The cell-surface properties of strains of Lactococcus garvieae were examined. Two capsular types were found, one with a highly developed capsule (KG9408) and one with a micro-capsule (MS93003) carrying fimbriae-like components projecting from the cell surface. One strain (NSS9310) had neither cell capsular nor fimbriae-like structures on its cell surface. The strains with the highly developed capsule were more virulent to fish than either the micro-capsular or non-capsular strains. The KG9408, MS93003 and NSS9310 strains could be clearly differentiated by their susceptibility to bacteriophages. Protection against L. garvieae infection was induced in the yellowtail Seriola quinqueradiata by immunization with formalin-killed L. garvieae KG9408 and MS93003 cells. Although protection was also induced by immunization with NSS9310, the level of protection was significantly lower than that with KG9408 and MS93003 vaccines. Passive immunization with yellowtail immune sera raised against KG9408 and MS93003 conferred strong protection on yellowtail with rapid bacterial clearance after challenge with L. garvieae. Immunoblotting analysis of protein antigens extracted from L. garvieae strains using rabbit anti-KG9408 and anti-MS93003 sera and yellowtail anti-KG9408 and anti-MS93003 sera indicated that some bands in KG9408 and MS93003 strains were not detectable in NSS9310.KEY WORDS: Immunogenicity ⋅ Lactococcus garvieae ⋅ Seriola quinqueradiata ⋅ Cell capsule ⋅ Fimbriae Resale or republication not permitted without written consent of the publisherDis Aquat Org 51: [169][170][171][172][173][174][175][176][177] 2002 thought to play roles in the pathogenicity of L. garvieae infection, possibly by increasing resistance to fish phagocytosis (Yoshida et al. 1996(Yoshida et al. , 1997.Control of Lactococcus garvieae infection in yellowtail culture has depended on chemotherapy with macrolides. The identification of multiple drug-resistant strains has indicated future problems in controlling the pathogen (Aoki et al. 1990). Recently, oral and injectable vaccines against L. garvieae infection in Seriola quinqueradiata have been developed and commercialized in Japan. Experimental vaccination against L. garvieae has been described (Iida et al. 1982, Sato et al. 1996 and has been reported to provide immunity and increased opsonic activity in the fish. However, no detailed information on the antigenicity of L. garvieae phenotypes nor the duration of the immunity was given. The protective mechanisms of the L. garvieae vaccine remains unknown.In a previous study, Ooyama et al. (1999) reported that formalin-killed Lactococcus garvieae KG-phenotype (capsulated phenotypes) cells and KG+ phenotype (noncapsulated) cells induced strong immunity in Seriola quinqueradiata against artificial infection and longlasting agglutinating titres against non-capsulated cells (avirulent KG+ phenotype). Furthermore, appendages (fimbriae-like structures) were seen extending from the cell surface of L. garvieae KG-phenotype, with some destruction of...

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White spot syndrome virus (WSSV) has caused mass mortality on tiger shrimp (Penaeus monodon) culture and adversely affects prawn industry worldwide including Indonesia. It is well known that the protein structure of WSSV plays an important role in the virus infection and morphogenesis process. A viral protein structure called VP-15 is located in the nucleocapsid of virion virus. The protein structure involves in the life cycle of WSSV in host cells. A gene encoding VP-15 could be involved in constructing the RNA interference (RNAi), so it is needed to isolate and characterize for RNAi technology purpose. The study was aimed to isolate and characterize the VP-15 from the infected WSSV tiger shrimp. The characterization of VP-15 was undertaken through assessment of nucleotide sequence, amino acid deduction, alignment nucleotide/protein searches using Genetyx and BLAST program, and dendrogram construction analysis. The results showed that VP-15 was successfully isolated in form of ORFDNA with a fragment size of 243 bp. The phylogenetic tree analysis revealed three clusters corresponding to the time (year) of isolates collection. The VP-15 consisted of 80 amino acids, two start codons (ATG), one stop codon (TAA), and one Kozak context (AAAATGG). Hydrophilic amino acid was the highest composition (44.2%), followed by neutral (31.2%) and hydrophobic (24.6%) amino acid groups. The VP-15 was rich in amino acid of lysine (21.3%), arginine (22.9%) and serine (24.6%). The successful isolation of VP-15 is a very important step in providing a basic yet suitable material in constructing the dsRNA vaccine to control shrimp diseases in aquaculture.

In Southern Thailand in 1996, intense luminescence in many shrimp rearing ponds was accon~panied by massive mortality resulting in total crop loss within 3 or 4 d. Mortality was correlated with gross signs which shrimp farmers called (English translation) tea-brown gill syndrome (TBGS). Histological examination of moribund shrimp revealed massive lesions of the hepatopancreas characterized by hemocytic infiltration and the presence of bacterial cells. Bactenal isolation yielded several strains tentatively identified as Vibrio harveyi on the basis of luminescence and growth on BTB Teepol agar. Representative isolate VH1039 was selected and identified by biochemical tests as harveyi. When strain VH1039 and the other luminescent isolates were injected into normal shrimp (1 X 10'cells shrimp-'), no significant mortality was observed in comparison with control s h r m p injected without bacteria. Nor was any significant mortality observed after injection of supernatant fluids from normal or sonicated bacterial cultures of VH1039 (1 X 108 cells ml-l). Transmission electron microscopy (TEM) of hepatopancreatic tissue from farmed TBGS shrimp revealed bacterial cells of Vibrio morphology together with large numbers of bacteriophage particles that had round to hexagonal heads of approximately 60 nm diameter and tails of approximately 100 nm length. These were either free, attached to cell walls of intact bacteria or in various stages of replication within bacterial cells. Gills of farmed TBGS shrimp were subsequently homogenized in lobster haemolymph buffer (LHB) and membrane filtered (0.22 pm). Compared to control shrimp injected with LHB, shrimp injected with the lOOOx diluted gill filtrate (DGF) showed no significant mortality. However, when DGF was injected together with 1 X 10' cells of strain VH1039, there was total mortality within 48 h. High and rapid mortality concurrent with brown gills was seen only in the mixed injection group. TEM of the artificially infected shrimp tissues did show the presence of bacterial cells, but no mature bacteriophage particles or lysed bacterial cells were found similar to those seen in farmed TBGS shrimp. In further tests, addition of DGF to cultures of VH1039 induced extreme but transitory toxicity of culture filtrates from 24 to 36 h. Treatment of DGF with a germicidal lamp (UV) for 30 min or with heat at 100°C for 15 min failed to stop shnmp mortality from mixed DGFNH1039 injections. However, mortality was stopped if the heated DGF was treated with DNase. The results suggested that a bacteriophage may sometimes mediate the toxicity of V harveyiin Penaeus monodon by the transfer of a toxin gene@) or a gene(s) controlling toxin production.

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