Protease and virulence of the extracellular products produced by Vibrio carchariae after growth on various media.

Protease and virulence of the extracellular products (ECP) of Vibrio carchariae strain EmI82KL, a causative agent of gastroenteritis in Epinephelus coioides, cultured on different media were studied. The bacteria grown on peptone agar, nutrient agar or brain heart infusion agar produced higher protease activities than that grown on tryptic soy agar (TSA) in terms of protein content. The addition of ethylenediamine di(o-hydroxyphenylacetic acid) or horse serum in TSA enhanced the ECP protease production while the addition of grouper serum apparently reduced the enzyme activity indicating the presence of protease inhibitor(s) in the fish serum. Furthermore, the use of grouper meat or peptone as a single nutrient source remarkably enhanced the production of ECP protease. Adding proteinaceous materials from animal sources (horse serum, grouper meat or peptone) on agar manifested higher ECP protease activity than that from plant source (TSA), indicating the intestine of carnivorous groupers might favour the existence, survival or infection of the bacterium. The protease was a metal-chelator-sensitive serine protease since it was inhibited by 3,4-dichloroisocoumarin and phenylmethanesulfonyl fluoride while about 80% of its activity inhibited by chelating agents (ethylene-diaminetetraacetic acid and ethylene glycol-bis(beta-amino-ethylether) N,N,N',N'-tetraacetic acid). The ECP obtained from each medium was not lethal to the groupers suggesting that the bacterium is low virulent. As grouper is carnivorous, therefore, the role of the protease played in the fish intestine probably is competing for nutrients and/or associated with the cause of edema leading to gastroenteritis.

Isolation and characterization of Vibrio carchariae, a causative agent of gastroenteritis in the groupers, Epinephelus coioides.

An outbreak of serious mortality among the cultured groupers Epinephelus coioides, characterized by a swollen intestine containing yellow fluid, occurred in the summer of 1993 in Taiwan. A motile strain EmI82KL was isolated from the intestinal yellow fluid of the moribund groupers with tryptic soy agar supplemented with 2% NaCl and/or thiosulfate citrate bile salt sucrose agar. This strain was characterized and identified as Vibrio carchariae and was susceptible to chloramphenicol, doxycycline-HCl, nalidixic acid, oxolinic acid, oxytetracycline, and sulfonamide while resistant to ampicillin and penicillin G. In addition, the strain was neither auto-agglutinating nor hemagglutinating, but it was hemolytic against erythrocytes from sheep, rabbit, tilapia, and grouper. The bacteria could be reisolated from kidney, liver, and the transparent yellow fluid of swollen intestine of moribund groupers after bacterial challenge and re-identified as the same species. The LD50 value was 2.53 x 10(7) colony forming units/g grouper body weight.

Effects of extracellular products of Vibrio vulnificus on Acanthopagrus schlegeli serum components in vitro and in vivo.

A Vibrio strain Ls001, originally isolated from a body surface lesion of a moribund black porgy (Acanthopagrus schlegeli) in 1994 in Taiwan, was identified as Vibrio vulnificus. The extracellular products (ECP) of the strain were lethal to the fish, and its effects on fish serum in vitro and in vivo are described in the present study. Nine major precipitation arcs were visualized in normal fish serum in a crossed immunoelectrophoresis (CIE) gel using rabbit antiserum to the fish normal serum and staining with Coomassie brilliant blue. Only four and six of the nine major arcs could be tentatively identified by CIE following in vivo and in vitro ECP treatment, respectively. The same two major arcs were both missing following either in vivo or in vitro treatment with ECP. These complex events may significantly contribute to the pathogenesis of V. vulnificus in A. schlegeli.

Isolation of Vibrio harveyi from diseased kuruma prawns Penaeus japonicus.

Outbreaks of high mortality among the cultured kuruma prawn Penaeus japonicus without overt gross signs occurred during August and December of 1994 in I-Lan, Taiwan. Eleven luminous bacterial strains were isolated from the hepatopancreas of moribund prawns from five different farms by use of tryptic soy agar (TSA, supplemented with 2% NaCl) and/or thiosulfate citrate bile salt sucrose (TCBS) agar. These strains, together with our two previously unpublished isolates, were characterized and identified to be Vibrio harveyi in comparison with two ATCC Type strains and one strain previously isolated from the tiger prawn, P. monodon.