washed bacteria and a suspension of heat- identical wells, 35 mm in diameter and 18 mm deep. killed cells (turbot experiment only) were made Each well contained 11 m1 SSW. One egg was trans- immediately before infection of the fish eggs. The sus- ferred to each well. Two groups were included in this pensions of washed bacterial cells were obtained by experiment. Group As was exposed to 100 p1 of the sus- centrifugation of a volume of the culture and resuspen- pension of washed cells, equivalent to a concentration sion of the cells in an identical volume of 25%0 auto- of bacteria in the wells of 106 CFU ml-l, approximately claved seawater (SSW). The suspension of heat-killed 24 h after transfer to the wells. In addition, there was a cells was obtained by incubating a volume of the sus- control group in which nothing was added to the wells. pension of washed cells at 60°C for 30 min. One day after hatching, 10 m1 of water was removed Infection of turbot. One female turbot from the from each well and 10 m1 SSW was immediately added broodstock at the Austevoll Aquaculture Research in order to remove the remains of the egg and eggshell Station was stripped and the resulting eggs were fer- from the wells. The eggsAarvae were incubated in tilized with sperm from a single male. The entire ex- darkness. Handling of the multiwell dishes, including periment was carried out in a climate-controlled room inspection for mortality, was carried out in red light at 16°C. The fertilized eggs were incubated in the with an intensity of 2.5 lux. From each group, 10 multi- dark for 2 d in stagnant seawater which had been well dishes (60 individual larvae) were randomly stored and aerated at ambient temperature for at least selected and checked for mortality every second day 1 wk prior to the experiment. The eggs were trans- until termination of the experiment at Day 29 after ferred to 24-well polystyrene multidishes (Nunc, Ros- hatching. The remaining 180 individual larvae were kilde, Denmark) as described by Skiftesvik 8 Bergh used for sequential samplings for histological examina- (1993). One egg was transferred to each well. There- tion and attempts to re-isolate the pathogen. after, the entire experiment was carried out in white Histological examinations. Apparently live samples light with an intensity of 117.4 lux. Approximately 24 h of 6 to 8 larvae per group were fixed in 3.7 % (vol/vol) after transfer of the eggs to the multiwell dishes, the phosphate buffered formaldehyde at Days 1, 2, and 3 wells were inoculated with selected concentrations of after hatching (turbot experiment) and at Days 1, 3, 7, the suspensions of the bacterium. 13, and 15 after hatching (halibut experiment). The Three different amounts of the suspension of washed larvae were embedded in paraffin, sectioned at 3 pm, bacterial cells were added directly to the wells of 3 stained with Giemsa, and examined using a light groups of eggs, 200 p1 (group As-A), 20 p1 (group microscope. As-B), and 2 yl (group As-C), equivalent to final con- For immunohistochemical examinations of the sec- centrations in the wells of 1.0 X 107, 1.0 X 106,and 1.0 X tions, a standard avidin-biotin-peroxidase complex 105 CFU ml-l, respectively. Three groups of eggs were (ABC) method was used (Hsu et al. 1981), modified as exposed to equivalent amounts of a suspension of heat- described by Evensen & Rimstad (1990). Aeromonas killed cells. These groups were designated DC-A, salmonicida was identified by using a monoclonal anti- DC-B, and DC-C (equivalent to As-A, As-B, and As-C, body (D-5,National Veterinary Institute, Oslo, Nor- Bergh et al.. Infection of turbot and hallbut Idrvae

way), reactive with A. salmonicida lipopolysaccharide, (Fig. 3a, b). These cell-like structures were associated diluted 1:100. Tris with 2.5% Bovine Serum Albumin with positive immunohistochemical staining in 3 of the (BSA) was used as diluent. In the next 2 sequences, turbot larvae (Fig. 3c). The attempts to re-isolate the rabbit-anti-mouse immunoglobulins conjugated to pathogen from the larvae failed, as no colonies resem- biotin (Dakopatts) were used. The sections were then bling those of Aeromonas salmonicida subsp. salmoni- incubated with New Fuchsin substrate system, washed cida and producing pigment were found. in tap water, and counterstained with Harris haema- In the halibut experiment, no mortality took place toxylin. Sections were mounted in an aqueous mount- during the egg stage, and the mortality of the larvae ing medium. Staining controls were performed on was low the first 3 d after hatching (Fig. 4). Thereafter, tissue specimens from apparently healthy larvae the mortality increased in the challenged group, but it Re-isolation of bacteria from larvae. In an attempt was lower in the control group. The difference be- to re-isolate Aeromonas salmonicida subsp. salmoni- tween the control group and the challenged group was cida from the larvae, groups of 5 to 8 larvae were significant from Day 5 onwards (p < 0.05). Three out washed 3 times in SSW and homogenized. A dilution of 9 of the halibut larvae from the challenged group series was made from the homogenate and plated out sampled at Day 13 were characterized by degenerative on Tryptone Soya Broth and checked for growth of changes (pycnotic nuclei) in the skeletal muscle. How- colonies resembling those of A. salmonicida subsp. ever, similar changes were also found in 3 out of 6 salmonicida in pigment production and colony mor- halibut larvae from the control group at Days 13 and phology. 15. No pathological changes were found among hal- Statistical analysis. For both experiments, differ- ibut larvae sampled earlier. As with the the case of ences between the experimental groups were tested turbot, Aeromonas salmonicida subsp. salmonicida for statistical significance by x2analysis, followed by was not re-isolated from the larvae, but unlike the comparison of groups with the Tukey-type multiple result from the turbot experiment, no halibut larvae comparison of proportions (Zar 1984). gave positive immunohistochemical reactions for A. salmonicida subsp. salmonicida. The results demonstrate a significantly increased RESULTS AND DISCUSSION mortality of both species of fish larvae following expo- sure to Aeromonas salmonicida subsp. salmonicida, In the turbot experiment, a dose-response in cumula- compared to the uninfected control groups. The tive mortality was found for the groups exposed to absence of increased mortality in the groups of turbot washed bacteria (Fig. 1).No significant mortality took larvae that were exposed to heat-killed cells rules out place at the egg stage, but after hatch- ing, the mortality increased in group As- A from Day 1 onwards. The difference - + - Control As-A between group As-A and the control ' D- DC-A -+AS-B group was significant from Day 1 80 -- - - o -. DC-B --t AS-C onwards (p < 0.025).None of the remain- 5 --A-- DC-C -- ing groups showed mortalities signifi- -.g 70 cantly different from the control group. 60 -- Pathological changes were found in 3 0 E 50 -- out of 11 turbot larvae from group As-A a, sampled on Days 2 and 3. The changes 2 40.- were evident as sloughing of the in- testinal epithelium and degenerative changes in the skeletal muscle (Fig. 2). No such changes were found in the 4 turbot larvae sampled from this group on Day l. No changes were found in a -1 0 1 2 3 4 5 total of 5 larvae from the control group or Days after hatching-- in 7 larvae from group DC-A that were checked. In 1 of the turbot larvae with Fig. 1 Cumulative mortality (",I) in the experiment with turbot larvae chal- changes, and in 4 of the lenged with Aeromonas salmonjcida subsp salmonidda. The groups are- As-A, As-B, and As-C (exposed to various levels of viable, washed cells); other larvae from As-A' DC-A, DC-B, and DC-C (exposed to equvalent amounts of heat-killed cells); structures associated with bacteria were and control (nothlng added to the wells). Each group consisted of 72 indi- seen in the intestine and the oesophagus vldual eggs/larvae v------., '

Fig. 2. Semi-thin sections of turbot larvae, stained with Giemsa, from (a, b) group As-A, challenged with Aeromonas salmonicida subsp. salmonicida and (c, d) the control group. The sections from the infected larva (a, b) demonstrate the typical pathological changes found in turbot larvae: sloughing of the intestinal epithelium (arrows) and degenerative changes in the skeletal muscle (open arrows). Scale bars are (a, c) 80 pm and (b, d) 20 pm L I 7 - .D ;+ p-:P L P #? J h. t -.- L!xder;

Fig. 3. Semi-thin sections from a turbot larva of (a, b) group As-A, chal- lenged with Aerornonas salmonicida subsp. sal- monicida stained with Giemsa or (c) wlth the immunohistochemical stain to visualize this bacterium. The arrow in (a) points out the area of the oesophagus enlarged in (b) and (c). Cell-like structures can be seen (b, c), associ- ated with positively red-stained [arrow in (c)] bacteria-like structures [arrow in (b)]. Scale bars are (a) 40 pm or (b, c) 10 pm Dis Aquat Org 29: 13-20, 1997

100 - 1992, Pedersen & Larsen 1996). Thus, it -m- As seems that differences in host susceptibility 90 -- - - .n - - Control to a given pathogen could be present as -g 80-- early as the yolk sac stage. 70 -- .-Y The differences in rearing temperature - -- between the turbot and halibut experi- g 60 ments undoubtedly also contributed to the 50 -- results. In our experiments, both species of 0 > 40 -- larvae were reared close to the optimal .C- -a3 30 -- .* temperature of the species. Halibut larvae, which are commonly reared at 5 to 8"C, .a. BT - - . would not have been able to survive at the a--m--*- .Iy - - iy. - temperatures used in the experiment with turbot (Pittman et al. 1990). This necessi- -5 -3 -1 1 3 5 7 g 11 13 75 17 19 21 23 25 27 29 tated a temperature lower than the temper- Days after hatching atures at which epizootics with Aeromonas salmonicida subsp. salmonicida in salmon Fig. 4. Cumulative mortality (%) in the experiment with halibut larvae occur, typically 12 to 18°C (~j~l~~~~et challenged with Aeromonas salmonicida subsp. salmonicida. The groups are As (exposed to viable, washed cells), and control (nothing added to 1995). Thus, turbot larvae have a greater the wells). Each group consisted of 60 individual eggsllarvae overlap in temperature range with A. sal- monicida subsp, salrnonicida than halibut larvae. the possibility that the mortality was due to the in- Turbot also have greater overlap in spatial distribu- creased amount of organic matter that was added to tion with the bacterium than halibut, as they occur the wells. The low mortalities in the control groups naturally in the upper 10 m of the water column indicate that the eggs were healthy from the outset. (Nellen & Hempel 1970) as a result of their specific The histological data are more difficult to interpret. density. They may thus experience contact with the Turbot larvae showed degeneration of muscular tissue lipid microlayer found at the air-water interface in in response to infection by the bacterium. The marine systems (Norkrans 1980, Dahlback et al. 1981, immunohistochemical analyses were positive only in 1982) where Aeromomas salmonicida subsp. salrnoni- the case of turbot, in which the bacterium was detected cida can occur in high numbers. It seems evident from in the gut of the larvae, but not in skeletal muscle. As buoyancy measurements in laboratory experiments the degenerative changes in skeletal muscle were (Skiftesvik & Bergh 1993) that halibut larvae have found in halibut larvae from the control group as well higher specific density than turbot larvae. Due to its as in challenged groups, and because no halibut larvae high hydrophobicity, A. salmonicida subsp. salmoni- were positive in the immunohistochemical analysis, we cida aggregates in the surface layer, and an average conclude that there was no cause-effect relationship of 4.3 X 103 A. salmonicida subsp, salmonicida X ml-' between the challenge and the histological alterations was reported by Enger & Thorsen (1992) for a sampling in the halibut. Reduced prevalence of degenerative station within a furunculosis-affected salmon farm. changes in skeletal muscle of halibut larvae in re- This was 1 to 2 orders of magnitude higher than in the sponse to surface disinfection of the eggs has been water column immediately below the surface layer. In demonstrated by Bergh & Jelmert (1996), indicating samples from the sediment beneath, an average of that this condition may be related to microorganisms 2.2 X 106 A. salmonicida subsp. salmonicida X ml-' was naturally present on halibut eggs. found. Although such microenvironments might pos- The apparent difference in susceptibility to infection sess relatively high concentrations of A. salmonicida by Aeromonas salrnonicida subsp. salmonicida between subsp. salmonicida, it should be noted that all con- turbot and halibut seems to correspond to similar dif- centrations measured by immunofluorescence counts ferences between adult stages of these fish species. In in the fleld study by Enger & Thorsen (1992) were an earlier experiment (Hjeltnes et al. 1995), we found lower than our measurements by viable counts in that halibut survived injection challenge with 3 X 103 or group As-A at the time of infection. 3 X 104 CFU A. salmonicida subsp. salmonicida (4 indi- It is possible that the ability to occur in association viduals per group). In contrast, adult turbot appear to with fish larvae constitues a survival strategy for be relatively more susceptible to furunculosis, as Aeromonas salmonicjda subsp. salrnonicida. The bac- significant mortalities have been reported in farmed terium is highly hydrophobic and has been isolated specimens (Nougayrede et al. 1990, Toranzo & Barja from zooplankton and salmon lice (Nese & Enger Bergh et al.: Infection of turbot and halibut larvae 19

1993). Effendi & Austin (1994) noted that the shortest LITERATURE ClTED survival (measured as CFU) of starved cells of A. sal- monicida subsp. salmonicida was in seawater con- Bergh 0, Hansen GH. Taxt RE (1992) Experimental infection of eggs and yolk sac larvae of halibut, H~ppoglossus trols, whereas cells attached to wood, seaweeds or in- hippoglossus L J Fish Dis 15.379-391 vertebrate survived for comparatively longer Bergh 0, Jelmert A (1996) lodophor disinfection of eggs of periods. A. salmonicida subsp. salmonicida may also Atlantic halibut. J Aquat Anim Health 8:135-145 become associated with wild fish in the vicinity of fish Dahlback B, Gunnarson LA,Hermannsson M, Kjelleberg S (1982) Microbial investigations of surface microlayers, farms affected by furunculosis. The bacterium has water column, Ice and sediment in the Arctic Ocean. Mar been isolated from wild cod Gadus morhua and coal- Ecol Prog Ser 9:101-109 fish (Pollachius virens) captured within a range of Dahlback B, Hermannsson M. Kjelleberg S, Norkrans B 200 m from fish farms affected by furunculosis (Wil- (1981) The hydrophobicity of bacteria-an important fac- lumsen 1990). Samuelsen et al. (1992) demonstrated tor in the initial addition at the air-water interface. Arch Mlcrobiol 128:267-270 the presence of the bacterium in the gut of wild Effendi I, Austin B (1994) Survival of the fish pathogen salmon and coalfish in the vicinity of a fish farm. A Aerornonas salmonicida in the marine environment. J Fish special case is the outbreak of disease in goldsinny Dis 1?:375-385 Ctenolabrus rupestris and rock cook C, exoletus Enger 0, Gunnlaugsdottir B, Thorsen BK, Hjeltnes B (1992) Infectious load of Aeromoas salmonicida subsp. saln~oni- used as for salmon infested with lice c~daduring the initial phase of a cohabitant infection and furunculosis (Treasurer & Laidler 1994). These experiment wlth Atlantic salnlon (Salmo salar). J Fish DIS studies demonstrate the capability of the bacterium to 15:425-430 persist, at least for some time, in the gastrointestinal Enger 0. Thorsen BK (1992) Possible ecological implica- tract of certain fish species. Adult and larval stages tions of the high cell surface hydrophobicity of the fish pathogen Aeromonas salmonicida. Can J M1crobiol38(10). of several fish species may thus be vectors for the 1048-1052 bacterium. Evensen 0. Rimstad E (1990) Immunohistochernical identifi- Our attempts to re-isolate the bacterium failed, and it cation of infectious pancreatic necrosis virus in paraffin- is generally considered difficult to isolate Aeromonas embedded tissues of Atlantic salmon (Saho salar). J Vet Diagn Invest 2:288-293 salmonicida subsp. salmonicida from planktonic or- Hjeltnes B, Bergh 0, Wergeland H, Holm JC (1995) Susceptl- ganisms (Nese & Enger 1993) or from samples con- bility of Atlantic cod Gadus morhua, halibut Hippoglossus taining other fast-growing bacteria . The immunohisto- hippoglossus and wrasse (Labridae) to Aerornonas salrno- chemical signal was relatively weak, indicating a low nicida subsp. salmonicida and the possibility of transmis- slon of furunculosis from farmed salmon Salnlo salar to number of A. salmonicida subsp. salmonicida present manne fish. DIS Aquat Org 23:25-31 in the infected larvae. In addition, as evident by cul- Hsu SM, Raine L, Fanger H (1981) Use of an avidin-biotin- ture, a variety of different bacteria were isolated from peroxidase complex. (ABC) in immunoperoxidase tech- the larvae, probably originating from the gastrointesti- niques. A comparison between ABC and unlabelled nal tract and the skin. It is possible that the use of more antibody (PAP) procedures. J Histochem Cytochem 29: 577-580 sophisticated diagnostic methods, like isolation of the Nellen W, Hempel G (1970) Beobachtungen am Icthyo- bacterium by use of immunomagnetic beads would neuston der Nordsee. Ber Dt Wiss Komm Meeresforsch have detected the pathogen. 21:311-348 In conclusion, the results indicate that high concen- Nese L, Enger 0 (1993) Isolation of Aeromonas salrnonicida trations of Aeromonas salmonicida subsp. salmonicida from salmon llce Lepeophtheirus salmonis and manne plankton. Dis Aquat Org 16.79-81 during outbreaks of furunculosis in salmon farms Norkrans B (1980) Surface mlcrolayers in aquatic environ- might affect marine fish larvae in the vicinity of the ments. Adv Microb Ecol4:51-85 farms. The turbot larvae seemed susceptible to infec- Nougayrede P, Sochon E, Vuiiaume A (1990) Isolation of tion by A. salmonicida subsp. salmonicida. Although Aeromonas subspecies salmonicida in farmed turbot (Psetta maxima) In France. Bull Eur Ass Fish Pathol 10: pathological changes and increased mortality were 139-140 found for both species, evidence implicating A. salmo- Pedersen K, Larsen JL (1996) First report on outbreak of nicida subsp, salmonicida as the cause was stronger for furunculosis in turbot, Scophthalmus maximus caused by turbot than for halibut. Aeromonas salmonjcida subsp. salrnonjcida in Denmark. Bull Eur Ass Flsh Pathol 16(4)-129-133 Pittman K, Bergh 0, Opstad I, Skiftesvik AB, Skjolddal L, Strand H (1990) Development of eggs and yolk sac larvae Acknowledgements. Funds for this study were provided by of halibut (H~ppoglossushjppoglossus L.). J Appl Ichthyol the Norwegian Research Council, Grant Nos. 1203-701 433 6:142-160 and 1401-701.424.Technical assistance by Laila Baardset and Samuelsen OB, Lunestad BT, Husevdg B, H~llelandT, Ervik A Ingrid Uglenes is highly appreaated. We also thank Dr 0ys- (1992) Residues of oxolinic acld in wild fauna following tein Evensen of the National Veterinary Institute, who pro- medication in fish farms. DIS Aquat Org 12 111-119 vided the monoclonal antibody, and Dr Johan Glette for his Skiftesvik AB, Bergh 0 (1993) Changes in behaviour of comments on the manuscript. Atlantic halibut (Hippoglossus hippoglossus) and turbot Dis Aquat Org 29 13-20, 1997

(Scophthalmus maximus) yolk-sac larvae Induced by Atlantic salmon, Salmo salar L., farm J Fish Dis 17 bacterial infections ( an J Flsh Aquat Sci 50 2552-2557 155-161 Toranzo AE, Earja .TL (1992) First report of furunculos~sIn M'illumsen B (1990) A salmonicida subsp salmonicida ISO- turbot reared In tlodtlng cages In northwest of Spaln Bull ldted from atlantic cod and coalflsh Bull Eur Ass F~sh Eur Ass Fish Pathol 12 147-149 Pathol 10:62-64 Treasurer JW, Ladder LA (1994) Aeromonas salmonicida Zar JH (1984) Biostatistical analysis, 2nd edn. Prentice-Hall infection in wrasse (Labr~dae)used as cleaner flsh, on an Inc, Englewood Cliffs, NJ

Responsible Subject Editor T P T Er n, hranalmo, Manuscript first received. March 14, 1995 Bntish Columbia, Canada Revised verslon accepted: September 26, 1996