Kuo-Kau Lee

Pathogenicity of different isolates of Vibrio harveyi in tiger prawn, Penaeus monodon

P.‐C. LIU, K.‐K. LEE AND S.‐N. CHEN. 1996. The pathogenicity of six Vibrio harveyi strains in tiger prawn, Penaeus monodon, was studied, using both live bacteria and extracellular products (ECP). The organisms originally isolated from diseased penaeids were more virulent using both live bacteria and ECP (LD50, 4.87–8.65 times 104colony‐forming units (cfu) and 1.20–1.51 μg protein g‐1body weight) than the two reference strains originally isolated from either sea water (ATCC 25919; LD50, 3.18 times 106cfu and 2.70 μg protein g‐1body weight) or diseased Talorchestia sp. (ATCC 14126, 0.418 times 106cfu and 2.34 μg protein g‐1body weight). Each strain was reisolated from the haemolymph and the hepatopancreas of moribund prawns following each bacterial challenge. Both the live bacteria and the ECPs of the penaeid isolates exhibited stronger proteolytic (caseinase), phospholipase and haemolytic activities than those of the reference strains. These results indicate that there are differences between penaeid and non‐penaeid isolates of V. harveyi in pathogenicity and reveal that proteases, phospholipases, haemolysins or exotoxins might play leading roles in the pathogenicity of V. harveyi in the tiger prawn, Penaeus monodon.

Virulence of Vibrio parahaemolyticus isolated from cultured small abalone, Haliotis diversicolor supertexta, with withering syndrome

Outbreaks of mass mortality among cultured small abalone Haliotis diversicolor supertexta with withering syndrome occurred in May and September 1998 in Kao‐Hsiung, Taiwan. Bacterial strains CH‐1 and B4 were isolated from the haemolymph of the moribund small abalone using tryptic soy agar supplemented with 3% NaCl and/or thiosulphate citrate bile salt sucrose agar. These two strains were characterized and identified as Vibrio parahaemolyticus on the basis of various biochemical tests. The B4 strain and its extracellular products were virulent to small abalone with LD50 values of 1·6 × 105 colony‐forming units and 7·58 µg protein g−1 body weight, respectively.

Pathogenesis of Gastroenteritis Caused by Vibrio carchariae in Cultured Marine Fish

Serious mortality among the cultured grouper Epinephelus coioides, characterized by a swollen intestine containing yellow fluid (gastroenteritis), occurred in 1993 in Taiwan. A bacterium isolated from the intestinal fluid and head kidney of moribund groupers was identified as Vibrio carchariae. Since then, the same Vibrio species has also been isolated from moribund black sea bream Acanthopagrus schlegeli, yellowfin sea bream A. latus, Japanese sea bass Lateolabrax japonicus, and red drum Sciaenops ocellatus suffering from the same syndrome. Each isolate was virulent to the respective fish. Recently, a similar syndrome, flounder infectious necrotizing enteritis, also caused by V. carchariae in summer flounder Paralichthys dentatus, was reported in Rhode Island. The extracellular products (ECPs) of V. carchariae strains EmI82KL (from grouper), Rd (from red drum), and SfUSA (from summer flounder, U.S.A.) were virulent to the grouper or red drum. A 33-kDa serine protease partially purified from the ECP of strain EmI82KL was lethal to the fish. All the moribund or killed fish exhibited gastroenteritis except those killed within 12 hours. This report is the first to show that intraperitoneal injection of the ECP or protease in the fish is virulent and can reproduce gastroenteritis. The serine protease was suggested as a major toxin in the grouper or red drum secreted by V. carchariae.

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.

The implication of ambient temperature with the outbreak of vibriosis in cultured small abalone Haliotis diversicolor supertexta Lischke

Abstract (1) Outbreaks of mass mortality among cultured small abalone Haliotis diversicolor supertexta Lischke with vibriosis occurred in May and September of 1998 in Kao-Hsiung, Taiwan. (2) The effect of different temperature treatments on the susceptibility of small abalone to vibrio and its extracellular products (ECP) was investigated. (3) Two bacterial strains, Vibrio alginolyticus H11 and V. parahaemolyticus B4 originally isolated from the haemolymph of the moribund small abalone at 28 and 32°C, respectively, were used in this study for susceptibility tests. (4) The results reveal that at higher temperatures, the small abalone were more susceptible to vibrio and ECP challenge indicating that the outbreak of vibriosis is associated with warm water conditions.

A comparison of three methods for assaying hydrophobicity of pathogenic vibrios

The surface hydrophobicity of strains of Vibrio alginolyticus, V. carchariae, V. damsela, V. harveyi and V. vulnificus, isolated from either diseased cultured grouper (Epinephelus malabaricus) or penaeids (Penaeus monodon and P. japonicus) was determined using three different methods:the salt aggregation test (SAT), bacterial adhesion to hydrocarbons test (BATH), and adherence to nitrocellulose filters test (NCF). The results obtained indicate that all the strains tested showed some degree of hydrophobicity with the type strain of V. harveyi (ATCC 25919) showing strong hydrophobic properties in all the methods. The SAT method used in the present study was modified to a microtitre tray test, an easier test to read than the conventional in glass slide methodology. All the 13 test strains were positive in the BATH test when n‐octane was used as the solvent, but only one strain was positive when n‐hexadecane was used as the solvent. It is suggested that this method using n‐octane as solvent is suitable for assaying the hydrophobicity of pathogenic vibrios isolated from diseased aquatic animals.

Withering syndrome of the small abalone, Haliotis diversicolor supertexta, is caused by Vibrio parahaemolyticus and associated with thermal induction.

Abstract The susceptibility of the small abalone Haliotis diversicolor supertexta to Vibrio parahaemolyticus 880915 strain and its extracellular products (ECP) at different temperatures was investigated. The strain was previously isolated from the haemolymph of the moribund small abalone with withering syndrome during an outbreak of mass mortality among the cultured animals in September 1999 in I-Lan, Taiwan. The bacterium and its ECP were lethal to the small abalone. Onset of the withering syndrome in the moribund or dead animals could be observed at 4 -7 d post-bacterial challenge. The same bacterial strain could be isolated from the haemolymph of the moribund animals with or without the syndrome post-bacterial challenge. This syndrome could not be observed in the moribund or dead animals post-ECP challenge. The animals were more susceptible to the bacterium and ECP challenge at higher temperature (28 °C) indicating that the outbreak of the disease in warmer season is associated with thermal induction.

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.