Sung-Ju Jung

Toxicogenomic analysis of immune system-related genes in Japanese flounder (Paralichthys olivaceus) exposed to heavy oil.

Heavy oil contamination is one of the most important environmental issues. Toxicities of polycyclic aromatic hydrocarbons (PAHs), including immune toxicities, are well characterized, however, the immune toxic effects of heavy oil, as a complex mixture of PAHs, have not been investigated. In the present study, we selected Japanese flounder (Paralichthys olivaceus) as a model organism, and observed alteration of immune function by the exposure to heavy oil. To analyze the expression profiles of immune system-related genes, we selected 309 cDNAs from our flounder EST library, and spotted them on a glass slide. Using this cDNA array, alteration of gene expression profiles was analyzed in the kidneys of flounders exposed to heavy oil. Six Japanese flounders (mean body weight: 197 g) were acclimated to laboratory conditions at 19-20 degrees C. Three fish were exposed to heavy oil C (bunker C) at a concentration of 3.8 g/L for 3 days, and the others were kept in seawater without heavy oil and used as the control. After the exposure period, the fish were transferred into control seawater and maintained for 4 days, and then they were dissected and their kidneys were removed. Total RNA was extracted from the kidney samples to use in gene expression analyses. The microarray detected alteration of immune system-related genes in the kidneys of heavy oil-exposed flounders, including down-regulation of immunoglobulin light chain, CD45, major histocompatibility complex class II antigens and macrophage colony-stimulating factor precursor, and up-regulation of interleukin-8 and lysozyme. These results suggest that pathogen resistance may be weakened in heavy oil-exposed fish, causing a subsequent bacterial infection, and then proinflammatory genes may be induced as a defensive response against the infection. Additionally, we found candidate genes for use as biomarkers of heavy oil exposure, such as N-myc downstream regulated gene 1 and heat shock cognate 71 kDa proteins.

Pathogenicity of Miamiensis avidus (syn. Philasterides dicentrarchi), Pseudocohnilembus persalinus, Pseudocohnilembus hargisi and Uronema marinum (Ciliophora, Scuticociliatida)

The scuticociliates Miamiensis avidus (syn. Philasterides dicentrarchi), Pseudocohnilembus persalinus, Pseudocohnilembus hargisi and Uronema marinum were cloned and identified using morphological characteristics and the small subunit ribosomal RNA gene (SSU rRNA). M. avidus strains YS1, WS1, YK1 and JJ3 from southern coastal areas and Jeju Island in Korea were pathogenic to olive flounder Paralichthys olivaceus (80 to 100% mortality in 8 to 10 g fish) when inoculated intraperitoneally (i.p.) with 1.0 to 1.4 x 10(6) ciliates fish(-1). Mortality was lower (10 to 45%) when the inoculum was 1.0 to 1.4 x 10(4) ciliates fish(-1) in the i.p.-injected group. The M. avidus strains of YS1, WS1, YK1 and JJ3 caused 60 to 100% mortality by immersion infection with 3.2 to 4.2 x 10(3) ml(-1) in 8 to 10 g fish and 3.0 to 4.0 x 10(3) ml(-1) in 30 to 40 g fish. M. avidus strain Mie0301 from the Mie prefecture in Japan caused 70% mortality by immersion infection with 4.4 x 10(3) ml(-1) in 30 to 40 g fish. The predominant sign was severe abdominal distension in i.p.-injected fish, and extensive ulcer lesions in the skeletal muscle in immersion-infected fish. Numerous ciliates were observed in the ascetic fluid, ulcers, haemorrhagic lesions, gills and brain of infected fish. However, P. persalinus (strain SCL-A), P. hargisi (strain SCL-B) and U. marinum (strain JK3) showed less than 30% mortality from both i.p. and immersion challenges, with no ciliate invasion in the skin, gills or brain. M. avidus-infected fish showed many ciliates in gills, fins, skin muscle, brain and intestine accompanied by necrosis and haemorrhages. However, no histological changes were observed in P. persalinus-, P. hargisi- or U. marinum-infected fish.

Molecular characterization and expression analysis of Cathepsin B and L cysteine proteases from rock bream (Oplegnathus fasciatus).

Cathepsins are lysosomal cysteine proteases of the papain family that play an important role in intracellular protein degradation and turn over within the lysosomal system. In the present study, full-length sequences of cathepsin B (RbCathepsin B) and L (RbCathepsin L) were identified after transcriptome sequencing of rock bream Oplegnathus fasciatus mixed tissue cDNA. Cathepsin B was composed of 330 amino acid residues with 36 kDa predicted molecular mass. RbCathepsin L contained 336 amino acid residues encoding for a 38 kDa predicted molecular mass protein. The sequencing analysis results showed that both cathepsin B and L contain the characteristic papain family cysteine protease signature and active sites for the eukaryotic thiol proteases of cysteine, asparagine and histidine. In addition, RbCathepsin L contained EF hand Ca(2+) binding and cathepsin propeptide inhibitor domains. The rock bream cathepsin B and L showed the highest amino acid identity of 90 and 95% to Lutjanus argentimaculatus cathepsin B and Lates calcarifer cathepsin L, respectively. By phylogenetic analysis, cathepsin B and L exhibited a high degree of evolutionary relationship to respective cathepsin family members of the papain superfamily. Quantitative real-time RT-PCR analysis results confirmed that the expression of cathepsin B and L genes was constitutive in all examined tissues isolated from un-induced rock bream. Moreover, activation of RbCathepsin B and L mRNA was observed in both lipopolysaccharide (LPS) and Edwardsiella tarda challenged liver and blood cells, indicating a role of immune response in rock bream.

Genetic variation and geographic distribution of megalocytiviruses.

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae have caused mass mortalities in marine and freshwater fish in Asian countries. In this study, partial major capsid protein (MCP) gene of seven Japanese and six Korean megalocytiviruses was sequenced and compared with the known megalocytiviruses to evaluate genetic variation and geographic distribution of the viruses. Comparison of MCP gene nucleotide sequences revealed sequence identity of 92.8% or greater among these 48 isolates. A phylogenetic tree clearly revealed three clusters: genotype I including nine Japanese isolates, thirteen Korean isolates, one Chinese isolates, one Thailand isolate and one South China Sea isolate; genotype II including five freshwater fish isolates in Southeast Asian countries and Australia; and the remaining genotype III mainly consisted of flatfish isolate in Korea and China. This suggests that viruses belonging to the genotype I widely distribute among various fish species in many Asian countries. Conversely, the epidemic viruses belonged to genotype II and III are may be still locally spreading and constrained in their prevalence to the limited host fish species, i.e., genotype II viruses mainly distribute in Southeast Asian countries, whereas genotype III viruses distribute in flatfish species in Korea and China.

Toll-like receptors and interferon associated immune factors in viral haemorrhagic septicaemia virus-infected olive flounder (Paralichthys olivaceus)

Pattern recognition receptor (PRR) toll-like receptors (TLRs), antiviral agent interferon (IFN) and the effector IFN stimulated genes (ISGs) play pivotal role in antiviral innate immunity of a host. The present in-vivo experiment was conducted to investigate the role of these innate immune factors in early phase as well as during recovery of viral haemorrhagic septicaemia virus (VHSV) infection by quantitative real-time reverse transcriptase polymerase chain reaction. A less lethal VHSV infection was generated in olive flounder (Paralichthys olivaceus) and was sampled at 3, 6, and 12h post infection (hpi), and 1, 2, 4, and 7 days post infection (dpi). At 3 hpi, the VHSV N gene was detected in three out of five fish and all five fish showed a relative fold increase of TLR 2, TLR 7, interleukin 8 (IL 8), IFN regulatory factor 3 (IRF 3), IRF 7, and ISG 15. Viral copies rapidly increased at 12 hpi then remained high until 2 dpi. When viral copy numbers were high, a higher expression of immune genes IL 1β, IRF 3, IRF 7, Type I IFN, ISG 15 and Mx was observed. Viral copies were drastically reduced in 4 and 7 dpi fish, and also the immune response was considerably reduced but remained elevated, except for ISG 15 which found equal to control in 7 dpi fish. A high degree of correlation was observed between immune genes and viral copy number in each of the sampled fish at 12 hpi. A fish with ascites sampled at 7 dpi displayed high viral copy but under-expressed immune genes except for Mx. When viral copies were high at 1 and 2 dpi, both TLR 2 and TLR 7 were down-regulated, perhaps indicating immune suppression by the virus. The quick and prolonged elevated expression of the immune genes indicates their crucial role in survival of host against VHSV.

Atypical Aeromonas salmonicida infection in the black rockfish, Sebastes schlegeli Hilgendorf, in Korea.

Cultured black rockfish, Sebastes schlegeli, suffered mass mortalities during winter 2008 and spring 2009 in Korea, showing clinical signs of ulcer lesions and haemorrhages over their body surface. The aetiological agent was identified as Aeromonas salmonicida (strains RFAS-1, -2 and -3), which is a non-pigmented, slow-growing bacterium. Phenotypes of RFAS strains showed variation, while 16S rRNA, gyrB, rpoD, dnaJ and recA gene sequences of all the strains were affiliated to A. salmonicida. In particular, vapA gene sequences of the strains were most closely related to one of the five subspecies of A. salmonicida subsp. masoucida (=KCCM 40239(T) ). LD(50) values of RFAS-1 for intraperitoneal and intramuscular injection were 1.5 × 10(5.25) and 1.5 × 10(6.4) cfu/rockfish, respectively. However, A. salmonicida strains KCCM 40239(T) and SAS-1, which originate from masou and chum salmon, respectively, were not pathogenic to black rockfish. RFAS strains, possessing A-layer protein on their surface, exhibited β-haemolytic activity against rockfish erythrocytes and capability to survive in rockfish serum, which seem to be associated with virulence.

Temperature-dependent viral replication and antiviral apoptotic response in viral haemorrhagic septicaemia virus (VHSV)-infected olive flounder (Paralichthys olivaceus).

The olive flounder (Paralichthys olivaceus) shows a high rate of mortality to viral haemorrhagic septicaemia virus (VHSV) in the winter and spring but has zero mortality over 20 °C. In this experiment, we studied the effect of rearing temperature on viral replication, viral transcription and antiviral apoptotic immune response in VHSV-infected olive flounder by real-time polymerase chain reaction. Olive flounder were given intra-peritoneal injections of VHSV (10(7.8) TCID(50)/ml) and were reared at 15 °C or 20 °C. Five fish were randomly sampled for head kidney at 3, 6 and 12 h post-infection (hpi) and 1, 2, 4 and 7 days post-infection (dpi). Total RNA extracted from the tissue was reverse transcribed and used as template for real-time PCR. In the 15 °C group, the number of viral gRNA copies peaked after 2 dpi and remained high through 7 dpi, while in the 20 °C group, the copy number was at the highest at 1 dpi but drastically declined at later stages. Viral mRNA levels in the 15 °C group gradually increased starting at 3 hpi to reach their maximum value at 12 hpi and remained high until 2 dpi, whereas the other group showed much lower copy numbers that were undetectably low at 4 and 7 dpi. Type II IFN expression increased as the viral copies increased and the 20 °C group showed quicker and stronger expression than the 15 °C group. The MHC class I and CD8 expression was high in both the groups at early stage of infection (3-6 hpi) but at later stages (2-7 dpi) in 15 °C group expression reduced below control levels, while they expressed higher to control in 20 °C group. The expression of granzyme in 15 °C fish showed a single peak at 2 dpi, but was consistently expressing in 20 °C fish. Individuals expressed very high levels of perforin expressed very high levels of caspase 3. In 15 °C fish, TNFα, FasL and p53 expressed significantly higher than 20 °C only at initial stages of infection (3-6 hpi). Caspase 3 expression found to be low in 15 °C fish whereas it was significantly elevated in 20 °C group. Interestingly individual fish with high caspase 3 expression contained very low viral RNA. Thus, from our experiment, we can conclude that an effective apoptotic immune response in VHSV-infected olive flounder plays a crucial role in the survival of the host at higher temperatures.

Phylogenetic analysis of lymphocystis disease virus from tropical ornamental fish species based on a major capsid protein gene

Lymphocystis disease (LCD) occurs in approximately 100 marine and freshwater fish species (Wolf 1988) and is characterized by clusters of enlarged hypertrophied dermal cells on the skin and fins of affected fish (Wolf 1988). In Korea, LCD is a common fish disease, especially in aquaculture of Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel) and rockfish, Sebastes schlegeli (Hilgendorf) (Kitamura, Jung, Kim, Nishizawa, Yoshimizu & Oh 2006a; Kitamura, Jung & Oh 2006b). The disease rarely causes death but affected fish appear unsightly and lose commercial value. Additionally, in Korean aquarist shops, the disease has been observed in ornamental fish species such as painted glass fish, Chanda baculis (Hamilton), golden gourami, Trichogaster trichopterus (Pallas) and pearl gourami, Trichogaster leeri (Bleeker). The causative agent is lymphocystis disease virus (LCDV), belonging to the genus Lymphocystivirus within the family Iridoviridae, and is characterized by an icosahedral capsid approximately 200 50 nm in diameter, a double-layered capsid with an outer envelope and a fringe of fibril-like external protrusions (Walker 1962; Zwillenberg & Wolf 1968; Wolf 1988). The LCDV genome is a single linear dsDNA molecule, in which the structure is circularly permuted and terminally redundant (Darai, Anders, Koch, Delius, Gelderblom, Samalecos & Flügel 1983; Darai, Delius, Clarke, Apfel, Schnitzler & Flügel 1985; Schnitzler & Darai 1993). Recently, complete genome sequences of two LCDV strains, LCDV-1 from flounder, Platichtys flesus (L.), in Europe and LCDV-C from Japanese flounder in China, were determined. The former is 102 653 bp encoding 195 potential ORFs, while the latter is 186 250 bp with 240 potential ORFs (Tidona & Darai 1997; Zhang, Xiao, Xie, Li & Gui 2004). Comparing the LCDV-1 and LCDV-C genomes, the highest nucleotide sequence identity was observed in a major capsid protein (MCP) gene (78.9%) encoding a single polypeptide with a molecular mass of approximately 50 kDa (Schnitzler & Darai 1993; Tidona & Darai 1997; Zhang et al. 2004). The MCP gene is one of the most important for analysis of genetic relationships among iridoviruses, because its nucleotide sequence is relatively conserved within the family Iridoviridae (Tidona, Schnitzler, Kehm & Darai 1998; Williams, Barbosa-Solomieu & Chinchar 2005). Recently, we reported that at least three genotypes were present in the genus Lymphocystivirus, based on MCP gene sequence and the pathogenicity of lymphocystiviruses: genotype I, consisting of LCDV-1; genotype II, consisting of Japanese flounder isolates and a sea bass isolate; and genotype III, consisting of rockfish isolates (Kitamura et al. 2006a,b). Despite its global importance in freshwater ornamental fish, no genetic information Journal of Fish Diseases 2008, 31, 473–479 doi:10.1111/j.1365-2761.2008.00917.x

Impaired TLR2 and TLR7 response in olive flounder infected with viral haemorrhagic septicaemia virus at host susceptible 15 °C but high at non-susceptible 20 °C

The olive flounder (Paralichthys olivaceus) is susceptible to viral haemorrhagic septicaemia virus (VHSV) at 15 °C but no mortality is observed at 20 °C even though the virus can grow profusely in vitro. Thus, we designed an experiment to better understand the immune response of olive flounder to VHSV when the host reared at 15 °C or 20 °C and infected with the virus. Olive flounder (18-22 g) reared at 15 ± 0.5 °C or 20 ± 0.5 °C were intra-peritoneally injected with VHSV (10(7.8) TCID50/fish) and sampled (n = 5) for head kidney at 3, 6, 12 hpi, 1, 2, 4 and 7 dpi; similarly, mock injected control groups (n = 5). Real-time PCR-based absolute quantification method was followed to quantify copies of VHSV gRNA and mRNA, while the immune gene expression of the olive flounder was quantified relative to internal control, β-actin. Viral infection resulted in a cumulative mortality of 24% in olive flounder reared at 15 °C, but no mortality was recorded in the 20 °C group or control groups. TLR2 and TLR7 expression at 15 °C was enhanced during early-infection phase (3-6 hpi) and recovery phase (4-7 dpi) when viral transcription was low, but expression was significantly reduced (12 hpi-1 dpi) at peak-infection period. However, the 20 °C group showed low viral transcription and expressed high level of TLR7 and a moderately higher unchanged level of TLR2. In both the groups, TLR3 expression was unaffected. Nevertheless, expression of MDA5 and LGP2 increased significantly irrespective of rearing temperature at the time of peak infection, hence at 15 °C VHSV down-regulated expression of TLR2 and TLR7 but not MDA5 or LGP2. Comparatively, at 15 °C IRF3 expressed high but IRF7 remained very low. Interleukins (IL-1β, IL-6 and IL-8) were significantly elevated in both the groups, but quicker and for a shorter period at 20 °C. In the 15 °C group, an extended period of expression of ILs could create an unsafe prolonged inflammatory condition. The olive flounders expressed high ISGs at 15 °C but were lagging by 12 h than 20 °C group. Based on these findings, we concluded that viral-mediated disruption of TLR2 and TLR7 expression in the 15 °C group could have delayed the host interferon response and provided a window for high viral growth. However, an effective host immune response at 20 °C contained VHSV from reaching the critical limit.

Does heavy oil pollution induce bacterial diseases in Japanese flounder Paralichthys olivaceus

As basic research for the effect of heavy oil on the fish immune system, in this study, the number of leukocyte was counted in Japanese flounder Paralichthys olivaceus, after exposure to heavy oil at a concentration of 30 g/8L for 3 days. To compare the numbers of bacteria in the skin mucus between oil-exposed and control fish, viable bacteria were enumerated by counting colony forming unit (CFU). Compared with 5.79+/-1.88 x 10(7)leukocytes/mL in the controls, the exposed fish demonstrated higher counts, averaging 1.45+/-0.45 x 10(8)cells/mL. The bacterial numbers of control fish were 4.27+/-3.68 x 10(4)CFU/g, whereas they were 4.58+/-1.63 x 10(5)CFU/g in the exposed fish. The results suggest that immune suppression of the fish occurred due to heavy oil stressor, and bacteria could invade in the mucus, resulting in the increasing leukocyte number to prevent infectious disease.