Comparative study of phenotypic and virulence properties in Vibrio vulnificus biotypes 1 and 2 obtained from a European eel farm experiencing mortalities

Abstract

Strains of Vibrio vuln~flcus biotype 2 were recovered from diseased European eels Angujlla anguilla during 2 epizootics These stralns were biochemically and serologically identical to the ATCC 33149 strain, which was lsolated from the Japanese eel Angujlla japonica. The isolates of biotype 2 from European eels were more virulent for elvers (LD50 < 10' cfu) than the Japanese strain used as control (LDS0 = 104 cfu). All strains of biotype 2 were able to adhere to human and fish cell lines and were also strongly cytotoxic. The extracellu1a1-products (ECPs) of the Japanese strain exhibited lipolytic, phospholipolytic, proteolytic, and haernolyt~c activities, whereas the ECPs of the Spanish isolates displayed only proteolytic activity. Strains of biotype 1 were recovered from tank water and healthy eels during the second outbreak. These isolates were biochemically similar to the type strain ATCC 27562, and serologically distinct from the isolates of biotype 2. Strains of biotype 1 produced proteases, haemolysins, and cytotoxins and showed specific adhesion to human cells, but they were non-pathogenic for eels. The strains of biotype 2 were more virulent for mice (LDS" = 10' to 106cfu) than the strains of biotype 1 (LDS(, = 10%fu), suggesting that this eel pathogen may also represent a health hazard problem for fish farmers.

Phenotypic and genotypic characterization of a new fish-virulent Vibrio vulnificus serovar that lacks potential to infect humans

“…This finding was confirmed after LPS extraction and analysis by immunoblotting. Thus, all SerA strains exhibited identical LPS with a ladder-like structure that differed from that found in the control SerE strain and from those previously published for BT1, BT2-SerE and BT3 strains (Amaro et al, 1992b;Biosca et al, 1996;Bisharat et al, 2007). In addition, no cross-reactions were detected between LPS from SerA and SerE strains, although some cross-reactions with antiSerO3, -SerO3/O4 and -BT3 sera that affected the lipid A and core region of SerA-LPS were detected.…”

“…Bacterial strains were routinely grown on Tryptic Soy Agar (Oxoid) or Tryptic Soy Broth (Oxoid) supplemented with 0.5 % (w/v) NaCl (TSA-1 or TSB-1) at 28 uC for 24 h. The strains were maintained both as lyophilized stocks and as frozen stocks at 280 uC in marine broth (Difco) plus 20 % (v/v) glycerol. (Amaro et al, 1992b;Sanjuán & Amaro, 2004). Motility was assayed on TSA-1 motility plates containing 0.3 % (w/v) Bactoagar (Difco) (Gardel & Mekalanos, 1996).…”

“…Fresh and heated (100 uC for 2 h) whole-cell suspensions containing 10 8 c.f.u. ml 21 of each strain were used as antigens (Amaro et al, 1992b). A distinct and immediate agglutination was registered as positive.…”

“…V. vulnificus isolates were serologically characterized by slide-agglutination with rabbit polyclonal antisera according to the previously described procedure (Amaro et al, 1992b). Antisera were prepared by intravenous injection of New Zealand rabbits with formalin-killed cells from strains of SerE (CECT 4604), SerA (CECT 5198), BT1 (ATCC 27562) and BT3 (VV12) according to the procedure described by Sorensen & Larsen (1986).…”

“…Motility was assayed on TSA-1 motility plates containing 0.3 % (w/v) Bactoagar (Difco) (Gardel & Mekalanos, 1996). Antibiotic susceptibility was checked by the disc diffusion method on Mueller-Hinton agar (Difco) supplemented with 1 % (w/v) NaCl (Amaro et al, 1992b) using tetracycline (20 mg per disc), flumequine (30 mg per disc), sulfamethoxazole/trimethoprim (25 mg per disc) and oxolinic acid (2 mg per disc) (Oxoid). Inhibition zones were read following incubation at 28 uC overnight.…”