Ping-Chung Liu

News & Notes: Virulence of Vibrio alginolyticus Isolated from Diseased Tiger Prawn, Penaeus monodon

Abstract. Outbreaks of mass mortality among cultured tiger prawns (Penaeus monodon) with white spotted syndrome (WSS) in the carapace occurred in the summer of 1994 in I-Lan, Taiwan. A swarming strain Val was isolated from hemolymph of the moribund prawns with tryptic soy agar (TSA, supplemented with 1% NaCl, Oxoid) and/or thiosulfate citrate bile salt sucrose (TCBS, Difco) agar. This strain was characterized and identified to be Vibrio alginolyticus. The strain was susceptible to antibiotics such as chloramphenicol, ciprofloxacin, doxycycline hydrochloride, nalidixic acid, oxolinic acid, and oxytetracycline while resistant to ampicillin, novobiocin, penicillin G, sulfisoxazole, and sulfonamide. The bacteria and their extracellular products (ECP) were lethal to both tiger prawns (P. monodon) and kuruma prawns (P. japonicus) with LD50 values of 1.13 × 105, 2.46 × 105 CFU/g, and 0.23, 0.63 μg protein/g prawn body weight, respectively.

Purification and characterization of a cysteine protease produced by pathogenic luminous Vibrio harveyi.

Abstract. The purification and characterization of an extracellular protease produced by pathogenic luminous Vibrio harveyi strain 820514, originally isolated from diseased tiger prawn (Penaeus monodon), was presented in this paper. The purification steps included ammonium sulfate precipitation, with columns of hydrophobic interaction chromatography and anion exchange on fast protein liquid chromatography. The protease is an alkaline cysteine protease, heat labile, inhibited by iodoacetamide, iodoacetic acid, N-ethylmaleimide, p-chloromercuribenzoate, and p-chloromercuribenzene-sulfonic acid, and showed maximal activities at pH 8 and 50°C, having a molecular mass of 38 kDa as estimated by SDS-PAGE and gel filtration column. In addition, the protease was also completely inhibited by CuCl2 and HgCl2, but not or only partially inhibited by other inhibitors tested. Furthermore, 2-mercaptoethanol was the most effective reducing agent in the activation of the enzyme. The present protease is the first cysteine protease found in Vibrio species.

Alkaline serine protease is an exotoxin of Vibrio alginolyticus in kuruma prawn, Penaeus japonicus.

Abstract. An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn (Penaeus japonicus) was partially purified by Fast Protein Liquid Chromatography with hydrophobic interaction (Phenyl Sepharose High Performance) chromatography and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF), and showed maximal activity at pH 10, having a molecular weight of about 33 kDa estimated by SDS-PAGE and gel filtration chromatography. In addition, the toxin was also completely inhibited by FeCl2 but partially inhibited by CaCl2, CuCl2, CoCl2, MnCl2, and ZnCl2, and not inhibited by ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(β-amino-ethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecyl sulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK). Both the crude extracellular products (ECP) and the partially purified toxin are lethal for kuruma prawn at LD50 values of 0.30 and 0.27 μg protein/g body weight, respectively. The addition of PMSF completely inhibited the lethal toxicity of both the ECP and the partially purified toxin, indicating that this serine protease is a lethal factor produced by the bacterium. The 33-kDa protease is, therefore, suggested to be a new toxic protease produced by V. alginolyticus strain Swy.

Cysteine protease is a major exotoxin of pathogenic luminous Vibrio harveyi in the tiger prawn, Penaeus monodon

The role of an extracellular cysteine protease, produced by pathogenic luminous Vibrio harveyi strain 820514 originally isolated from diseased tiger prawn (Penaeus monodon), in the disease process in the prawns was studied. The protease was lethal to P. monodon with an LD50 value of 0·3 μg protein g−1 prawn. The lethal toxicity of the extracellular products (ECP) of the bacterium was neutralized by pre‐incubation of the ECP with rabbit antiserum to the cysteine protease. Pre‐incubation of ECP with CuCl2 (an inhibitor of cysteine protease) also inhibited toxicity. This suggests that cysteine protease is the major toxin produced by the bacterium. The present protease is the first toxic cysteine protease to be found in Vibrio species.

Lethal attribute of serine protease secreted by Vibrio alginolyticus strains in kuruma prawn Penaeus japonicus.

Abstract Toxicity of the extracellular products (ECP) and the lethal attribute of serine protease secreted by five pathogenic Vibrio alginolyticus strains from various sources in kuruma prawn Penaeus japonicus were studied. The ECPs of organisms originally isolated from diseased kuruma prawn or small abalone Haliotis diversicolor supertexta were more lethal (LD50 value of 0.48 or 0.41 μg protein/g prawn) than those from diseased tiger prawn P.monodon, yellowfin porgy Acanthopagrus latus or horse mackerel (LD50 value of 0.98 -1.17 μg protein/g prawn). All the ECPs manifested strong, weak and no activities against gelatin, sheep erythrocytes and chitin, respectively. In immunodiffusion tests using rabbit antiserum to a purified 33 kDa serine protease of strain Swy against ECP of each tested strain produced one single precipitation band in each treatment. Furtherm ore, the serine protease was suggested to be the dominant protease secreted by V. alginolyticus strains tested since the majority of enzymatic activity of the respective ECP was inhibited by phenylmethanesulfonyl fluoride (PMSF). A higher inhibition of serine protease activity by PMSF resulted in lower mortality rate of the ECPs injected into the prawns suggesting that the protease is one of the major lethal factor(s) secreted by Valginolyticus.

Dual challenges of infectious pancreatic necrosis virus and Vibrio carchariae in the grouper, Epinephelus sp.

The grouper industry in Taiwan faces serious threats from various disease problems. The present study investigated dual challenges with infectious pancreatic necrosis virus (IPNV) and Vibrio carchariae in the grouper (Epinephelus sp.). The fish were infected with IPNV for 2 weeks prior to a secondary infection with the bacteria, or vice versa, by either immersion (10(3)-10(4) TCID50 IPNV per ml, 10(6)-10(7) colony forming units (CFU) Vibrio per ml) or by intraperitoneal injection (10(3)-10(4) TCID50 IPNV per g fish or 10(7) CFU Vibrio/g fish) challenges. Mass mortalities occurred in fish infected with IPNV for 2 weeks prior to the infection with the bacteria, or vice versa, in either immersion or intraperitoneal injection challenges. The bacterium could only survive in seawater or brackish water similar to that of cultured groupers.

Effects of extracellular products of Vibrio alginolyticus on penaeid prawn plasma components

The effects of both crude extracellular products (ECP) and a partially purified protease of Vibrio alginolyticus on the plasma components of kuruma prawn (Penaeus japonicus) and tiger prawn (P. monodon) were studied using crossed immunoelectrophoresis (CIE). A component of the plasma, tentatively identified as coagulogen, apparently disappeared after incubation with the ECP, while the amount of a component tentatively identified as haemocyanin decreased. The coagulogen and an unknown component (component 1) in the penaeid plasma showed an increased migration rate after incubation with a partially purified 33 kDa protease of the bacterium. In contrast, incubation with protease had no detectable effect on the amount of haemocyanin. These events may significantly contribute to the pathogenicity of Vibrio alginolyticus in penaeids.

Passive immunization of the tiger prawn, Penaeus monodon, using rabbit antisera to Vibrio harveyi

Passive immunization, toxicity neutralization and the persistence of passive protection in the tiger prawn (Penaeus monodon) were investigated using rabbit antisera to the formalinized extracellular products (ECP) (RαECP) and/or formalinized bacterial cells (RαBC) of luminescent Vibrio harveyi strain 820514 originally isolated from diseased tiger prawns. Rabbit antiserum to bovine serum albumin (RαBSA) or phosphate‐buffered saline (PBS, pH 7·2) both served as controls. The toxicity of ECP to prawns was neutralized by pre‐incubation with RαECP. Passive immunization by pre‐injection of RαBC or RαECP into prawns 3 d in advance protected against a lethal dose challenge of bacteria. To determine the persistence of passive protection by rabbit antiserum in tiger prawns, the RαBC, RαECP, RαBSA or PBS were injected into prawns. At 10, 17 or 24 d post‐immunization, groups of prawns were given a lethal dose challenge of bacteria. The prawns in the two control groups were all killed within the first 2 d following challenge at all three challenge dates, Pre‐injection with RαBC and RαECP provided total protection for 10 and 17 d, respectively, with all treated prawns surviving for at least 2 weeks post‐challenge. This is the first study using mammalian antisera to investigate toxicity neutralization, passive immunization and persistence of passive protection by rabbit antisera in prawns. The results could be useful in future studies on virulence mechanisms and disease control of vibriosis in cultured prawns.

Protease and Virulence of the Extracellular Products Produced by Vibrio carchariae after Growth on Various Media

Abstract Protease and virulence of the extracellular products (ECP) of Vibrio carchariae strain EmI82KL, a causative agent of gastroenteritis in E pinephelus 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(y3-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.