Molecular cloning and characterization of chemokine C-C motif ligand 34 (CCL34) genes from olive flounder (Paralichthys olivaceus).

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 (ß), 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-1ß, IL-6, and CXCL8), but the overall expression of interferon-related genes (IFN-α/ß, IFN-γ, Mx, and ISG15) did not increase. Olive flounder injected with recombinant PoCCL34 demonstrated increased expression of pro-inflammatory cytokines (IL-1ß and IL-6) in the head kidney. However, there was no increase in the expression of interferon-related genes (IFN-α/ß, 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.

Modifications of the nucleoprotein of viral haemorrhagic septicaemia virus showed gain of virulence in intraperitoneally infected rainbow trout.

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.

The susceptibility of silver crucian carp (Carassius auratus langsdorfii) to infection with koi herpesvirus (KHV).

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.

First Report of Clavinema mariae (Nematoda: Philometridae) in Cultured Rockfish, Sebastes schlegeli, in Cheonsuman (Bay), the Republic of Korea.

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.

Comparison of the efficacy of Poly(I:C) immunization with live vaccine and formalin-killed vaccine against viral hemorrhagic septicemia virus (VHSV) in olive flounder (Paralichthys olivaceus).

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.

Development of a rapid method for identifying carryover contamination of positive control DNA, using a chimeric positive control and restriction enzyme for the diagnosis of white spot syndrome virus by nested PCR.

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.

Effects of Low-Lipid Diets on Growth, Haematology, Histology and Immune Responses of Parr-Stage Atlantic Salmon (Salmo salar).

Lipids in fish diets provide energy and play important roles in immunity and metabolism. Atlantic salmon, a species that migrates from freshwater to seawater, requires high energy, especially during smoltification. Juvenile teleosts have low lipid requirements, and a high dietary lipid content is known to have negative effects on their growth and digestion. Therefore, this study evaluated the effect of two commercial rainbow trout feeds (low-lipid, 13.41% and 14.6%) on the growth and immune responses of early parr-stage Atlantic salmon compared to commercial salmon feed (high-lipid, 29.52%). Atlantic salmon parr (weight: 14.56 ± 2.1 g; length: 11.23 ± 0.44 cm) were randomly divided into three groups and fed either one of two commercial rainbow trout feeds (RTF1 and RTF2) or the commercial salmon feed (ASF) for 12 weeks. At the end of the feeding trial, growth, haematology, histology and gene expression analyses were performed. There were no significant differences in weight gain rates or feed efficiency between the groups (p > 0.05). Superoxidate dismutase, glutathione peroxidase, lysozyme and immunoglobulin M activities were not different among the experimental groups (p > 0.05). A histological examination of the liver and intestinal tissues showed no pathological symptoms of inflammatory response or lipid accumulation in any of the groups. In an intestinal transcriptome analysis using RNA-seq, the expression levels of several genes linked to lipids, immune-related proteins, cytokines and chemokines did not differ significantly between the groups (p > 0.05). Commercial rainbow trout feed with low lipid content has no clear negative impact on the development of Atlantic salmon during the early parr stage (14.5 to 39.6 g). This study provides basic information for the development of economical feed for early parr-stage Atlantic salmon.

Evidence for two koi herpesvirus (KHV) genotypes in South Korea.

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.

Pathogenicity of two lineages of infectious hematopoietic necrosis virus (IHNV) to farmed rainbow trout (Oncorhynchus mykiss) in South Korea.

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.

DNA vaccine dual-expressing viral hemorrhagic septicemia virus glycoprotein and C-C motif chemokine ligand 19 induces the expression of immune-related genes in zebrafish (Danio rerio).

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 (105 TCID50/10 µl/fish) was intraperitoneally injected. A survival rate of 68% was observed in the pVG + CCL19 group but this was not significantly different from the survival rate of fish treated with pVG alone, that is, without the adjuvant. However, the expression of interferon- and cytokine-related genes in the spleen and kidney tissues of zebrafish was significantly increased (p < 0.05) on days 1, 3, 7, and 14 after immunization. Thus, CCL19a.2 induced an initial immune response as a molecular adjuvant, which may provide initial protection against virus infection before vaccination-induced antibody formation. This study provides insights on the functions of CCL19a.2 adjuvant in DNA vaccines.

Evaluation of the Antimicrobial Effect of Graphene Oxide Fiber on Fish Bacteria for Application in Aquaculture Systems.

The growing importance of the domestic aquaculture industry has led not only to its continuous development and expansion but also to an increase in the production of wastewater containing pathogenic microorganisms and antibiotic-resistant bacteria. As the existing water purification facilities have a high initial cost of construction, operation, and maintenance, it is necessary to develop an economical solution. Graphene oxide (GO) is a carbon-based nanomaterial that is easy to manufacture, inexpensive and has excellent antimicrobial properties. In this study, the antimicrobial effect of GO polyester fibers on seven species of fish pathogenic bacteria was analyzed to evaluate their effectiveness in water treatment systems and related products. As a result of incubating GO polyester fibers with seven types of fish pathogenic bacteria for 1, 6, and 12 h, there was no antimicrobial effect in Vibrio harveyi, V. scopthalmi, and Edwardsiella tarda. In contrast, GO fibers showed antimicrobial effects of more than 99% against A. hydrophila, S. parauberis, S. iniae, and P. piscicola, suggesting the potential use of GO fibers in water treatment systems.

Protection of Japanese flounder Paralichthys olivaceus from viral hemorrhagic septicemia (VHS) by Poly(I:C) immunization.

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.

Establishing the optimal fetal bovine serum concentration to support replication of cyprinid herpesvirus 3 in CCB and KF-1 cell lines.

Koi herpesvirus (KHV) disease is a serious disease in cultured carp (Cyprinus carpio). CCB and KF-1 cell lines are commonly used for virus isolation and observation of cytopathic effects (CPE) in carp and koi samples. The purpose of this study was to determine the optimal concentration of fetal bovine-serum (FBS) to use for supporting the replication of cyprinid herpesvirus 3 CyHV-3 in CCB and KF-1 cell lines. The following concentrations were tested: 0%, 2%, 5%, and 10% FBS. At 7 days post-viral inoculation (dpi), CPE with clear vacuolation was observed in both cell lines when supplemented with 0 and 2% FBS, but not in those supplemented with 5% or 10% FBS. At 14 dpi, CPE was observed in both cell lines supplemented with FBS at any of the tested concentrations when a high virus titer was inoculated. However, CPE was indistinguishable between cell lines supplemented with 10% FBS when a low virus titer was inoculated. Results of qPCR indicated that the number copies of the viral genome tended to be larger in both cell lines supplemented with 10% FBS than the corresponding number in cell lines supplemented with 0%, 2%, or 5% FBS, at 7 dpi. In conclusion, we recommend using 2% FBS as supplement for isolation and diagnosis of CyHV-3 viral infection in carp samples.

Molecular cloning, functional characterization and localization of an annexin from a fish gill fluke Microcotyle sebastis (Platyhelminthes: Monogenea).

The full cDNA of an annexin gene from Microcotyle sebastis (MsANX) was cloned for the first time in monogeneans. The cDNA of MsANX comprises 1199bp with a 29bp 5' untranslated region, an open reading frame of 1062bp, and a 108bp 3' untranslated region. The recombinantly produced MsANX bound phosphatidylserine vesicles in the presence of Ca2+, whereas no MsANX was precipitated in the absence of free Ca2+. 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 naïve fish with the recombinant MsANX can induce protective immune responses against M. sebastis infection.

Generation of Vibrio anguillarum ghost by coexpression of PhiX 174 lysis E gene and staphylococcal nuclease A gene.

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.

Generation of safety enhanced Edwardsiella tarda ghost vaccine.

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.

Development of a stringent ELISA protocol to evaluate anti-viral hemorrhagic septicemia virus-specific antibodies in olive flounder Paralichthys olivaceus with improved specificity.

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.

Inhibition of chemiluminescent response of olive flounder Paralichthys olivaceus phagocytes by the scuticociliate parasite Uronema marinum.

Experiments were conducted to evaluate the in vitro capacity of the scuticociliatian parasite Uronema marinum to inhibit chemiluminescence (CL) of olive flounder Paralichthys olivaceus phagocytes. Luminol-enhanced CL was used to measure the production of reactive oxygen intermediates (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.

Pseudocohnilembus persalinus (Ciliophora: Scuticociitida) is an additional species causing scuticociliatosis in olive flounder Paralichthys olivaceus.

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.

Comparison of toxic effects of nitric oxide and peroxynitrite on Uronema marinum (Ciliata: Scuticociliatida).

To discover the effects of nitric oxide (NO) and peroxynitrite on Uronema marinum (a ciliate responsible for systemic scuticociliatosis in cultured olive flounder Paralichthys olivaceus), the dose-dependent inhibitory effect of NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) on the proliferation and survival of U. marinum was investigated. The inhibitory effects of exogenous superoxide dismutase (SOD) and catalase on the toxicity of SIN-1 were also investigated. After 24 h of incubation in the presence of 0.2 mM SNAP, the number of ciliates was not statistically different from that of the controls, whereas incubation in the presence of 0.5 mM SNAP reduced the number of parasites significantly to 59.1% of controls. Concentrations of SNAP higher than 0.5 mM resulted in greater reductions in the number of ciliates, but levels of generated NO far exceeded physiological ranges. The number of viable ciliates incubated for 24 h with 0.2 mM SIN-1 was reduced significantly to 25.0%, and all ciliates were killed by incubation in concentrations above 0.5 mM SIN-1. Although SOD decreased the toxic effect of SIN-1 on U. marinum, protection was not complete and did not improve after increasing the SOD concentration from 50 to 400 U ml(-1). Addition of catalase ranging from 500 to 10000 U ml(-1) completely protected U. marinum from SIN-1 toxicity. Ciliates exposed to catalase alone or catalase plus SIN-1 showed significantly higher and dose-dependent proliferation rates compared to controls. Addition of haemoglobin, ranging from 0.5 to 2.0 mg ml(-1), also protected U. marinum from SIN-1 toxicity, and increased the proliferation rate dose-dependently. In conclusion, resistance of U. marinum to oxidative and nitrative stress may allow this pathogen to withstand the NO- and oxygen-radical-dependent killing mechanisms of phagocytic cells.