demonstrated that V. damsela is a common member of the .... United States. 2 ..... Grimes, D. J., D. Jacobs, D. G. Swartz, P. R. Brayton, and R. R. Colwell. 1993.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1997, p. 3711–3715 0099-2240/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 63, No. 9
Vibrio damsela Associated with Diseased Fish in Denmark KARL PEDERSEN,1* INGER DALSGAARD,2
AND
JENS LAURITS LARSEN1
Laboratory of Fish Diseases, Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University,1 and Fish Disease Laboratory, Danish Institute for Fisheries Research,2 DK-1870 Frederiksberg C, Denmark Received 27 September 1996/Accepted 27 June 1997
A total of 26 Vibrio damsela strains were isolated in Denmark. Fifteen strains were isolated from the head kidney of rainbow trout (Oncorhynchus mykiss) in aquaculture, eight were from organs of two stingrays (Dasyatis pastinaca) held in captivity, two were from organs and pathological material of a nurse shark (Orectolobus ornatus) held in captivity, and one was from the water in the aquarium where the rays and shark were kept. This is the first report on the isolation of V. damsela in Denmark and the first report on this organism associated with diseased farmed rainbow trout. Ecological and epizootiological aspects of the observation are discussed on the basis of phenotypic and genotypic properties of the isolated strains. examined bacteriologically during the period 1993 to 1996. All fish and water samples were sent, on the day of sampling, on ice or similar material at a temperature of 0 to ;10°C. This procedure ensured that the fish had not undergone significant autolysis upon arrival at the laboratory. Fish were aseptically opened, after which initial cultures from abdominal organs were made on marine agar (Difco, Detroit, Mich.) supplemented with 5% calf blood (blood agar [BA]) and incubated at 20°C for 1 to 2 days. Water samples were spread on BA and on thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Difco) and incubated at 20°C for 1 to 2 days. Colonies from organs of fish and dominant colonies from water samples were selected and identified. V. damsela was identified by its reaction in oxidase, catalase, arginine dihydrolase, lysine decarboxylase, and ornithine decarboxylase, by Gram staining and the VogesProskauer test, and by its sensitivity to O/129 (2,4-diamino-6,7diisopropylpteridine), acid and gas from glucose, o-nitrophenyl-b-D-galactopyranoside, indole, and urease. The V. damsela isolates were further characterized according to the methods of Fouz et al. (8) as outlined by Barrow and Feltham (2). For plasmid profiling, bacteria were propagated overnight at 20°C in veal infusion broth (Difco) supplemented with 0.5% NaCl. Plasmid DNA was extracted by the method of Kado and Liu (15) as described by Pedersen et al. (26), separated by gel electrophoresis in 0.8% agarose gels (SeaKem GTG; FMC BioProducts, Rockland, Maine) in Tris-acetate-EDTA buffer (40 mM Tris, 5 mM sodium acetate, 1 mM EDTA [pH 8.0]), stained with ethidium bromide (2 mg/ml), and photographed in UV light. Isolation of total DNA for ribotyping was performed as described by Pedersen and Larsen (24). Cells from 1.5 ml of overnight broth cultures were harvested by centrifugation, resuspended in 500 ml of Tris-EDTA buffer (50 mM Tris, 50 mM EDTA [pH 8.0]), and lysed with 20 ml of 10% sodium dodecyl sulfate. The lysates were incubated at 56°C for 30 min, after which cell debris, etc., was extracted with 700 ml of phenolchloroform-isoamyl alcohol (25:24:1) and then with 700 ml of chloroform-isoamyl alcohol (24:1). DNA was precipitated with 300 ml of isopropanol and washed twice with 500 ml of 70% ethanol. Finally, the DNA was dissolved in 50 ml of Tris-EDTA (10:1) buffer, pH 7.6. Restriction enzyme digestion was performed as follows. Five microliters (;5 to 10 mg) of DNA solution was digested with HindIII (Boehringer, Mannheim, Germany) in a total volume of 50 ml. Digested DNA was precipitated with 22 ml of 7.5 M ammonium acetate and 130 ml of 96% ethanol, centrifuged, and resuspended in 20 ml of
Vibrio damsela is a relatively recently described Vibrio species, first isolated from damselfish (Chromis punctipinnis) and described by Love et al. in 1981 (18). It has been described as a human pathogen (3, 19) that may cause fatal infections, and its pathogenicity has been partly attributed to the production of a powerful cytolysin (16). However, the organism has also been associated with disease in a number of marine animals. Thus, it has been described as the primary bacterium causing wound infections in bottlenose dolphins (Tursiops truncatus) (9); infection in leatherback turtles (Dermochelys coriacea) (22), damselfish (Chromis punctipinnis) (18), yellowtail (Seriola quinqueradiata) (28), sea bream (Sparus aurata) (29), barramundi (Lates calcarifer) (27), and brown shark (Carcharhinus plumbeus) (4, 11, 12); and, more recently, infections in farmed turbot (Scophthalmus maximus) (7, 8). In addition, it has been demonstrated that V. damsela is a common member of the natural microflora of healthy carcharhinid sharks (10). In Denmark, V. damsela has not previously been isolated either from fish or from any other sources. However, during the summer of 1994 and 1995, we experienced periods with unusually high temperatures from the beginning of July until mid-August. During these periods, the water temperatures were up to 5°C higher than normal (5) and increased mortality due to various bacterial infections was recorded among rainbow trout in aquaculture. The present paper reports the characteristics of a number of V. damsela strains isolated from diseased rainbow trout in aquaculture during these warm summer periods and from two rays and a shark held in captivity. A total of 26 Danish isolates of V. damsela were studied together with five reference strains (Table 1). Eight strains were isolated from organs of two different stingrays, two strains were isolated from a nurse shark, and one strain was isolated from the water in the nurse shark’s aquarium, and 15 strains were isolated from the head kidneys of 15 rainbow trout from five different fish farms. The salinity of the water where the fish were kept ranged from 20 to 25‰ for the rainbow trout and from 30 to 32‰ for the shark and rays. The fish were submitted to the laboratory as soon as possible after death, and the water samples from the aquaria were submitted immediately after sampling. A total of 16 water samples were submitted and
* Corresponding author. Mailing address: Laboratory of Fish Diseases, Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Bu ¨lowsvej 13, DK-1870 Frederiksberg C, Denmark. 3711
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PEDERSEN ET AL. TABLE 1. Vibrio damsela isolates from Denmark and five reference strains
Isolate no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Strain
Source
Yr of isolation or publication
Country
ATCC 33539 ATCC 35083 CDC 2227-81 CDC 1421-81 RM 71 94-11-229 94-11-230 94-11-231 94-11-232 94-11-233 95-2-28 95-2-29/1 95-2-29/2 940804-1/1 940804-1/2 940804-2/1a 940804-2/3 940804-2/4 940804-2/5a 950810-3/2 950810-3/4 950810-3/5 950823-1/3B 950823-1/5 950825-2/4A 950828-1/3 950901-2/2B 950901-2/5B 96-1-22/B 96-2-25/1 96-2-26
Damselfish (Chromis punctipinnis) Brown shark (Carcharhinus plumbeus) Human (Homo sapiens) Human (H. sapiens) Turbot (Scophthalmus maximus) Kidney, stingray (Dasyatis pastinaca) Spleen, stingray (D. pastinaca) Liver, stingray (D. pastinaca) Heart, stingray (D. pastinaca) Muscle, stingray (D. pastinaca) Liver, stingray (D. pastinaca) Muscle, stingray (D. pastinaca) Muscle, stingray (D. pastinaca) Kidney, rainbow trout (Oncorhynchus mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Kidney, rainbow trout (O. mykiss) Water from aquarium with sharks and rays held in captivity Liver, nurse shark (Orectolobus ornatus) Pathological material, nurse shark (O. ornatus)
1981 1984 1981 1981 1988 1994 1994 1994 1994 1994 1995 1995 1995 1994 1994 1994 1994 1994 1994 1995 1995 1995 1995 1995 1995 1995 1995 1995 1996 1996 1996
United States United States United States United States Spain Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark Denmark
distilled water. Ten microliters of loading buffer (2 mM EDTA [pH 8.0], 0.1% bromophenol blue, 30% glycerol) was then added to the resuspended material, and the mixture was subjected to electrophoresis in 0.8% agarose gels in Tris-acetateEDTA buffer. Subsequently, gels were stained and photographed as described above. DNA fragments were blotted onto nylon membranes (Hybond-N; Amersham) with a vacuum blotter (VacuGene XL; Pharmacia LKB, Uppsala, Sweden) and fixed to the membranes by baking at 80°C for 1 h. The membranes were hybridized overnight at 56°C with a digoxigenin-labelled DNA probe complementary to 16S and 23S rRNA. The probe was made by reverse transcription of 16S and 23S rRNA from Escherichia coli. Subsequent immunoenzymatic detection of hybridized fragments was carried out by incubation with alkaline phosphatase-labelled anti-digoxigenin antibody, followed by the addition of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate. From the organs of the rays and shark and from 12 of the 15 rainbow trout, V. damsela was recovered alone, whereas from the remaining 3 rainbow trout, V. damsela was recovered from the head kidneys in mixed culture with Aeromonas salmonicida, indicating a double infection. The bacteriological findings clearly demonstrated that V. damsela and A. salmonicida were the causes of death and that the fish had not been held too long from death to sampling. Examination of 16 water samples from the aquaria in which the rays and shark were kept detected V. damsela in one sample. However, in the water samples, mixed cultures were always found, including Vibrio anguillarum, Vibrio alginolyticus, and Vibrio harveyi or Vibrio carchariae. A total of 26 V. damsela isolates were collected and subjected to further phenotypic and genotypic investigation to-
gether with five reference strains. After 48 h at 20°C colonies of V. damsela were 2.5 to 3.0 mm in diameter, low convex, greyish white, entire, and smooth, and all of them displayed hemolytic activity on BA. The biochemical and physiological properties of these strains were very homogeneous irrespective of their origin (Table 2). Variation was recorded in only a few characteristics. The strains were all gram negative, motile but nonswarming rods that were catalase, oxidase, methyl red, and nitrate positive but indole and H2S negative, and they formed green colonies on TCBS agar. They produced arginine dihydrolase but not ornithine decarboxylase. Urease and chitinase were produced, but amylase, caseinase, and alginase were not produced. Glucose was degraded by the fermentative pathway under evolution of gas. Acid was produced from glucose and maltose but not from salicin, arabinose, rhamnose, inositol, or mannitol. o-Nitrophenyl-b-D-galactopyranoside and esculin were not degraded. The strains were able to grow at both 20 and 37°C and in 6% NaCl but not in 0 or 10% NaCl. Most strains were positive in the Voges-Proskauer test and for lipase, cellobiose, growth in 7% NaCl, and sensitivity to O/129 but negative for lysine decarboxylase and gelatinase; however, some variation was recorded for these characteristics. On the basis of these variable characteristics, nine different biotypes were recorded (Table 2). This biochemical and physiological homogeneity is in accordance with previous reports by Austin et al. (1) and Fouz et al. (8). In contrast, Grimes et al. (13) found variation in a number of characteristics among 52 V. damsela strains. Most reports (8, 11–13) found V. damsela urease positive. Urease activity, which is also present in V. carchariae, is believed to be important for its pathogenicity to elasmobranch fish. Interestingly, four
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TABLE 2. Biochemical and physiological characteristics of Vibrio damsela reference strains and Danish isolates Property of biotype no. (isolate no.): Characteristic
Voges-Proskauer Lysine decarboxylase Gelatinase Lipase Cellobiose Growth in 7% NaCl Sensitivity to O/129 (10 mg and 150 mg)a a
1 (1, 3, 4, 11– 19, 29, 30)
2 (2)
3 (5)
4 (6–10)
5 (20, 21, 26)
6 (22, 25)
7 (23, 24)
8 (27, 28)
9 (31)
1 2 2 1 1 1 S
1 2 2 2 1 1 S
1 2 2 1 1 2 S
2 2 2 1 1 1 S
1 1 2 1 2 1 S
1 2 2 1 2 1 S
1 1 1 1 1 2 R
1 2 1 1 1 1 R
1 2 1 1 1 1 S
S, sensitive; R, resistant.
strains in the present study were resistant to a vibriostatic agent, O/129. These strains were also resistant to trimethoprim, whereas all other strains were sensitive. This crossresistance between O/129 and trimethoprim has previously been described for V. anguillarum by Muroga et al. (20) and Pedersen et al. (25). A total of 17 different ribotype patterns were found (Fig. 1 and Table 3). These patterns were given numbers from 1 to 17. The results demonstrated that ribotyping was a good method for subtyping of V. damsela in ecological and epizootiological studies. Several different ribotype patterns were found among the strains from rainbow trout, indicating that there has not been a clonal spread of a single strain but the simultaneous occurrence of several clones. This situation is parallel to a number of human cases of infection with Vibrio vulnificus that also occurred in Denmark during the summers of 1994 and 1995. Some of these infections were fatal (6). The sources of the infections are unknown for both organisms. However, because of the presence of several different clones of the bacterium, it may be suggested that the bacterium is normally present in Danish coastal waters but in low numbers that will
not cause any disease problems. Further investigations of the prevalence of V. damsela in Danish coastal waters will be carried out, but they are still hampered by the lack of selective media and species-specific probes. It is possible that the simultaneous occurrence of V. damsela and V. vulnificus in 1994 and 1995 is associated with the unusually high (for Danish conditions) water temperatures (5). It is a well-known fact that changes in temperature or climate in a specific area may affect the infectious disease pattern and cause the multiplication or emergence of pathogens not usually seen there. This point has most recently been emphasized by Holzman (14), but Larsen and Mellergaard (17) also found a close relationship between water temperature and the incidence of vibriosis and furunculosis in rainbow trout in Danish marine aquaculture facilities. The occurrence of several clones and, indeed, strains displaying two different ribotype patterns from the same fish (strain 11 versus 12 and 13) and the isolation of the same clone from a water sample and 1 month later from the organs of a dead fish from the same aquarium (strains 29 and 30) also suggest that the infectious agents were already present in the environment and were able to cause infection under stressful conditions,
FIG. 1. Ribotype patterns of V. damsela isolates. Among 31 strains, a total of 17 different patterns were identified. (A) Lanes 1 and 17, HindIII-digested l DNA as molecular size markers (in kilobases); lane 2, pattern 1, strain 1; lane 3, pattern 2, strain 2; lane 4, pattern 3, strain 3; lane 5, pattern 4, strain 4; lane 6, pattern 5, strain 14; lane 7, pattern 6, strain 25; lane 8, pattern 7, strain 21; lane 9, pattern 8, strain 6; lane 10, pattern 9, strain 27; lane 11, pattern 10, strain 23; lane 12, pattern 11, strain 5; lane 13, pattern 12, strain 15; lane 14, pattern 13, strain 16; lane 15, pattern 14, strain 11; lane 16, pattern 15, strain 12. (B) Lane 1, HindIII-digested l DNA (arrows correspond to molecular sizes in panel A); lane 2, pattern 16, strain 29; lane 3, pattern 17, strain 31.
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APPL. ENVIRON. MICROBIOL.
TABLE 3. Ribotypes and plasmid content of Vibrio damsela strains Isolate no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 a
Strain
ATCC 33539 ATCC 35083 CDC 2227-81 CDC 1421-81 RM 71 94-11-229 94-11-230 94-11-231 94-11-232 94-11-233 95-2-28 95-2-29/1 95-2-29/2 940804-1/1 940804-1/2 940804-2/1a 940804-2/3 940804-2/4 940804-2/5a 950810-3/2 950810-3/4 950810-3/5 950823-1/3B 950823-1/5 950825-2/4A 950828-1/3 950901-2/2B 950901-2/5B 96-1-22/B 96-2-25/1 96-2-26
Ribotype patterna
Plasmid content (kb)
1 2 3
16, 190 No plasmids 170
4
170
11 8 8 8 8 8 14 15 15 5 12 13 13 13 13 7 7 7 10 10 6 7 9 9 16 16 17
170 5.8, 41, 47, 62 5.2, 5.8, 41, 47, 62 5.8, 41, 47, 62 5.2, 5.8, 41, 47, 62 5.2, 5.8, 41, 47, 62 41, 47, 62, 76, 103 47, 76 47, 76 25 4.1 3.0, 4.1 3.0, 4.1, 170 3.0, 4.1 3.0, 4.1 No plasmids No plasmids No plasmids No plasmids No plasmids 25 38 170 170 67, 155 67, 155 5.2, 29, 45
Ribotype patterns were given arbitrary pattern numbers from 1 to 17.
such as high temperatures, introduction of new fish or fish species, transport, the presence of other pathogens, etc. The finding of two different ribotype patterns from the same fish was most likely caused by a double infection by two different strains, since ribotype patterns have been shown to be stable during an outbreak (24). The results thus give evidence to suggest that V. damsela should not be considered a primary fish pathogen but more an opportunistic pathogen attacking the stressed hosts. This is also in accordance with the disease patterns on the fish farms in the present study. Thus, on one farm, there was an outbreak of disease among rainbow trout where the only pathogenic agent that was found was V. damsela. On a second farm, strains were isolated from fish during periods of increased mortality. Finally, the majority of the V. damsela isolates were recovered during periods of outbreaks of furunculosis caused by Aeromonas salmonicida subsp. salmonicida. Here, V. damsela could be isolated in pure culture from some fish and as a mixed culture together with A. salmonicida in other fish. Most strains (25 of 31) carried one or more plasmids, and in all, 14 different plasmid profiles were recorded (Table 3). The plasmids were heterogeneous in size, varying from very small (3.0 kb) to very large (;190 kb). No single plasmid was present in all strains, but some of the plasmids were detected in several strains. Plasmids have previously been reported to be very common in V. damsela (1, 8). In the present study, 25 of the 31 strains contained plasmids. There was considerable diversity in the plasmid profiles, although some plasmids were found in
more than one strain. The strains from stingrays all had a 47-kb plasmid, and most strains also had a 5.8-kb plasmid, a 41-kb plasmid, and a 62-kb plasmid. Likewise, some strains from rainbow trout carried an ;170-kb plasmid, suggesting an epidemiological relationship among some of the strains. However, as more than one plasmid profile was recorded on each farm and even among isolates from the same stingray, the results also indicate that plasmid profiles are diverse among V. damsela and that no common virulence plasmid is present. This is in accordance with previous observations by Austin et al. (1) and was also recently reported for atypical A. salmonicida (23). Fish in marine aquaculture are usually vaccinated against vibriosis caused by V. anguillarum and in certain areas are also vaccinated against Vibrio salmonicida. In many countries, such vaccination procedures have reduced the number of outbreaks of disease caused by these bacteria dramatically. However, this has given other bacterial species the opportunity to emerge and cause infections (21). Although V. damsela has not previously been recognized as a pathogen in salmonid fish, challenge experiments with both Atlantic salmon (Salmo salar) (1) and rainbow trout (8) have shown that this organism is highly virulent for both species by intraperitoneal injection. These and other reports suggest that V. damsela is not a speciesspecific fish pathogen. It may therefore be anticipated that more outbreaks of disease caused by this organism in salmonid fish in aquaculture will occur in the future, depending on the water temperature during the summer season. The technical assistance of Maj-Britt Højgård and Kirsten Kaas is gratefully acknowledged. The financial support of the Danish Agricultural and Veterinary Research Council, grant no. 9503658, is appreciated. REFERENCES 1. Austin, B., D. A. Austin, A. R. Blanch, M. Cerda ´, F. Grimont, P. A. D. Grimont, J. Jofre, S. Koblavi, J. L. Larsen, K. Pedersen, T. Tiainen, L. Verdonck, and J. Swings. 1997. A comparison of methods for typing of fish-pathogenic Vibrio spp. Syst. Appl. Microbiol. 20:89–101. 2. Barrow, G. I., and R. K. A. Feltham (ed.). 1993. Cowan and Steel’s manual for the identification of medical bacteria. Cambridge University Press, Cambridge, England. 3. Clarridge, J. E., and S. Zighelboim-Daum. 1995. Isolation and characterization of two haemolytic phenotypes of Vibrio damsela associated with a fatal wound infection. J. Clin. Microbiol. 21:302–306. 4. Colwell, R. R., and D. J. Grimes. 1984. Vibrio diseases of marine fish populations. Helgol. Meeresunters. 37:265–287. 5. Dalsgaard, A., I. Dalsgaard, L. Høi, and J. L. Larsen. 1995. Prevalence and significance of Vibrio vulnificus in Danish coastal waters. Evaluation of API 20E and a DNA probe for the identification of V. vulnificus. Dan. Veterinærtidsskr. 78:496–501. 6. Dalsgaard, A., N. Frimodt-Møller, B. Bruun, L. Høi, and J. L. Larsen. 1996. Clinical manifestations and molecular epidemiology of Vibrio vulnificus infections in Denmark. Eur. J. Clin. Microbiol. Infect. Dis. 15:227–232. 7. Fouz, B., J. L. Larsen, and A. E. Toranzo. 1991. Vibrio damsela as a pathogenic agent causing mortality in cultured turbot (Scophthalmus maximus). Bull. Eur. Assoc. Fish Pathol. 11:80–81. 8. Fouz, B., J. L. Larsen, B. Nielsen, J. L. Barja, and A. E. Toranzo. 1992. Characterization of Vibrio damsela strains isolated from turbot Scophthalmus maximus in Spain. Dis. Aquat. Org. 12:155–166. 9. Fujioka, R. S., S. B. Greco, M. B. Cates, and J. P. Schroeder. 1988. Vibrio damsela from wounds in bottlenose dolphins Tursiops truncatus. Dis. Aquat. Org. 4:1–8. 10. Grimes, D. J., P. Brayton, R. R. Colwell, and H. Gruber. 1985. Vibrios as autochthonous flora of neritic sharks. Syst. Appl. Microbiol. 6:221–226. 11. 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. Vibrio species as agents of elasmobranch disease. Helgol. Meeresunters. 37:309–315. 12. Grimes, D. J., J. Stemmler, H. Hada, E. B. May, D. Maneval, F. M. Hetrick, R. T. Jones, M. Stoskopf, and R. R. Colwell. 1984. Vibrio species associated with mortality of sharks held in captivity. Microb. Ecol. 10:271–282. 13. Grimes, D. J., D. Jacobs, D. G. Swartz, P. R. Brayton, and R. R. Colwell. 1993. Numerical taxonomy of gram-negative, oxidase-positive rods from carcharhinid sharks. Int. J. Syst. Bacteriol. 43:88–98. 14. Holzman, D. 1996. Global changes affect infectious disease patterns. ASM News 62:238–239.
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