Isolation of Oxidase-Negative Aeromonas Salmonicida from Diseased Turbot Scophthalmus Maximus

DISEASES OF AQUATIC ORGANISMS Vol. 18: 149-154, 1994 ' Published February 24 Dis. aquat. Org. l

NOTE

Isolation of oxidase-negative Aeromonas salmonicida from diseased turbot Scophthalmus maximus

Karl Pedersenl,Hans Kofodl, Inger Dalsgaard2,Jens Laurits Larsen'

'RoyalVeterinary and Agricultural University,Section of Fish Diseases.13 Bulowsvej,DK-1870 Frederiksberg C, Denmark 'Danish Institute forFisheries and Marine Research.Laboratory of Fish Diseases,13 Biilowsvej,DK-1870 Frederiksberg C, Denmark

ABSTRACT: The first outbreak of dlsease due to an atyplcal isolation of oxidase-negative but otherwise typical Aeromonas salmonicida among turbot Scophthalmus maxi- A. salrnonicida. mus (L.)in Denmark is reported. The causal organism was This paper describes the outbreak of an ulcerative oxidase-negative, non-pigmented, and slow-growing. Additi- onally it differed from the typical strains in a number of bio- disease caused by an atypical Aeromonas salmoni- chemical characters. The mortality among turbot in the farm cida strain among turbot in a Danish salt water fish was high but was responsive to antlmlcrobial therapy. farm. Materials and methods. Turbot: The main produc- KEY WORDS: Atypical . Aeronlonas salmonicida . Turbot. Ulcerative lesions tion on the farm was rainbow trout Oncor-hynchus my- kiss. Turbot production occurred on the facility on an experimental basis. The number of turbot before the Aeromonas salmonicida is the causative agent of outbreak of disease was approximately 3200, distrib- furunculosis, a disease generally restricted to salmo- uted in twelve 1000 1 tanks, and with an average body nids in fresh water (Austin & Austin 1987). However, weight of approximately 100 g. an increasing number of atypical A. salmonicida have Bacteriological and serological examination: Initial been isolated from diseased fish from various parts of cultures were made on marine agar (Difco, Detroit, MI, the world. These strains have been isolated from fresh- USA) supplemented with 5 %calf blood (BA) and incu- water fish as well as marine fish and are not restricted bated at 20 OC for 4 d. Bacteria were identified accord- to salmonids. Atypical A. salmonlcida have been re- ing to Popoff (1984) using methods outlined by Cowan ported to have caused ulcerative lesions or other clini- (1974). Vibrio angujllarum strains were serotyped as cal signs in carp Cyprinus carpio (McCarthy 1977), described by Ssrensen & Larsen (1986), while serolog- minnow Phoxinus phoxinus (Hgstein et al. 1978), ical examination of Aeromonas was performed as Atlantic salmon Salmo salar (Paterson et al. 1980), described by Dalsgaard & Paulsen (1986). goldfish Carassius auratus (Elliott & Shotts 1980, Protein staining: The reaction of Aeromonas colo- Whittington et al. 1987), Atlantic cod Gadus morhua nies with Coomassie blue and Congo red was exam- (Cornick et al. 1984), eel Anyuilla anguilla (Kitao et al. ined as described by Evenberg et al. (1985) and Ishi- 1985), sand-eels Ammodytes Jancea and Hyperoplus guro et al. (1985), respectively. lanceolatus (Dalsgaard & Paulsen 1986), pike Esox SDS-PAGE and western blotting: Protein samples lucjus (Wiklund 1990), and flounder Platichthys flesus were prepared from stationary phase broth cultures. (Wiklund & Bylund 1991). Cultures were transferred to Eppendorf microfuge Atypical Aeromonas salmonicida strains may differ tubes and centrifuged at 8000 rpm for 10 min. Pellets in various characters such as fermentation of carbohy- were washed in phosphate buffered saline (PBS),pH drates (McCarthy 1977, Wiklund 1990), amino acid 7.3, resuspended in 20 ,p1 distilled water, and 300 p1 decarboxylation (McCarthy 1977), oxidase reaction sample buffer [0.0625 M Tris (pH 6.8), 5 % mercaptoe- (Wiklund & Bylund 1991), growth intensity and thanol, 10 % glycerol, and 2 % SDS] was then added. requirements for growth factors (McCarthy 1977, Ishi- The mixtures were boiled for 5 min, diluted with 320 p1 guro et al. 1986). Chapman et al. (1991) reported the of distilled water, boiled again for 5 min. and centri-

O Inter-Research 1994 150 Dis. aquat. Org. 18: 14S154,1994

fuged at 13000 rpm for 5 min. Three hundred m1 of PaUlogenicity test: In order to determine an LDSo supernatant were transferred to new microfuge tubes value (Reed Pc Muench 1938) for the strain in sal- and 40 p1 of sample buffer containing 0.0001 % bro- monids, groups of 6 Atlantic salmon Salmo salar (L.) mophenol blue was then added. Purified protein A were injected with approximately, 1.3 X 108, 1.3 X 106, from A. salmonicida subsp. salmonicida was obtained 1.3 x 104, or 1.3 X 102 colony-forming units (CFU) in as a kind gift from Dr L. J. Reitan, Veterin~rinstituttet, PBS or with PBS alone as control. Bacteria for the Oslo, Norway. Bacterial protein preparations and puri- challenge were obtained from a broth culture by fied protein A were subjected to SDS-PAGE (Laemmli centrifugation; they were then resuspended in PRS. 1970) and blotted onto nitrocellulose membrane (west- Each group of fish was kept in a separate tank at ern blotting). Blocking and subsequent incubations 16 "C. were carried out at room temperature in a blocking Results and discussion. In mid June most of the tur- buffer consisting of PBS containing 1.0 % n.on-fat bot developed a disease with lethargy and skin lesions instant dry milk. The nitrocellulose membranes were as the predominant signs. The lesions usually started incubated with serum from infected and uninfected as erosions at the tip of the skin nodules, with a white turbot followed by rabbit anti-turbot antiserum (Kofod center surrounded by a thin hemorrhagic zone (Fig. 1) et ai. 1994), and peroxidase iabeled swine anti-rabb~t approximately 'l to 4 mm in diameter. It is uncertain IgG (Dako, Glostrup, Denmark). Between steps, mem- whether the erosions were part of the infection or branes were washed 4 times in PBS containing 0.05 '% whether they were caused by mechanical or other Twcen 80. Peroxidase activity was detected with damage and subsequently formed the port of entry for 10 mg diaminobenzidine (DAB; Sigma, St. Louis, MO, the pathogen. Some erosions developed into ulcers

USA) dissolved in 40 ml H20 containing 20 p1 30 O/o that varied in size from approximately 0.5 to 3 cm in H202. diameter and were distri.buted over the whole body, Antibiotic sensitivity: Antibiotic sensitivity testing the dorsal and the ventral surfaces being equally af- was carried out on BA using the agar diffusion method fected (Fig. 2).Typical ulcers had an umbonate appear- (Neo-Sensitabs, Rosco, Denmark). ence with a hemorrhagic center surrounded by a

Fig. 1.Scophthalrnus maximus. Small erosions and ulcers, 2 to 4 mm in diameter, at the tip of the skin nodules of a turbot. These erosions may have been caused by mechanical damage and may have been the port of entry of the Aerornonas strain. A small ulcer is recognized at the operculum. On several fish large, irregular ulcers were detected here as well as on various sites on the head or at the base of the fins Pedersen et al.: Alypicdl Aeromonas salmoniada infection in turbot

Fig. 2. Scophthalmus maximus. Typical ulcers on the dorsal, pig~nentedside of the turbot. Ulcers, approximately 0.5-3 cm in diameter, were found and were equally frequent on dorsal and ventral s~des.Ilecoloration of the skin surrounding some of the ulcers was evident. This decoloration was absent on some fish but was pronounced on others whitish, slightly elevated zone. Frequently, large irreg- metabolized glucose by the fermentative pathway, and ular ulcers were situated at the base of the fins, on they were considered to belong to the genus the operculum, or on the head, exposing the under- Aeromonas (Popoff 1984) although they were oxidase- lying bony tissue. Some ulcers were surrounded by negative. Further characterization and comparisons a larger zone of marked decoloration of the skin. with the type strains A. salmonicida subsp. salmoni- The number of lesions varied considerably from one cida NCMB 1102 and A. salmonicida subsp. achrom- fish to another, ranging from 1 to more than 20 ogenes NCMB 1110 were conducted as shown in per fish. Table 1. As a result, the strains were identified as atyp- The ulcers as well as the kidneys from several speci- ical A. salmonicida. mens, collected on 2 occasions within a 1'12 mo period, The bacteria showed a positive reaction in the slide were examined bacteriologically. Pin-point, non-pig- agglutination test with rabbit antiserum raised against mented colonies were cultured from all specimens. The Aeromonas salmonicida subsp. salmonicida, and colo- bacterium grew in very large numbers and in pure cul- nies were Coomassie blue- and Congo red-positive. In ture from the kidneys of all animals tested. However, western blots, serum from infected turbot reacted with Vibrio species were also cultured from the ulcers. The purified protein A and with an approximately 49 kDa small colonies together with some Vibrio anguillarum - protein from the atypical A. salmonicida strain. These like colonies were subcultured on blood agar to ensure results suggested that the isolate produced a protein that they represented pure cultures before studies to A-like compound. identify the cultures were undertaken. The strains were sensitive to sulphonamides + tri- The Vibrio anguillarum -like isolates proved to be methoprim (Tribrissena), oxolinic acid, nitrofurantoin, V a.nguillarum, strains representing 2 serotypes (01 and tetracycline. They showed intermediate sensi- and 02) being identified. tivity to sulphonamides but were resistant to tnme- The cultures derived from the small colonies were thoprim. subjected to biochemical tests. Cells from 48 h brain Onset and progression of the infection developed heart infusion (BHI) broth (Difco) cultures were short in parallel with a marked increase in the water temper- rods, 0.5 X 1-3 pm. The bacteria were Gram-negative, ature caused by a period of warm sunny weather non-motile, oxidase-negative, catalase-positive, and (Fig. 3). Mortality increased in the farm for 5 wk, Dis. aquat. Org. 18: 149-154, 1994

Table 1. Biochemical reactions of atypical Aeromonas salmonicida lsolated from turbot Scophthalmus maximus, compared with A. salmonicida subsp. salmonicida NCMB 1102 and A. salmonicida subsp. achromogenes NCMB 11 10. + and - indicate posltlve and negative reactions, respectively, while R indicates resistance and S sensitivity to the test antibiotic

Feature Strains from turbot A. salmonicida subsp. A. salmoniada subsp. (n = 5) salmonicida NCMB 1102 achromogenes NCMB 11 10

Cell morphology Short rods Short rods Short rods Gram reaction - - - Motility - - Oxidase + Catalase + Haemolysls - Brown p~gment - Arginine dihydrolase + Lysin decarboxylase Ornithin decarboxylase Indole Aesculin Voges-Proskauer Acid from glucose Gas from g!ucose Acid from: d-Arabinose l- Arabinose Salicin Sucrose Mannitol Trehalose Gelatine Penicillin. high, 62.5 pg Cephalotin 66 pg 0/129, 150 pg 0/129, 10 pg Novobiocin, 5 pg whereafter treatment with sulphonamides + trimetho- cally. This time, a Wbrio anguillarum, serogroup 01, prim (TribrissenB)was initiated and performed 3 times was isolated from all fish examined. The rainbow trout a week for 3 wk (Fig. 3). After the antibacterial treat- had been vaccinated against vibriosis and no vibriosis ment, the mortality decreased significantly. However, was recorded among them. The turbot, however, had the ulcers healed very slowly, leaving scars in the skin not been vaccinated against vibriosis. as well as in the muscle. A few weeks after this out- Due to the high mortality among the turbot during break, mortality suddenly increased again dramati- the 2 disease outbreaks and the poor quality of surviv-

% mortality water temperature, "C 20 1 120 X

Fig. 3. Scophthalmus maximus. Water temperature and turbot mortality (as a percentage of surviving fish) was recorded during the outbreak. The first peak, Weeks 25 to 32, wiis caused by the atypical Aeromonas salmonicida while the second peak, week \,Veeks 37 to 45, was caused by Vlbrio anyuillarum serotype 01 Antibiotic treatment is indicated by - % mortality + water temperature arrows Pedersen et al.. Atypical Aeromona s salmonicida infection in turbot 153

ing fish, the remaining turbot on the farm were de- LITERATURE CITED stroyed. The source of the atypical Aeromonas salmonicida Austln, B. (1993). Recovery of 'atypical' isolates of Aeromonas salmonicida, which grow at 37 OC, from ulcerated non- infection is unknown. The bacterium did not appear to salmonids in England. J. Fish Dis. 16: 165-168 cause problems among the rainbow trout on the farm. Austin, B., Austin, D. A. (1987). Bacterial fish pathogens: dis- However, because the fish farm had a large production ease in farmed and wild fish. Ellis Horwood Ltd, of ralnbow trout, it was decided to deteimine whether Chichester, p. 11 1-171 Austin, D. A., McIntosh, D., Austin, B. (1989). Taxonomy of the turbot pathogen was also pathogenic to salmonids. fish associated Aeromonas spp., with the description of However, in the LDS, test with the rainbow trout no fish Aeromonas salmonicida subsp. smithia subsp. nov. Syst. died during a 2 mo observation period. The challenged appl. Microbiol. 11: 277-290 fish were therefore killed and examined bacteriologi- Chapman, P. F., Cipriano, R.C., Teska, J. D. (1991). Isolation cally. Swabs collected from the pronephros were inoc- and phenotypic characterization of an oxidase-negatlve Aeromonas salmonicida causing furunculosis in coho sal- ulated onto BA plates and incubated at 20 "C. All spec- mon (Oncorhynchus kisutch). J. Wildl. Dis. 27: 61-67 imens proved to be sterile. It was therefore concluded Comick, J W., Morrison, C. M., Zwicker, B., Shum, G. (1984). that the strain had no or very low pathogenicity for sal- Atypical Aeromonas salmonicida infection in Atlantlc cod, monids. Gadus morhua. J. Fish Dis. 7: 495-499 Cowan, S. T. (1974). Cowan and Steel's manual for the iden- The epizootiology, clinical signs, and pathological tification of medical bacteria. Cambridge University Press, changes observed in the present case were similar to Cambridge those described by Devesa et al. (1989). However, Dalsgaard, I.,Paulsen, H. (1986). Atypical Aeromonas salmo- these authors reported the finding of Cryptocarion nicida isolated from diseased sand-eels, Ammodytes lan- spp., myxobacteria, and halophilic, urease-positive cea (Cuvier) and Hyperoplus lanceolatus (Lesauvage). J. Fish Dis. 9: 361-364 Vibrio strains in the lesions. No Aeromonas or Devesa, S., Barja, J. L., Toranzo, A. E. (1989). Ulcerative skin Aeromonas - like microorganisms were reported. In the and fin lesions in reared turbot Scophthalmus maximus present, study the microorganisms found by Devesa et (L.).J. Fish Dis. 12: 323-333 al. (1989) were not detected. Elliott. D. G., Shotts, E. B. Jr (1980). Aetiology of an ulcerative disease in goldfish Carassius auratus (L.): microbiological Reports on the existence of oxidase-negative forms examination of diseased fish from seven locations. J. Fish of Aeromonas salmonicida are relatively new and Dis 3. 133-143 therefore few in number. Chapman et al. (1991) de- Evenberg, D.,Versluis, R., Lugtenberg, B.(1985). Biochemical scribed the isolation from coho salmon Oncorhynchus and immunological characterization of the cell surface of kisutch of an oxidase-negative bacterium that other- the fish pathogenic bacterium Aeromonas salmonicjda. Biochim. biophys. Acta 81. 233-244 wise had the characteristics of A, salmonicida subsp. HAstein, T., Saltve~t,S. J., Roberts, R.J. (1978). Mass mortality salmonicjda. Thls was reported as the first isolation of among minnows Phoxinus phoxinus (L.) in Lake an oxidase-negative A. salmonicida, and such strains Tveitevatn, Nonuay, due to an aberrant strain of were considered to be extremely rare. Almost simulta- Aeromonas salmonicida. J. Fish Dis. 1: 241-249 Ishiguro. E. E., Ainsworth. T., Kay, W. W., Trust, T. J. (1986). neously, Wiklund & Bylund (1991) reported the isola- Heme requirement for growth of fastidious atypical strains tion of an oxidase-negative presumptively atypical A. of Aeromonas salmonicida. Appl. environ. Microbiol. 51: salmonicida from ulcers of flounders Platichthys flesus 668-670 in the Baltic Sea. The present strain, isolated from tur- Ishiguro, E. E., Ainsworth, T., Trust, T J., Kay, W. W. (1985). bot, is to our knowledge the first description of an oxi- Congo red agar, a differential medium for Aeromonas salmonicida, detects the presence of the cell surface dase-negative A. salmonicida from Denmark and from protein array involved in virulence. J. Bacteriol 164: turbot. The strain differed in various characters from 1233-1237 the subspecies of A. salmonicida listed by Popoff (1984) Kitao, T., Yoshida, T., Aoki, T., Fukudome, M. (1985). and from the A. salmonicida subsp, smithia, proposed Characterization of an atypical Aeromonas salmonicida strain causing epizootic ulcer disease in cultured eel. Fish by Austin et al. (1989) and Austin (1993). However, its Pathol. 20: 107-114 isolation should serve to alert fish health researchers to Kofod, H., Pedersen, K., Larsen, J. L., Buchmann, K. (1994). the existence of a hitherto unrecognized but poten- Purification and characterization of IgM-like immuno- tially important group of fish pathogenic bacteria - a globulin from turbot (ScophthalmusmaximusL.).Acta vet. group that will require more study to answer questions scand. (in press) Laemmli, U. K. (1970). Clevage of structural proteins during on its taxonomy, ecology, epizootiology, and pathoge- the assen~blyof the head of bacteriophage T4. Nature 227. nicity. 680-685 McCarthy, D. H. (1977). The identification and significance of atypical strains of Aeromonas salmonicida. Bull. off. Int. Acknowledgements. The technical assistance of Mrs Kirsten Epiz. 87: 459-463 Kaas and the support of The Danish Agricultural and Paterson, W.D., Douey, D., Desautels, D. (1980). Isolation and Veterinary Research Council grant no. 13-4710-1 and 13- identification of an atypical Aeromonas salmonicida strain 4508-1 are gratefully acknowledged. causing epizootic losses among Atlantic salmon (Salmo 154 Dis. aquat. Org. 18: 149-154, 1994

salar) reared in a Nova Scotian hatchery. Can. J. Fish. L. D., Thursten, S. J (1987). Clinical microbiological and Aquat. Sci. 37: 2236-2241 epidemiological findings in recent outbreaks of goldfish Popoff, M. (1984). Genus 111. Aeromonas. Kluyver and Van ulcer disease due to atypical Aeromonas salmonicida in Niel 1936,398"L In. Krieg, N. R.,Holt. J. G (eds.)Bergey's south-eastern Australia. J. Fish Dis 10. 353-362 manual of systematic bacteriology, Vol. 1 Williams and Wiklund, T. (1990). Atypical Aeromonas salmonicida ~solated Wilkins, Balt~more,p 545-548 from ulcers of pike, Esox lucius L. J Fish Dis. 13: 54 1-544 Reed, L. J., Muench, H (1938). A simple method of estimating Wiklund, T.,Bylund, G.(1991). A cytochrome oxidase nega- fifty per cent endpoints. Am. J. Hyg. 27: 493-497 tive bacterium (presumptivcly an atypical Aeromonas sal- Ssrensen, U. B, S., Larsen, J. L. (1986). Serotyping of Vibrio monicida) isolated from ulcerated flounders (Platichthys anguillarum. Appl. environ. Microbiol. 51: 593-597 flesus (L.))in the northern Baltic Sea. Bull. Eur. Ass. Fish Whittington, R. J., Gudkovs, N., Carrigan, M. J., Ashbum. Pathol. 11 74-76

Responsible Subject Editor: T Evelyn, Nanaimo, B.C., Manuscript first received: April 20, 1993 Canada Revised version accepted: November 3, 1993