The significance of Aeromonas hydrophila in association with disease outbreaks in aquaculture production in the Zhejiang province of China was investigated. Bacteriological examination of moribund fish and crabs resulted in 95 bacterial isolates: 88 bacterial isolates from fish and 7 isolates from crabs. PCR and traditional biochemical methods were used for identification of A. hydrophila. Out of 69 motile aeromonads, 35 isolates were identified as A. hydrophila by biochemical tests. However, 6 of those were not identified as A. hydrophila by a species specific PCR method. Serotyping revealed 2 dominant serotypes (O9 and O97) among A. hydrophila isolates. The data presented show that approximately 42% of the motile aeromonads isolated from disease outbreaks among various fish species were A. hydrophila. It is noteworthy that A. hydrophila accounted for more than 50% of the isolated aeromonands isolated from crucian carp Carassius carassius and Wuchang bream Megalobrama amblycephala with haemorrhagic septicaemia. Although this species was the most frequently isolated organism from internal organs of diseased fish and crabs in the present study, other motile Aeromonas spp. were also found. The PCR assay was useful in preventing misidentification of A. hydrophila, which may occur when only phenotypic tests are employed. KEY WORDS: Aeromonas hydrophila · PCR · China · Haemorrhagic septicaemia · Fish diseases Resale or republication not permitted without written consent of the publisherDis Aquat Org 46: [23][24][25][26][27][28][29] 2001 primary pathogen of freshwater fish or a secondary opportunistic pathogen of compromised or stressed hosts (Jeney & Jeney 1995). A. hydrophila has been associated with tail and fin rot, haemorrhagic septicaemia and epizootic ulcerative syndrome (EUS) (Austin & Adams 1996, Roberts 1997. A. hydrophila has also been described as the dominant infectious agent of 'fish-bacterial-septicaemia' in freshwater cultured cyprinid fishes, mainly crucian carp Carassius carassius, Wuchang bream Megalobrama amblycephala and silver carp Hypophthalmichthys molitrix in the Zhejiang province and other provinces in the Southeast of China between 1989to 1993(Qian et al. 1997. 'Fish-bacterial-septicaemia' occurs each summer in the Zhejiang province and results in significant losses for the fish farmers of the region; from 1989 to 1991 the losses were estimated to have been approximately 2200 tons of fish per year (D. Qian pers. comm.).The taxonomy of the genus Aeromonas has been revised, and new motile, mesophilic species have been identified: A. allosaccharophila, A. veronii biogroups sobria and veronii and A. encheleia have been reported as fish pathogens (Toranzo et al. 1989, Paniagua et al. 1990, Joseph & Carnahan 1994, Esteve et al. 1995. Still, A. hydrophila is regarded as the predominant fish pathogen within the mesophilic aeromonads, although its importance may have been overestimated in the past.Thus, the purpose of the present study was to estimate the actual significance of Aeromonas...

The clinical manifestations of and epidemiological data from 11 patients infected with Vibrio vulnificus admitted to Danish hospitals during the unusually warm summer of 1994 are reported. All patients contracted the disease after exposure to seawater; however, none had consumed seafood. Four patients developed bacteremia, one of whom subsequently died; nine patients, including the four with bacteremia, exhibited skin manifestations. Four patients contracted the disease while fishing; in at least one case the patient had handled eels. All Vibrio vulnificus strains were highly susceptible to 11 antimicrobial agents tested. Plasmid analysis revealed that 8 of 11 strains carried plasmids. Ribotyping using the enzyme HindIII on the 11 strains showed five different types, two of which comprised four strains each. The present study provides the first clinical and epidemiological data about a series of human Vibrio vulnificus infections from a temperate zone.

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.

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.