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

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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' 'Royal Veterinary and Agricultural University, Section of Fish Diseases. 13 Bulowsvej, DK-1870 Frederiksberg C, Denmark 'Danish Institute for Fisheries 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-hynchusmy- 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.
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