Vibrio salmonicida sp. nov., a New Fish Pathogen

INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, OCt. 1986, p. 518-520 OO20-7713/86/O40518-03$02.OO/O Copyright 0 1986, International Union of Microbiological Societies

Vol. 36, No. 4

Vibrio salmonicida s p . nov., a New Fish Pathogen E. EGIDIUS,' R. WIIK, K. ANDERSEN, K. A. HOFF, AND B. HJELTNES Institute of Marine Research, 5000 Bergen, Norway A disease persisting in Norwegian salmonid cultures since the late 1970s is caused by a Vibrio-like bacterium. This bacterium belongs to the genus Vibrio, but it differs from all previously described species of this genus. The name Vibrio salmonicidu sp. nov. is proposed. The type strain is strain NCMB 2262 (= HI 7751).

In 1979 a new disease appeared in Norwegian salmonid farms around the island of Hitra south of Trondheim. This disease persisted at Hitra and northward until the late autumn of 1983, when it struck southward to Stavanger. In particular, the Bergen region, which has the highest density of fish farms, was badly hit. The disease is characterized by anemia and extended hemorrhages, especially in the integument surrounding the internal organs of the fish. There is a general septicemia, often with large numbers of bacteria in the blood of moribund and newly dead fish. Isolations were attempted from the blood and kidneys. This disease occurs mainly in late autumn, winter, and early spring and has been called cold-water vibriosis or Hitra disease (7). The disease has also been described under the term hemorrhagic syndrome (13) with undefined etiology. We isolated a Vibrio-like bacterium from all of the fish which we examined. Tests with these isolates fullfil Koch's postulates and indicate the bacterial etiology of the disease (7). Characterization of the bacterium strongly indicated that it is a new species of Vibrio. MATERIALS AND METHODS Bacterial strains and growth. Unless otherwise stated, substrates were prepared with 1.5% NaCl, and incubation was at 15°C. The general substrates used throughout this work were nutrient agar (Oxoid Ltd., London, United Kingdom) supplemented with 5% human blood (NBA), and tryptone soy broth (TSB) (Oxoid) supplemented with NaCl to a final concentration of 1.5%. Similar bacteria were isolated from diseased fish all along the Norwegian west coast. Strain HI 7751, which was isolated from a farmed Atlantic salmon in the Bergen region in late 1983, was chosen as the type strain and is described here. Cultures were isolated from moribund or newly dead fish on NBA which was incubated at 15°C. Colonies were detected after 1to 5 days. Salinity optimum and range. The optimum salinity was determined in TSB, which basically contained 0.5% NaCl. NaCl was added to TSB to give final concentrations of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0%. Salt-free TSB was made from the standard ingredients by omitting NaCl. Growth was measured nephelometrically. To test the survival of the bacteria in the presence of different NaCl concentrations, the cultures were streaked onto NBA after 11days, and the plates were observed for 2 weeks. To test for an NaCl requirement, modified TSB was made with 1.5% KCl; TSB containing 1.5% NaCl was used as a control. Temperature optimum and range. The temperature optimum was determined on an NBA plate (50 by 10 cm) on

* Corresponding author.

which a temperature gradient was set up by using a thermostatic cooler at one side and a heater at the other. The growth limits were determined by inoculation onto NBA plates which were incubated at different temperatures. Sensitivity. Sensitivity disks were made by transferring 10 pl of a saturated solution of 2,4-diamino-6,7-di-isopropylpteridine phosphate (vibriostatic agent 01129) in water to a sterile disk, which was then placed in the center of an NBA plate covered with bacteria. Sterile antibiotic sensitivity disks (AB BIODISK; Solna, Sweden) were used to determine novobiocin sensitivity (5 pg/disk). Biochemical tests. Biochemical activity was tested by using the API 50CH system (API System S.A., La balme les Grottes, France) with sterile NaCl added to the fluid to a concentration of 1.5%. Strains were also tested by using the API 20B system supplemented with sterile NaCl at a final concentration of 2.0%. The presence of cytochrome oxidase was tested in 24-h NBA cultures, which were transferred to filter paper with 1%n,n-dimethyl-p-phenylendiaminehydrochloride (12). The reduction of nitrate was tested by using the method of Conn (5). Indole was tested in TSB after 48 h with Kovacs reagent (5). Fermentative metabolism and oxidative metabolism were tested in Hugh-Leifson medium supplemented with 1% glucose by using bromthymol blue as an indicator (4); tests were read after 10 days. Chitinase production was tested on chitin agar plates containing minerals and 0.4% dried, purified chitin by using the method of Hsu and Lockwood (11).Production of lipase was tested by the method of Harrigan and McCance (9), and the ability to decarboxylate arginine was tested on Mgller decarboxylase medium (4). Serology. Rabbit sera were produced against some of the bacterial strains (see Fig. 3). An enzyme-linked immunosorbent assay was performed by the method of Bratthall et al. (2), with some modifications. A 96-well microtitration plate (InterMed; Nunc, Roskilde, Denmark) was coated with bacterial cells. Rabbit sera were diluted 1:4,000 in phosphate-buffered saline (pH 7.2) containing 0.05% Tween 20 and 0.5% bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) . Goat anti-rabbit immunoglobulin G-horseradish peroxidase conjugate (Bio-Rad Laboratories, Richmond, Calif.) was diluted 1:3,000 in phosphate-buffered saline containing Tween 20 and bovine serum albumin. A 100-p1 portion of a solution of 40 mg of 0-diaminobenzene dihydrocloride [E. Merck AG, Darmstadt, Federal Republic of Germany] in 100 ml of phosphate-citrate buffer [pH 5.01 with 40 pl of water added immediately before use was added to each well. The reaction was stopped by adding 50 pl of 2.5 NHZS04 to each well after 3 min, and the optical density at 492 nm was read with a model MR 600 microplate reader (Dynatech Laboratories, Inc., Alexandria, Va.).

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VIBRIO SALMONICIDA SP. NOV.

VOL.36, 1986

FIG. 1. V. sulrnonicidu cell prepared for scanning electron microscopy by uranyl acetate staining , showing a polar tuft of nine sheathed flagella. Prepared by T. Rolfsen, University of Bergen, Bergen, Norway. ~ 7 , 1 2 0 Bar . = 1 Fm.

Pathogenicity tests. Tests for pathogenicity by intraperitoneal injection were performed by using the method of Egidius et al. (7). Tests for waterborne infection were performed as described by Egidius and Andersen (6) at 8 to 9°C. RESULTS AND DISCUSSION

Strain HI 7751T (T = type strain) is a gram-negative, facultatively anaerobic, curved, motile rod-shaped organism. It is oxidase positive and strongly susceptible to vibriostatic agent 0/129; Na+ is required for growth, and the guanine-plus-cytosine content is 42 mol% (14). Growth is oxidative or fermentative. These characteristics, together with its previously described morphology and its biochemical reactions, place this organism in the genus Vibrio (Pacini). The characteristics of this organism differ from those of previously described Vibrio species. We propose the new species described below. Vibrio salmonicida sp. nov. Vibrio salmonicida (sal. mon. i. ci' da. N.L.n. salmon salmon; L.v. caedo to kill; N.L. adj. salmonicida salmon killer). During initial isolation growth on artificial substrates may take 3 to 5 days. After a few passages on such substrates, good growth is obtained within 48 h. In many cases apparently pure cultures are obtained directly from the kidneys of moribund or newly dead fish. Cells from 24-h cultures on NBA are curved rods which are 0.5 by 2 to 3 pm. When they are isolated, strains may show a high degree of pleomorphism. Electron micrographs of cells from 24-h tryptone soy agar plates show the presence of at least nine polar sheathed flagella (Fig. 1). No lateral flagella are observed. Colonies on NBA are small, smooth with entire edges, and grayish; neither pigmentation nor swarming is observed. No hemolysis of either human or sheep blood. The optimum salinity for growth is 1.5% NaCl. Growth occurs at NaCl concentrations between 0.5 and 4.0% (Fig. 2). After 11 days growth is obtained only from cultures containing 3.0, 3.5, and 4.0% NaC1, which may indicate a higher survival capacity at higher salinities. Na' is essential for growth. Growth occurs between 1 and 22°C; optimum growth

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occurs at 15°C. A slight increase in maximal temperature can occur after many passages on an artificial substrate. Cultures are sensitive to vibriostatic agent 0/129, with zones of inhibition up to 30 mm in diameter, and are relatively resistant to novobiocin (diameter of inhibition zone, 18 mm). Biochemical reactions. Catalase and cytochrome oxidase are present. Citrate cannot be used as the only carbon source; nitrates are not reduced. Acetoin, arginine, H*S, and indole are not produced. Chitinase, P-galactosidase, gelatinase, lipase, and urease are not present. D-Fructose, Dgalactose, D-glucose, maltose, ribose, and trehalose are utilized, as are N-acetylglucosamine, gluconate, and mannitol. Glycerol is utilized slowly. The following compounds are not utilized: D-arabinose, L-arabinose, D-cellobiose, Dfucose, L-fucose, f3-gentibiose, lactose, D-lycose, D-mannose, melezitose, melibiose, D-raffinose, rhamnose, sucrose, Lsorbose, D-tagatose, D-turanose, D-xylose, L-xylose, adonitol, D-arabitol, L-arabitol, amygdalin, arbutin, dulcitol, erythritol, esculin, a-methyl-D-glucoside, 2-keto-gluconate, 5-keto-gluconate, glycogen, inositol, inulin, a-methyl mannoside, salicin, sorbitol, starch, xylitol, and p-methyl xyloside. The reactions of the type strain have been tested by using API systems repeatedly during the 2 years since its isolation and seem to be stable, even though the reactions tend to become weaker. Loss of the ability to use gluconate is the only exception. Seven other isolates from diseased fish from different localities all along the Norwegian west coast and one isolate from Shetland (3) have also been tested by using the API systems. The results are similar to those obtained with the type strain, with a few exceptions. Only four of the strains, including the one from Shetland, can utilize galactose. The Shetland strain can utilize D-mannose, and its utilization of glucose varies. Two of the strains do not utilize mannitol. The strains vary in their ability to utilize ribose, and the reaction usually is weak. Holm et al. (10) found the same biochemical pattern as described above for the type strain, with the exception of slow utilization of cellobiose and utilization of gentiobiose and D-mannose. Serology. No cross-reaction is observed with the enzymelinked immunosorbent assay between V. salmonicida and

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EGIDIUS ET AL.

outbreaks occur at lower water temperatures, which is in agreement with the temperature ranges which we found for growth. Disease outbreaks southward along the Norwegian Coast in 1983 might be related to generally lower water temperatures in this region during that year. V. salmonicida (type strain HI 7751) has been deposited with the National Collection of Marine Bacteria, Aberdeen, Scotland, as strain NCMB 2262.

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FIG. 3. Serological differences between V. anguillarum and V . salmonicida. Each group of bars represents the immunological (enzyme-linked immunosorbent assay) reactions between selected bacterial strains and rabbit antisera produced against strains NCMB 2129, NCMB 2133, NCMB ST, NCMB 2262, and HI 7953. The reactions were measured in absorbance units by using the enzymelinked immunosorbent assay technique. Strains NCMB 2129, NCMB 2133, and NCMB 6Tare V. anguillarum strains, and strains NCMB 2262 and HI 7953 are V. salmonicida strains.

three strains of Vibrio anguillarum (Fig. 3 ) . V . salmonicida is serologically distinct from V . anguillarum. Pathogenicity. Intraperitoneal injection of broth cultures of V . salmonicida into both rainbow trout and Atlantic salmon and challenge through water reproduce symptoms identical to those of natural outbreaks. In each instance the bacterium can be reisolated. Atlantic salmon appear to be more susceptible than rainbow trout to the disease. Of 22 previously described species, only Vibrio fisheri (l), Vibrio logei (l),Vibrio marinus (l), and Vibrio damsela (8) have guanine-plus-cytosine contents that are significantly lower than 44 mol%, as is the case with V. salmonicida (14). These four species also are the only species which have the same temperature pattern for growth as V . salmonicida. Pathogenicity may be lost by passage on artificial substrates but can be regained by passage through fish. Disease

LITERATURE CITED 1. Baumann, P., and R. H. W. Schubert. 1984. Family 11. Vibrionaceae Vernon, p. 516-538. In N. R. Krieg and J. G. Holt (ed.), Bergey’s manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 2. Bratthall, D., L. Gahnberg, and B. Krasse. 1978. Method for detecting LGA antibodies to Streptococcus mutans serotypes in parotid saliva. Arch. Oral Biol. 23:843-849. 3. Bruno, D. W., T. S. Hasting, A. E. Ellis, and R. Wotten. 1985. Outbreak of a cold water vibriosis in Atlantic salmon in Scotland. Bull. Eur. Assoc. Fish Pathol. 562-63. 4. Collins, G. H. 1964. Microbiological methods. Butterworths, London. 5. Conn, H. J. 1954. Routine tests for the descriptive chart, p. 13-15. In Society of American Bacteriologists manual of methods for pure culture studies of bacteria. Biotech Publications, New York. 6. Egidius, E., and K. Andersen. 1979. Bath-immunization4 practical and non-stressing method of vaccinating sea farmed rainbow trout (Salmo gairdneri Richardson) against vibriosis. J. Fish Dis. 2:405410. 7. Egidius, E., K. Andersen, E. Claussen, and J. Raa. 1981. Cold-water vibriosis or Hitra-disease in Norwegian salmonid farming. J. Fish Dis. 4:353-354. 8. Grimes, D. J., R. R. Colwell, J. Stemmler, H. Hada, D. Maneval, F. M. Hetrick, E. B. May, R. T. Jones, and M. Stoskopf. 1984. Vibriosis species as agents of elasmobranch disease. Helgol. Meeresunters. 32:309-3 15. 9. Harrigan, W. F., and E. McCance. 1966. Laboratory methods in microbiology. Academic Press, Inc., New York. 10. Holm, K. O., E. Strem, K. Stensvhg, J. Raa, and T. Jargemen. 1985. Characteristics of a Vibrio sp. associated with the Hitra disease of Atlantic salmon in Norwegian fish farms. Fish Pathol. 20: 124-1 29. 11. Hsu, S. C., and J. L. L. Lockwood. 1975. Powered chitin agar as a selective medium for enumeration of Actinomycetes in water and soil. Appl. Microbiol. 29:422426. 12. Kovacs, N. 1956. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature (London) 178:703. 13. Poppe, T. T., T. Hhtein, and R. Salte. 1985. Hitra disease (haemorrhagic syndrome) in Norwegian salmon farming: present status, p. 223-229. In A. E. Ellis (ed.), Fish and shellfish pathology. Academic Press, Inc., New York. 14. Wiik, R., and E. Egidius. 1986. Genetic relationships of Vibrio salmonicida sp. nov. to other fish-pathogenic vibrios. Int. J. Syst. Bacteriol. 36521-523.

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