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Glycoprotein (G protein)-based DNA vaccines are effective in protecting aquaculture fish from rhabdoviruses but the degree of immune response they elicit depends on plasmid concentration and antigen cassette. Here, we developed a DNA vaccine using the viral hemorrhagic septicemia virus G (VG) gene and chemokine (C-C motif) ligand 19 (CCL19)a.2 regulated by the CMV promoter as the molecular adjuvant. After transfection of the prepared plasmid (pVG + CCL19) into epithelioma papulosum cyprini cells, mRNA expression was confirmed through quantitative real-time polymerase chain reaction. The vaccine was intramuscularly injected into zebrafish (Danio rerio), and 28 days after immunization, viral hemorrhagic septicemia virus (10

In May 2015, a high mortality event in farmed rainbow trout occurred in Jeollabuk-do province in Korea. Histopathological analysis revealed necrosis in the kidney, liver, branchial arch, and gills of moribund fish, and infectious hematopoietic necrosis virus (IHNV) was detected in the lesions by immunohistochemistry. Cytopathic effects were observed in EPC, FHM, and RTG-2 cell lines after inoculation with kidney and spleen tissues and IHNV was detected by reverse transcription polymerase chain reaction (PCR). The amplified PCR product was sequenced, and phylogenetic analysis placed IHNV in the JRt Nagano group. Both in vivo and in vitro trials were performed to compare the virulence properties between RtWanju15 isolate, which causes 100% mortality in imported fry, and a previous isolate RtWanju09 of the JRt Shizuoka group isolated from eggs of healthy broodfish. In vivo challenge with high dose on specific pathogen free (SPF) rainbow trout fry performed in Denmark with isolates RtWanju09, RtWanju15 and DF04/99 isolates showed a survival rates of 60%, 37.5% and 52.5% (average), respectively without statistical difference. The replication efficiency of the two isolates in the in vitro challenge was similar.

Chemokines are a superfamily of chemotactic cytokines that regulate the migration and immune responses of leukocytes. Depending on the arrangement of the first two cysteine residues, chemokines are divided into four groups: CXC (α), CC (β), C (γ), and CX3C (δ). Chemokine C-C motif ligand 34 (CCL34) is a member of the CC chemokine family and is known as a fish-specific CC chemokine. In this experiment, we analyzed the molecular cloning and characterization of the PoCCL34 gene in olive flounder (Paralichthys olivaceus), including CCL34a.3 (PoCCL34a.3) and CCL34b.3 (PoCCL34b.3). The amino acid sequence of PoCCL34 has four highly conserved cysteine residues and it has a C-C motif. Phylogenetic analysis revealed that PoCCL34 was phylogenetically clustered in the fish CCL34 subcluster. Recombinant PoCCL34 induced chemotaxis of head kidney leukocytes in a dose-dependent manner. Head kidney leukocytes stimulated with PoCCL34 also exhibited significant respiratory burst activity and increased expression of pro-inflammatory cytokines (IL-1β, IL-6, and CXCL8), but the overall expression of interferon-related genes (IFN-α/β, IFN-γ, Mx, and ISG15) did not increase. Olive flounder injected with recombinant PoCCL34 demonstrated increased expression of pro-inflammatory cytokines (IL-1β and IL-6) in the head kidney. However, there was no increase in the expression of interferon-related genes (IFN-α/β, IFN-γ, Mx, and ISG15). Additionally, recombinant PoCCL34 induced high lysozyme activity in the serum of the flounder. These results indicate that although PoCCL34 is not involved in the antiviral response, it may play a significant role in the overall immune response of the flounder, particularly in mediating the inflammatory response.

International audience; Viral haemorrhagic septicaemia virus (VHSV) is the cause of an important listed disease in European rainbow trout (Oncorhynchus mykiss) aquaculture and can be present in a wide range of fish species, including marine fish, which can act as viral reservoir. Recent studies revealed putative genetic virulence markers of VHSV to rainbow trout highlighting the roles of the nucleoprotein, phosphoprotein and non-virion protein. Using reverse genetics, we produced recombinant viruses by introducing parts of or the entire nucleoprotein from a high-virulent isolate VHSV into a low-virulent backbone. Furthermore, we also made recombinant viruses by introducing residue modifications in the nucleoprotein that seem to play a role in virulence. Rainbow trout challenged with these recombinant viruses (rVHSVs) by intraperitoneal injection (IP) developed clinical signs and showed lower survival when compared to the parental rVHSV whereas fish challenged by immersion did not show clinical signs except for the high-virulent control. The mutations did not influence the viral growth in cell culture. The recombinant viruses and parental recombinant were unable to replicate and show cytopathic effect in EPC cells whereas the high-virulent control was well adapted in all the fish cell lines tested. We showed evidence that corroborates with the hypothesis that the nucleoprotein has virulence motifs associated with VHSV virulence in rainbow trout.

Koi herpesvirus (KHV) infections cause high mortality in carp (Cyprinus carpio). This study compared the susceptibility of silver crucian carp (Carassius auratus langsdorfii), also called ginbuna, and koi carp to KHV infection. Silver crucian carp and koi carp were challenged with KHV by both intraperitoneal injection and immersion, respectively, and kept in tanks at 22°C. All KHV-exposed koi carp died within 14 days post-infection (dpi), whereas no clinics nor mortality was observed in the KHV-exposed silver crucian carp. KHV DNA was detected in both koi and silver crucian carp shortly after infection. At 7 dpi, the copy numbers of KHV genome were increased in koi carp but decreased in silver crucian carp. Using reverse transcriptase PCR, KHV mRNA was detected in koi carp but not in silver crucian carp. Cell cultivation on common carp brain (CCB) cell samples from koi carp caused KHV-associated cytopathic effects in CCB cells. Therefore, we concluded that KHV replicated in koi carp but not in silver crucian carp and that silver crucian carp is not susceptible to infection with KHV.

In July 2012, philometrid nematodes were discovered in cultured rockfish (Sebastes schlegeli) in Cheonsuman (Bay), the Republic of Korea. The nematodes were detected in the epithelial tissues of the rockfish and were identified as Clavinema mariae based on morphological studies using light and scanning electron microscopy. They revealed the characteristics same as previously identified C. mariae, notably having a long body with narrow posterior half, no caudal projection, a cylindrical-shaped esophagus, a well-developed anterior bulbous part of the esophagus, cephalic papillae, and a dorsal esophageal gland. This is the first confirmation of C. mariae infection in rockfish in Korea.

Viral hemorrhagic septicemia (VHS) in olive flounder, Paralichthys olivaceus, causes significant economic loss for the flounder aquaculture industry in Korea. In this study, the immunogenicity of Poly(I:C) immunization with a live vaccine against the VHS virus (VHSV) was compared with that of a formalin-treated vaccine in the olive flounder. In vaccine trial I, fish pre-injected with Poly(I:C) were highly protected from VHSV infection 2 d later (survival rate: 96%) and the surviving fish (Poly(I:C)-VHSV group) showed a 100% survival rate against VHSV re-challenge. Mortality in fish pre-injected with diethylpyrocarbonate-treated water followed by injection with formalin-treated VHSV was only 2% (1 of 50 fish), whereas survivors (DEPC-FT VHSV group) showed an 80% survival rate. In vaccine trial II, 100% survival was observed in all Poly(I:C) vaccination groups-Poly(I:C)-VHSV 6, Poly(I:C)-VHSV 5, and Poly(I:C)-VHSV 4. In contrast, the survival rates of the groups administered the formalin-treated VHSV at a dose of 10(6), 10(5), and 10(4) TCID50 100 μL(-1) fish(-1) (DEPC-FT VHSV 6, DEPC-FT VHSV 5, and DEPC-FT VHSV 4) were only 8%, 12%, and 12%, respectively. The differences in the survival rates of the formalin-treated vaccine groups in trial I and trial II were attributed to the difference in the formalin-treatment period: the formalin-treated VHSV administered in trial I was not completely inactivated and worked as a live vaccine, which explains the 80% survival rate against VHSV challenge. Specific antibodies against VHSV were detected in sera from all vaccinated survivors, except the DEPC-VHSV 4 group. Furthermore, the specific antibody titers of fish vaccinated with the live and dead VHSV vaccines were similar, but the protective effects of the live and dead vaccines varied considerably. Our findings show that Poly(I:C) immunization with the live vaccine offers better protection than the formalin-treated vaccine against VHS in olive flounder and revealed that antibody levels are not a reliable indicator of the protective effect of the vaccine against the pathogen. In the future, elements of T cell immunity may be used as a means of evaluating the protective efficacy of a vaccine against VHSV instead of ELISA.

We investigated a nematode infection in the epithelial tissue of rockfish Sebastes schlegeli cultured in Cheonsu Bay, western Korea, from May 2013 to April 2014. Nematodes infected the epithelial tissues of various external organs, including the fins, operculum, nares, mouth, and head. Over a 1-year period, the overall nematode infection rate in rockfish was 55% (n

The geographic distribution of koi herpesvirus (KHV) has recently been analyzed by polymerase chain reaction (PCR, based on the alleles of 3 domains) and sequence analysis using 3 regions of KHV genomic DNA (SphI-5, 9/5, and the thymidine kinase gene). In this study, samples from 6 carp showing symptoms of KHV infection in 2008 were examined for the presence of KHV by using PCR and cell culture isolation methods. KHV was detected in 2 (Pyeongtaek and Buan) of the samples. Sequence analysis revealed that the genotype of the KHV PT-08 isolate was Asia genotype variant 1 (A1), and the genotype of the KHV BA-08 isolate was European genotype variant 4 (E4). In addition, PCR patterns and sequence analysis based on the alleles of 3 domains of an alternate KHV classification system confirmed that the genotype of the KHV PT-08 isolate was CyHV3-J, and the genotype of the KHV BA-08 isolate was CyHV3-third genotype. To our knowledge, this is the first study to demonstrate the presence of 2 genotypes of KHV (genotype A1/CyHV3-J; genotype E4/CyHV3-third genotype) in South Korea.

Saprolegnia isolate from wild brook lamprey was identified on the basis of its morphological and molecular characteristics. The isolates showed aseptic hyphae and clavate zoosporagium. Zoospores discharge was typically saprolegnoid. Neither oogomia nor antheridia was observed in this study. ITS sequence obtained from the isolate was compared with other Saprolegnia spp. to analyse their phylogenetic relationships. Results showed that the isolate belongs to clade I including Saprolegnia parasitica. Based on the asexual organs, zoospore discharge manner and ITS sequence analysis, the isolate was identified as S. parasitica.

Vibrio vulnificus ghosts (VVG) were generated using a mobilizable vector including a thermosensitive expression cassette by conjugation. The vaccine potential of VVG was investigated in mouse. Mice immunized with VVG showed significantly higher antibody titer than those with formalin-killed V. vulnificus. The present study supports the conceptive usefulness of bacterial ghosts as vaccine candidates.

Gene expressions of cytochrome P4501A (CYP1A), aryl hydrocarbon receptor (AhR) and vitellogenin (Vg) by endocrine disruptors, benzo[α]pyrene (B[a]P) and tributyltin (TBT) were examined in cultured eel hepatocytes which were isolated from eels treated previously with B[a]P (10 mg/kg) or estradiol-17β (20 mg/kg) in vivo, and the relationship between CYP1A, AhR and Vg genes were studied. When the cultured eel hepatocytes were treated with B[a]P (10(-6)-10(-5) M) the gene expressions of CYP1A and AhR were enhanced in a concentration-dependent manner. However, when treated with TBT (10(-9)-10(-5) M) the gene expressions of CYP1A and AhR were suppressed at high concentrations (10(-6)-10(-5) M), while having no effects at low concentrations (10(-9)-10(-7) M). Gene expression of Vg was also suppressed by TBT in a concentration-dependent manner in cultured eel hepatocytes which was previously treated in vivo with estradiol-17β.

In July 2012, philometrid nematodes were discovered in cultured rockfish (Sebastes schlegeli) in Cheonsuman (Bay), the Republic of Korea. The nematodes were detected in the epithelial tissues of the rockfish and were identified as Clavinema mariae based on morphological studies using light and scanning electron microscopy. They revealed the characteristics same as previously identified C. mariae, notably having a long body with narrow posterior half, no caudal projection, a cylindrical-shaped esophagus, a well-developed anterior bulbous part of the esophagus, cephalic papillae, and a dorsal esophageal gland. This is the first confirmation of C. mariae infection in rockfish in Korea.

Major capsid protein (MCP), the adenosine triphosphatase (ATPase), and the PstI fragment genes from five Japanese and three Korean megalocytivirus isolates were sequenced and phylogenetically analyzed with known megalocytiviruses. Phylogenetic trees formed three major clusters (M1, M2, and M3 or P1, P2, and P3), and genogroup I was divided into two minor clusters (M1a/M1b and P1a/P1b) using three target genes. Sequence identity was >97% within each cluster, except cluster II of the PstI fragment (>94% of sequence identity). Interestingly, different genotyping patterns were observed for the same isolates depending on the gene analyzed. The JPN-YelTail and JPN-BfTuna isolates located in the minor M1a cluster, based on MCP and ATPase nucleotide sequences, appeared in the minor P1b cluster based on the PstI fragment, suggesting a shift of phylogenic position in megalocytiviruses. Further study will be conducted to compare the viral antigenicity and pathogenicity between the two isolates showing the shift of phylogenic position and the other isolates clustered within genogroup I.

On September in 2010, myxosporidian parasites were found on the fins of Chinese muddy loach, Misgurnus mizolepis, Several cysts of approximately 2 mm diameter were observed on the fins. Numerous spores were found in the cyst. The total length of the spore was 13.0 to 15.8 , and the width of spore was 6.0 to 7.5 . As the results, the myxosporidians were finally identified as Thelohanellus misgurni (Kudo, 1919) on the basis of the presence of characteristic cysts and size of spores.

In immunization of fish with polyinosinic-polycytidylic acid (poly[I:C], a synthetic double-stranded RNA, injection of Poly(I:C) followed by challenge with a live virus induces a transient, non-specific antiviral state by interferon activity. When exposed to a virus while in this antiviral state, the fish acquire a specific and protective immunity against the corresponding viral disease and survive. In the present study, the effiacy of Poly(I:C) immunization was investigated in japanese flounder Paralichthys olivaceus using viral hemorrhagic septicemia virus (VHSV) as a model; the minimum dose of Poly(I:C) required for inducing protection and the duration of the antiviral state were determined, and a potentially curative effect of Poly(I:C) administration was assessed. The antiviral state was induced by administration of Poly(I:C) doses ranging from 12.5 to 200 microg fish(-1). Minimum dose to induce the antiviral state (relative percentage survival, RPS: 90%) was 12.5 microg fish(-1). No curative effect of Poly(I:C) was observed in fish pre-infected with VHSV. Fish injected with 200 microg Poly(I:C) fish(-1) were highly protected (RPS: 100%) from an artificial challenge with VHSV, and specific antibodies against VHSV were detected. The corresponding high level of antiviral state against VHSV was attained 1 d post Poly(I:C) injection, lasted for 6 d and susequently decreased. Moreover, the surviving fish were highly protected from re-challenge with VHSV (RPS: 100%). Thus, it was considered that an immunity against viral hemorrhagic septicemia was induced in the Japanese flounder by injecting live VHSV following Poly(I:C) administration.

FBS components in viral culture fluid interfere with the detection of specific antibody in immunized fish. In the present study, we developed a method to eliminate the interference of FBS using a model of red sea bream iridovirus (RSIV) vaccine. In an experiment using yellowtail antiserum against BSA, which is a major component of FBS in cell culture media, antibodies against BSA were completely absorbed via incubation of antiserum in 50% FBS at 25 degrees C for 1 h. In antibody detection, ELISA with the sera of yellowtail surviving RSIV-challenge, antibodies against RSIV were detected, but those against FBS disappeared via the pre-treatment of the sera. Therefore, antibodies against RSIV are effectively detected using ELISA with the pre-treatment of fish sera., RSIV培養液を接種したブリからの抗RSIV抗体検出ELISA法について検討した。培養液に含まれるFBSの主要成分であるBSAに対する抗ブリ血清を用いた場合、血清に終濃度50％（v／v）となるようFBSを添加し25℃で1時間処理することで、血清中の抗BSA抗体が完全に吸収された。培養RSIV液で攻撃した生残ブリから得た血清を同様に処理することで、抗体検出ELISAにより抗RSIV抗体が検出できることが明らかになった。

The full cDNA of an annexin gene from Microcotyle sebastis (MsANX) was cloned for the first time in monogeneans. The cDNA of MsANX comprises 1199 bp with a 29 bp 5′ untranslated region, an open reading frame of 1062 bp, and a 108 bp 3′ untranslated region. The recombinantly produced MsANX bound phosphatidylserine vesicles in the presence of Ca 2+ , whereas no MsANX was precipitated in the absence of free Ca 2+ . Phylogenetically, MsANX formed a cluster with human annexin A13, known as the earliest annexin in vertebrates and expressed mainly in the intestine. The localization of MsANX in M. sebastis was analyzed by Western blotting and immunohistochemistry using the antiserum raised against the recombinant MsANX. In Western blot analysis, rat antiserum bound to a protein corresponding to the MsANX in size when worm crude extracts were used as antigens, but no bands were detected by the antiserum when the excretory/secretory proteins of worms were used as antigens. In immunohistochemistry analysis, significant antibody binding annexin was found in the ovarian region, the pharynx and the intestinal caecum of the worm. Interestingly, the alimentary canal location of MsANX was similar to the location of human annexin A13, and further research is needed to trace evolutionary relationship among helminthic annexins and human annexin A13. Also it remains to be investigated whether immunization of naive fish with the recombinant MsANX can induce protective immune responses against M. sebastis infection.

A dual vector expressing the ghost-inducing PhiX174 lysis E gene and the bacterial DNA degrading staphylococcal nuclease A (SNA) gene was constructed to solve the problem of remnant antibiotic resistance genes and genomic DNA with intact pathogenic islands in the final product of Edwardsiella tarda ghosts (ETG). The SNA (devoid of secretion signal sequence and the nuclease B amino terminus sequence), fused with the 26 amino acid N-terminal sequence of the lambda phage Cro gene, showed successful degradation of bacterial nucleic acids. Furthermore, the nuclease activity of SNA in E. tarda was enhanced by codon optimization of the SNA gene using site-directed mutagenesis. ETG were generated via coexpression of the SNA gene and lysis gene E under the control of each lambdaP(R) promoter. The ghost bacteria generation system we describe is advantageous as it allows the use of a single plasmid, improves safety and vaccine purity by limiting residual genetic content from the ghost bacteria, and reduces production costs through cheap means of induction that use only temperature shifts.

To investigate the pathogenicity of marine birnavirus (MABV) on rock bream (Oplegnathus fasciatus), two different sized rock bream fingerlings were artificially infected with MABV. The experimental MABV strain previously isolated from asymptomatic olive flounder (Paralichthys olivaceus), was not pathogenic for olive flounder. Experimental infection of rock bream fingerlings with the virus elicited typical signs of birnaviral disease and yielded high cumulative mortality. The presence of MABV in infected rock bream fingerlings was confirmed by electron microscopy. These results indicate that rock bream is susceptible to MABV isolated from asymptomatic olive flounder. There were no differences in gene sequence encoding VP5-pVP2 region between the viruses isolated from diseased rock bream and the virus cultured in fish cell line. These results indicate that the virulence of the present MABV strain is variable according to host fish species, and the gene sequence encoding VP5-pVP2 region is not related at least by itself to the susceptibility of rock bream to the disease caused by the present MABV strain.

Olive flounder were vaccinated with polyinosinic:polycytidylic acid [Poly (I:C)] to prevent viral hemorrhagic septicemia (VHS). Vaccine efficacy was verified by detection of anti- VHS virus (VHSV) antibodies using enzyme-linked immunosorbent assay (ELISA). In the study, ELISA absorbance values of the negative control group [Poly (I:C)-MEM10] were saturated when an ELISA protocol, that includes pretreatment of the fish sera with 5% skim milk, was used. However, the saturated OD values in the negative control did not correlate with a specific immune response against VHSV, because the group showed low survival rate (only 10%) following the VHSV challenge. Also, OD values of Poly (I:C)- VHSV group were high, and the group showed high survival rate (97.5%) against VHSV challenge test. It was suggested that the high OD values were possibly due to the presence of anti-fetal bovine serum (FBS) cross-reactivity. To compensate this, we subtracted the absorbance of infectious hematopoietic necrosis (IHNV)-Ag plates from those of the VHSV-Ag plates. However, the average value for the Poly (I:C)-VHSV group (0.167) was lower than expected even though high survival rate. We used an advanced ELISA system to pre-treat fish sera with 5% skim milk and two novirhabdoviruses as capture antigens as well as 50% FBS. The corrected absorbance values for pre-treated fish sera from the negative control Poly (I:C)-MEM10 and experimental Poly (I:C)-VHSV groups averaged 0.033 and 0.579, respectively. The specific VHSV antibody response of the vaccinated group was assessed using fish sera pretreated with skim milk and FBS and by calculating the corrected absorbance values from ELISA with two novirhabdovirus capture antigens.

The recombinant β-tubulin of Pseudocohnilembus persalinus was investigated to test whether tubulin could be a suitable antigenic target against scuticociliatosis. The cDNA of P. persalinus β-tubulin has an open reading frame of 1332 bp and had 15 TAA triplets code for glutamine (Q). All 15 TAA/Q triplets of the β-tubulin cDNA were mutated to universal CAA/Q triplets by site-directed mutagenesis and the mutated β-tubulin was expressed in Escherichia coli as a fusion with glutathione S-transferase (GST-BTU). In Western blot analysis, rat sera immunized with recombinant GST-BTU reacted with reduced β-tubulin of P. persalinus. The immunized rat sera showed higher parasiticidal activity than those of control. Ciliates preincubated with heat-inactivated immune sera showed significantly lower proliferation than ciliates preincubated with control sera when exposed to naive rat sera as a complement source. The present results suggest that the recombinant β-tubulin of scuticociliates may be used as a target antigen for development of vaccine against scuticociliatosis.

Most diagnostic studies of red sea bream iridovirus (RSIV) disease in fish have been conducted on the acutely infected fish, and spleen has been used commonly as a target organ for diagnosis. However, little information about persistent RSIV infection is available, and the distribution of RSIV in organs and tissues in persistently infected fish has not been systematically determined. In the present study, organ distribution of RSIV in asymptomatic carriers of yearling and fingerling rock bream was analyzed using PCR, and transition possibility of RSIV into acute phase in both age groups by elevating water temperature was investigated. RSIV DNA was predominantly detected in heart, stomach, intestine, muscle, eye and gill by nested-PCR from asymptomatic yearling rock bream. Strikingly, the detection frequency of RSIV DNA in spleen was very low. In fingerling rock bream, consistent with the result of yearling, the frequency of RSIV DNA detection in spleen was low. Fingerling rock bream showed a high cumulative mortality by water temperature elevation, and RSIV DNA was detected by primary PCR from dead fish. In yearling rock bream, however, there were no mortality and no detection of RSIV DNA by primary PCR during water temperature elevation period.

The vaccine potential of Edwardsiella tarda ghosts produced by gene E mediated lysis was investigated using tilapia (Oreochromis mosambicus). Tilapia immunized with E. tarda ghosts (ETG) and formalin killed E. tarda (FKC) vaccines showed significantly higher serum agglutination titers than control fish. Fish immunized with ETG showed no significant differences with fish immunized with FKC in serum agglutination titers, but showed significantly higher bactericidal activity than fish immunized with FKC. Furthermore, fish immunized with ETG showed higher protection than fish immunized with FKC. As this promising type of a non-living whole cell envelope preparation seems to be favorable over conventional vaccines, we suggest E. tarda ghosts as a new vaccine candidate.

Bacterial ghosts generated by the controlled expression of the PhiX174 lysis gene E in Gram-negative bacteria have been known to retain the functional and antigenic determinants of the envelope with their living counterparts and thus represent ideal vaccine candidates. The objective of this study was to examine the potential utility of the PhiX174 E gene driven by the P R /cI regulatory system for the generation of Edwardsiella tarda ghosts. The lysis kinetics in E. tarda were also compared with those in Escherichia coli . Generation of ghosts in the transformants of E. coli and E. tarda carrying plasmid pλP R -cI-Elysis was performed successfully by increasing the incubation temperature up to 42 °C. Compared to E. coli , in which lysis was observed within 30 min and was completed 2 h after induction of E gene expression, onset of E. tarda lysis occurred 2 h after temperature elevation and the lysis process was completed 16 h after induction. At the end of the lysis process, the efficiency of ghost induction in non-lyophilized E. tarda was 99.99 ± 0.01%, as the results of 10 replicate experiments showed. However, no bacterial growth was detected in lyophilized E. tarda . Holes ranging from 80 to 400 nm in diameter were observed in E. tarda ghosts by scanning electron microscopy. The present advance, especially regarding the tight control of ‘ E -lysis’ and complete cell killing activity, will provide a new opportunity to improve the efficiency of vaccination against edwardsiellosis.

Differences between short-term (STC) and long-term (LTC) in vitro cultures of Uronema marinum in suppression of olive flounder (Paralichthys olivaceus) phagocytes' chemiluminescent (CL) response, antioxidative enzymes and protease activity were investigated. Luminol-enhanced CL was used to measure the production of reactive oxygen intermediates (ROIs) generated by respiratory burst of phagocytes using zymosan as a stimulant. Crude extracts of both STC and LTC of U. marinum inhibited CL responses of zymosan-stimulated phagocytes in a dose-dependent manner. The reduction of CL response was significantly higher in phagocytes exposed to extracts from STC as compared to those from LTC. Both crude extracts of U. marinum showed activities of superoxide dismutase (SOD) and catalase (CAT). Protease activity of the STC crude extract was significantly higher than that of the LTC extract. Results of this study suggest that antioxidative enzymes and proteases of U. marinum may participate in protection of the parasite against host phagocyte mediated oxidative damage.

Chimeric positive plasmids have been developed to minimize false-positive reactions caused by polymerase chain reaction (PCR) contamination. Here, we developed a rapid method for identifying false-positive results while detecting white spot syndrome virus (WSSV) by nested PCR, using chimeric positive plasmids. The results of PCRs using WSSV diagnostic primer sets showed PCR products of a similar size (WSSV 1st PCR product, 1,447 bp; WSSV 2nd PCR product, 941 bp) using WSSV chimeric plasmids or DNA from shrimp infected with WSSV. The PCR products were digested with DraI for 1 h at 37 °C. The digested chimeric DNA separated into two DNA bands; however, the WSSV-infected shrimp DNA did not separate. Thus, chimeric plasmid DNA may be used as positive control DNA instead of DNA from WSSV-infected shrimp, in order to prevent PCR contamination. Thus, the use of restriction enzyme digestion allowed us to rapidly distinguish between WSSV DNA and WSSV chimeric plasmid DNA.

To elucidate whether daily water temperature changes influence chemiluminescent response, plasma glucose level and mortality of cultured rockfish ( Sebastes schlegeli ), three experimental temperature groups were established. The fish in group I were maintained constantly at 27°C, and those in groups II and III were exposed to daily elevation of water temperature from 27°C to 28°C and 20°C to 22°C for 5 h, respectively. After 1 week of being maintained in each experimental tank, the fish in groups II and III showed significantly lower CL peak values ( P P

Vibrio anguillarum ghosts (VAG) were generated, for the first time, using a conjugation vector containing a ghost bacteria inducing cassette, pRK-lambdaP(R)-cI-Elysis, in which the expression of PhiX174 lysis gene E was controlled by the P ( R )/cI regulatory system of lambda phage. By scanning electron microscopy, holes ranging 80-200 nm in diameter were observed in the VAG. To avoid the presence of bacterial genomic DNA and an antibiotic resistance gene in the final VAG product, we constructed a new dual vector, pRK-lambdaP(R)-cI-E-SNA, containing the E-mediated lysis cassette and the staphylococcal nuclease A (SNA)-mediated DNA degradation cassette, and generated safety-enhanced VAG for use as a fish vaccine.

In the present study, Pseudocohnilembus persalinus was first reported as a species causing scuticociliatosis in olive flounder Paralichthys olivaceus. Based on the stained specimens, P. persalinus was clearly differentiated from Uronema marinum, which has been shown to be a cause of scuticociliatosis in farmed olive flounder in Korea from its characteristic oral infraciliature structure. The 1754 bp small subunit ribosomal RNA (SS rRNA) gene sequence of P. persalinus showed 95% homology with the partial sequence of P. hargisi SS rRNA. Moreover, multiplex PCR based on the species-specific amplification of the SS rRNA gene sequence enabled us to distinguish P. persalinus from U. marinum in a simple and rapid manner. P. persalinus was clearly differentiated from U. marinum even when the host was infected simultaneously with both species. These data suggest that the multiplex PCR procedure would make it possible to avoid the cumbersome and time-consuming procedures of morphological analysis for the definitive identification of ciliates.

Experiments were conducted to evaluate the in vitro capacity of the scuticociliatian par- asite Uronema marinum to inhibit chemiluminescence (CL) of olive flounder Paralichthys olivaceus phagocytes. Luminol-enhanced CL was used to measure the production of reactive oxygen interme- diates (ROIs) generated by respiratory bursts of phagocytes using zymosan as a stimulant. Cytotoxic and antioxidative activities of excretory-secretory (ES) products of the parasite were evaluated as well. Live U. marinum and its ES products had a negative and dose-dependent effect on luminol- enhanced CL responses of zymosan-stimulated phagocytes of olive flounder. After CL assay, the number of phagocytes showing viability was significantly reduced in the cells incubated with live U. marinum at ratios of 2:1 and 1:1 phagocytes:ciliates or ES products with 0.3 mg protein ml -1 compared to controls. Lysis of phagocytes by exposure to ES products was observed also. ES products from U. marinum showed considerably high activities of superoxide dismutase (SOD) and catalase. The results of this study suggest that U. marinum can protect itself against host's phagocytes mediated oxidative damage by destroying phagocytes and scavenging ROIs.

The effect of cimetidine on the treatment efficacy of praziquantel against Microcotyle sebastis infestation in cultured rockfish Sebastes schlegeli was investigated. Juvenile rockfish were divided into 7 groups, and orally administered praziquantel alone (50, 100 and 200 mg kg(-1) body wt, BW) or in combination with cimetidine at a dose of 200 mg kg(-1) BW for each praziquantel dose. The fish in the control group were administered only saline. The results clearly showed that coadministration of cimetidine with praziquantel led to a significantly increased treatment efficacy of the latter drug, and consequently would lead to a lowering of the total dose of praziquantel, and a reduction in the administration times and costs for the treatment of M. sebastis infestation in cultured rockfish.