V~b n o vuln~ficus was isolated in 1996 f~o m 2 d~s e a s e outbreaks on a Danish eel f a~m lvhich used brackish water A charactenstic clinical sign was extensive deep muscle necrosis in the head region V vulnlficus was isolated from k~d n e y mucus spleen gill and intestlne of diseased eels Th~rty-two isolates were examined phenotypically and serologically for pathogenic~ty to eels and for correlat~on to nbotype and plasmid profile B~ochemically, the ~solates showed propert~es slm~lar to those described previously for eel-pathogenic strains of V i/ulnlficus w~t h the exception of lndole prod u c t~o n Virulence was evaluated by LDSo (the 50 '0 lethal dose) which ranged from < 9 4 X 103 to 2 3 X 105 CFU (colony-formng units) per f~s h The isolates which were lethal for eels showed identical nbotypes and serotypes A relat~onship between certain plasm~ds and virulence \4as not found A serotyping system based on l~popolysacchande (LPS)-associated O antigen type and on carbohydrate capsule antigens showed that the eel-virulent ~solates shared a common LPS-based homogeneous O serogroup and a capsule antigen V rulnlficus serovar 0 4 and capsule type 9 was identical serologically to the Japanese isolate ATCC 33149 and was the agent responsible for the disease outbreaks that occurred on the Danish eel farm Despite absence of ant~biotic resistance treatment had llttle effect and disease reoccurred

During the unusually warm summer in Denmark in 1994, 11 clinical cases of Vibrio vulnificus infection were reported. These reports initiated an investigation of the occurrence of V. vulnificus biotypes in Danish marine environments. Samples of coastal water, sediment, shellfish, and wild fish were analyzed by preenrichment in alkaline peptone water amended with polymyxin B (2.0 × 104 U/liter) followed by streaking onto modified cellobiose-polymyxin B-colistin agar. V. vulnificus-like colonies were tested with a V. vulnificus-specific DNA probe. Low densities of V. vulnificus were detected in water (0.8 to 19 CFU/liter) from June until mid-September and in sediment (0.04 to >11 CFU/g) from July until mid-November. The presence of V. vulnificus was strongly correlated with water temperature. However, we isolatedV. vulnificus from water from a mussel farm at a lower temperature than previously reported (7°C). In 1 of the 13 locations studied, V. vulnificus was found in mussels in 7 of 17 samples analyzed; this is the first report of V. vulnificusin European shellfish. V. vulnificus was also isolated from gills, intestinal contents, and mucus from wild fish. Although biotyping of 706 V. vulnificus strains isolated during our investigations revealed that the majority of the strains (99.6%) belonged to biotype 1, biotype 2 was detected in seawater at a low frequency (0.4%). Our findings provide further evidence that seawater can serve as a reservoir and might facilitate spread of V. vulnificus biotype 2 to eels, with subsequent spread to persons handling eels. In conclusion, our data demonstrate that V. vulnificus is ubiquitous in a temperate marine environment and that V. vulnificus biotype 2 is not strictly confined to eels.

No abstract

It is well known that Aeromonas salmonicida attacks a variety of freshwater fish (McCarthy 1977). However, there have only been two reports of its presence in marine species, the sablefish, Anoplopoma fimbria (Pallas), (Evelyn 1971) and Atlantic cod, Gadus morhua L., (Cornick, Morrison, Zwicker & Shum 1984). Both of these bioisolates were chemically atypical.This paper describes the first reported infection of sand-eels, Ammodytes lancea (Cuvier) and Hyperoplus lanceolatus (Lesauvege), with Aeromonas salmonicida. In September 1984, approximately 500 sand-eels were caught in a pound net in the northern Kattegat. The fish were placed in partly recirculating seawater (34%^) tanks (2x2x0-5 m) at the Hirtshals Laboratory. The sand-eel catch consisted of both Ammodytes lancea and Hyperoplus lanceolatus. The mean length of the Ammodytes was 12 cm and of the Hyperoplus about 16 cm. The catch also contained 15 herring, Clupea harengus L., with a mean length of 10 cm.A few days after capture, some sand-eels were observed to have haemorrhages on the snout. However, this was at first attributed to stress induced by the restrictive conditions of the tanks.The condition developed quickly and small haemorrhages appeared on the caudal fin with subsequent necrosis of the tail. Some of the sand-eels had lesions on the flank with haemorrhagic patches and typically umbonate furuncles (Fig. 1). Affected fish sometimes died within a week but often the disease progressed more slowly.At post-mortem, the main pathological features were haemorrhages in the musculature and focal haemorrhages in the liver and intestine. The mortality increased during the month after capture to a maximum of 5% per day, and then gradually declined to less than 0 5% per day.Thirty-six sand-eels were examined bacteriologically during the 10 months following capture. The bacteriological samples were taken from the kidneys and sent in Stuart's transport medium (Oxoid) to the Fish Disease Laboratory. The initial cultures were made on marine agar (Difco) and blood agar base (Difco) both with 5% citrated calf blood and incubated at 20°C for 48 h. The inoculated plates showed a pure growth of one colony type, which grew best on blood agar. Biochemical tests were carried out on Correspondence: Dr I. Dalsgaard, Danmarks Fiskeri-og HavundersOgelser, Fiskepatologisk Laboratorium, c/o Den

Methods for the identification and isolation of environmental isolates of Vibrio vulnificus were evaluated. Alkaline peptone water supplemented with polymyxin B and colistin-polymyxin B-cellobiose agar were employed for the isolation of suspected V. vulnificus from water, sediment and shellfish samples. When comparing the identification of putative V. vulnificus obtained with the API 20E assay and an oligonucleotide probe, 29 API 20E profiles were obtained with only four profiles (representing 20 isolates) reaching the identification threshold of V. vulnificus among a total of 66 isolates hybridizing with the probe. The results indicated that, compared with colony hybridization, the API 20E assay was not adequate for the identification of environmental isolates of V. vulnificus.