Cloning and expressional analysis of secretory and membrane-bound IgM in rock bream (Oplegnathus fasciatus) under megalocytivirus infection and vaccination.

In this study, for better understanding the humoral immunity of rock bream (Oplegnathus fasciatus), 2 transcripts of immunoglobulin M (IgM) heavy chain gene including membrane bound (m-IgM) and secretory (s-IgM) forms were sequenced and analyzed their tissue distribution and differential expression in rock bream under rock bream iridovirus (RBIV) infection and vaccination since RBIV has caused mass mortality in rock bream aquaculture in Korea. Consequently, s-IgM cDNA was 1902 bp in length encoding a leader region, a variable region, four constant regions (CH1, CH2, CH3, CH4) and a C-terminal region while m-IgM cDNA was 1689 bp in length encoding shorter three constant regions (CH1, CH2, CH3) and two transmembrane regions. The predicted s-IgM and m-IgM represent a high structural similarity to other species including human. In tissue distribution analysis in healthy fish, the highest expression of s-IgM was observed in head kidney followed by body kidney, spleen, and mid gut whereas m-IgM expression was the highest in blood followed by head kidney and spleen. In vitro, s-IgM expression was up-regulated by LPS in head kidney and spleen cells at 24 h with no change of m-IgM expression. In vivo upon vaccination, s-IgM expression was up-regulated in liver and blood but not in head kidney while m-IgM expression was only up-regulated in head kidney. After challenge with RBIV, s-IgM expression level was higher in vaccinated fish than in unvaccinated fish and m-IgM expression was up-regulated in head kidney of vaccinated group. In conclusion, differential expression of m-IgM and s-IgM may indicate their differential functions to produce the most effective IgM during adaptive immune response. Although it is not able to assess specific IgM at protein level due to a lack of antibody against rock bream IgM, the present study on s-IgM and m-IgM gene expressions upon infection and vaccination will be useful in developing efficient vaccines in the future.

Complete genome sequence and pathogenic analysis of a new betanodavirus isolated from shellfish.

We determined the complete genomic RNA sequence of a new type of betanodavirus Korea shellfish nervous necrosis virus (KSNNV) isolated from shellfish. Compared with other isolates representing four genotypes of betanodaviruses, the identity of the whole nucleotide sequence of the virus was in the range of 76%-83% with the presence of specific genetic motifs and formed a separate new branch in the phylogenetic analysis. In pathogenic analysis by immersion method, KSNNV-KOR1 shows 100% cumulative mortality like SFRG10/2012BGGa1 (RGNNV) in newly hatched sevenband grouper and mandarin fish, which is clearly different from those found in negative control groups. There were no significant differences in increasing rates of mortality and viral intra-tissue concentration of larval fishes infected with KSNNV-KOR1 at both 20 and 25°C water temperature. Histopathological examination of each fish species in the moribund stage revealed the presence of clear vacuoles in both brain and retinal tissues similar to typical histopathology features of RGNNV. In the present study, we first report a new betanodavirus from shellfish as the aetiological agent of viral nervous necrosis disease in fish with complete genomic nucleotide sequence and pathogenic analysis.

Evaluation of blue mussel Mytilus edulis as vector for viral hemorrhagic septicemia virus (VHSV).

When viral diseases occur in aquaculture farms, the virus released into the seawater from infected animals can re-infect other susceptible species or accumulate in filter-feeding organisms. We conducted a viral hemorrhagic septicemia virus (VHSV) survivability analysis of blue mussel Mytilus edulis digestive enzymes, viral depuration, and infectivity tests via in vitro and in vivo inoculation to evaluate the infectious state. VHSV particles were not completely digested within 24 h in vitro and were maintained for 7 d in the mussel digestive gland. Mussels cohabitating with naturally VHSV-infected olive flounder Paralichthys olivaceus could accumulate the viral particles. Although the viral particles in the gill as the entrance of filter-feeding organisms are infectious, the presence of these particles in the digestive gland were not able to induce cytopathic effects in vitro. Viral particles detected by RT-PCR from bivalve mollusks (Pacific oyster Crassostrea gigas and mussel) from the field did not produce cytopathic effects in cell culture and did not replicate after intraperitoneal injection into olive flounder. Therefore, VHSV particles in blue mussel might be in a non-infectious stage and the possibilities of VHSV transmission to fish under field conditions are scarce.

Analysis of proinflammatory gene expression by RBIV infection in rock bream, Oplegnathus faciatus.

Early induction of proinflammatory cytokines is known to regulate the later immune responses to inhibit the progress of infectious diseases. In this study, proinflammatory cytokine gene expression has been studied in immune tissues to understand the early immune response induced by megalocytivirus in rock bream (Oplegnathus faciatus). For this, we have cloned interleukin (IL)-1ß and IL-8 gene and performed the phylogenetic and structural analysis. Also the constitutive gene expressions of IL-1ß and IL-8 were assessed in 12 organs and found to be the highest expression in tail fin and liver, respectively. The expressions of proinflammatory cytokine genes including IL-1ß, IL-8, TNFα and Cox-2, and antiviral genes like Mx and IFN1 were analysed by stimulation with PAMPs and RBIV infection. In vitro study showed the highly up-regulated proinflammatory gene expressions in head kidney and the moderate up-regulation in spleen by LPS. Same concentration of polyI:C moderately upregulated IL-1ß gene expression in head kidney but down-regulated IL-8 and TNFα gene expression in head kidney and spleen at 8 h. Mx and IFN1 gene expressions were highly upregulated by polyI:C in head kidney and spleen cells in vitro. By RBIV infection, proinflammatory gene expressions were initially up-regulated and later down-regulated in head kidney. In spleen, although mostly not significant, proinflammatory cytokine gene expressions were down-regulated by RBIV infection except up-regulation of Cox-2 gene expression by low concentration of RBIV at 24 h. Mx and IFN1 gene expressions were down-regulated by high dose of RBIV infection in vitro. In vivo study revealed that IL-8, TNFα, and IFN1 gene expressions were down-regulated in brain, head kidney, spleen, and gill while up-regulated in heart and liver, indicating differential proinflammatory and antiviral responses in the organs. It is supposed that down-regulation of proinflammatory gene expression in the immune organs may result in the failure of antiviral immune responses, causing high mortalities by megalocytivirus infection in rock bream.

Evaluation of Formalin-Inactivated Vaccine Efficacy against Red Seabream Iridovirus (RSIV) in Laboratory and Field Conditions.

Red seabream iridovirus (RSIV) is a major cause of marine fish mortality in Korea, with no effective vaccine available since its first occurrence in the 1990s. This study evaluated the efficacy of a formalin-killed vaccine against RSIV in rock bream under laboratory and field conditions. For the field trial, a total of 103,200 rock bream from two commercial marine cage-cultured farms in Southern Korea were vaccinated. Farm A vaccinated 31,100 fish in July 2020 and monitored them for 18 weeks, while farm B vaccinated 30,700 fish in August 2020 and monitored them for 12 weeks. At farm A, where there was no RSIV infection, the vaccine efficacy was assessed in the lab, showing a relative percentage of survival (RPS) ranging from 40% to 80%. At farm B, where natural RSIV infections occurred, cumulative mortality rates were 36.43% in the vaccinated group and 80.32% in the control group, resulting in an RPS of 54.67%. The RSIV-infectious status and neutralizing antibody titers in serum mirrored the cumulative mortality results. This study demonstrates that the formalin-killed vaccine effectively prevents RSIV in cage-cultured rock bream under both laboratory and field conditions.

Effects of RNA interference-mediated knock-down of hypoxia-inducible factor-α on respiratory burst activity of the Pacific oyster Crassostrea gigas hemocytes.

In mammals, hypoxia-inducible factor-1 α (HIF-1α) is known to play important roles not only in oxygen homeostasis but also in innate immune responses. In this study, to assess the functional role of HIF-α in respiratory burst activity of Crassostrea gigas hemocytes, oysters were injected with HIF-α- or green fluorescent protein (GFP)-targeted-long double-stranded RNAs (dsRNAs), and at 1, 3, and 7 days post-injection, knock-down of C. gigas HIF-α expression and production of reactive oxygen species (ROS) were analyzed. Expression of HIF-α in mantle, gill, and hemocytes of C. gigas was clearly down-regulated by injection of the HIF-α-targeted-long dsRNA, but was not inhibited by the GFP-targeted-long dsRNA, indicating that HIF-α expression was suppressed through sequence-specific and systemic RNA interference (RNAi). Respiratory burst activity of hemocytes was significantly increased by administration of GFP-targeted-long dsRNA. However, knock-down of HIF-α expression led to significant decrease of chemiluminescence (CL) response of C. gigas hemocytes at 3 and 7 days post-administration of HIF-α-targeted-long dsRNA, indicating the critical role of HIF-α in activation of respiratory burst activity of oyster hemocytes.

Microbacterium oxydans, a novel alginate- and laminarin-degrading bacterium for the reutilization of brown-seaweed waste.

There is a growing demand for the efficient treatment of seaweed waste. We identified six bacterial strains from the marine environment for the reutilization of brown-seaweed waste, and the most potentially useful strain, Microbacterium oxydans, was chosen and further investigated. Plate assays indicated that this bacterial isolate possessed both alginate lyase and laminarinase activities. The optimal inoculum size, pH, temperature and substrate concentration for the degradation of brown-seaweed polysaccharides by the isolate were as follows: 20% (v v(-1)), pH 6.0, 37 °C, and 5 g L(-1) for alginate and 20% (v v(-1)), pH 6.0, 30 °C, and 10 g L(-1) for laminarin, respectively. During 6 d in culture under the optimal conditions, the isolate produced 0.17 g L(-1) of reducing sugars from alginate with 11.0 U mL(-1) of maximal alginate lyase activity, and 5.11 and 2.88 g L(-1) of reducing sugars and glucose from laminarin, respectively. In particular, a fair amount of laminarin was degraded to glucose (28.8%) due to the isolate's exolytic laminarinase activity. As a result, the reutilization of brown-seaweed waste by this isolate appears to be possible for the production of reducing sugars as a valuable resource. This is the first study to directly demonstrate the ability of M. oxydans to degrade both alginate and laminarin.

Identification of Various InDel-II Variants of the White Spot Syndrome Virus Isolated from Frozen Shrimp and Bivalves Obtained in the Korean Commercial Market.

White spot syndrome virus (WSSV) poses a significant threat to the global shrimp industry. We investigated the presence of WSSV in frozen shrimp (n = 86) and shellfish (n = 185) from the Korean market (2010-2018). The detection rate of first-step polymerase chain reaction (PCR) in domestic shrimp was 36.8% (7/19), whereas that in imported shrimp was 0.01% (1/67). Furthermore, the WSSV genome was amplified from domestic bivalve mollusks by first- and second-step PCR with accuracies of 3.4% (5/147) and 15.6% (23/147), respectively. The genetic relatedness of InDel-II regions among WSSVs detected in domestic shrimp groups revealed four variants (777, 5649, 11,070 and 13,046 bp insertion or deletion), and imported shrimp groups had four variants (10,778, 11,086, 11,500 and 13,210 bp) compared with the putative ancestor WSSV strain. The 5649 bp variant was the dominant type among the WSSV variants detected in domestic shrimp (54.5%, 6/11). Notably, bivalve mollusks exhibited six variants (777, 5649, 5783, 5876, 11,070 and 13,046 bp), including four variants detected in shrimp, indicating that bivalve mollusks could facilitate WSSV tracking. In a challenge test, whiteleg shrimp (Litopenaeus vannamei) exhibited varying mortality rates, indicating a link between InDel-II deletion and viral replication. These findings highlight the complexity of WSSV transmission.

Fugu double U6 promoter-driven long double-stranded RNA inhibits proliferation of viral hemorrhagic septicemia virus (VHSV) in fish cell lines.

A long double-stranded RNA (dsRNA)-producing vector driven by fugu double U6 promotors, in which the two promoters were arranged in a head-to-head fashion, was newly constructed. To determine whether the DNA-vector-based long dsRNAs can induce sequence-specific RNA interference (RNAi), Epithelioma papulosum cyprini (EPC) cells and chinook salmon embryonic (CHSE-214) cells were transfected with the long dsRNA vector targeting the G gene of VHSV, and its effect on expression of the G gene and viral proliferation was investigated. The sequence-specific inhibitory effect was further confirmed by analysis of interferon (IFN)-triggered Mx1 gene expression and cross-protection against infectious hematopoietic necrosis virus (IHNV). The fugu double U6 promoter-driven vector successfully produced long dsRNAs in EPC cells, a system that allows continuous production of long dsRNAs in transfected cells. The plasmid-based long dsRNAs targeting the VHSV G gene effectively suppressed G gene expression, but control dsRNAs targeting the EGFP gene did not. Furthermore, there was no significant difference in Mx gene expression between cells transfected with the long dsRNA-producing vector and those transfected with the control empty vector. These results suggest that G gene expression was suppressed not by type-I-IFN-mediated nonspecific inhibition but in a sequence-specific manner. Both EPC and CHSE-214 cells transfected with plasmids producing long dsRNAs targeting the VHSV G gene were protected against VHSV infection but were not protected against IHNV infection, suggesting sequence-specific RNAi-mediated inhibition of viral proliferation. In conclusion, we show, for the first time, long-dsRNA-mediated RNAi in fish cells. The DNA-vector-based long dsRNAs may provide an efficient tool for analysis of gene function in fish cells without preliminary burdensome work for selection of effective siRNA clones, and it may be applied as an antiviral measure in cultured fish.

Emergence of Reassortment between a New and Reported Types of Betanodavirus in Shellfish.

Recently, three types of betanodavirus including red spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV), and Korean shellfish nervous necrosis virus (KSNNV) (proposed as a new fifth type) have been detected in shellfish in the marine environment around Korea. To investigate the presence of reassortment between betanodavirus types, the type based on the RNA2 segment of betanodaviruses carried in 420 domestic shellfish (n = 306) and finfish (n = 35), as well as imported shellfish (n = 79), was compared with the type identified by reverse-transcriptase polymerase chain reaction (RT-PCR) for RNA1 segment. Only five samples carrying reassortant betanodaviruses were found, appearing as RG/KSNNV (n = 2), KS/RGNNV (n = 1), and SJ/RGNNV (n = 2) types. From these samples, we successfully isolated two reassortant strains from Korean and Chinese shellfish in E-11 cells and called them KG1-reKS/RG and CM1-reRG/KS, respectively. In the full genome sequences, each RNA segment of the reassortant strains exhibited the same gene length and high sequence homology (≥98%) with the reference strains corresponding to the type of each segment. Both these reassortant strains induced high mortality to sevenband grouper (Epinephelus septemfasciatus) larvae with high viral concentrations in the body (109 viral particles/mg) and severe vacuolation in the retina and brain. These are the first results showing the involvement of the KSNNV type in the reassortment of RNA segments in the reported types of betanodavirus, which could represent a new potential risk in fish.

Development and characterization of megalocytivirus persistently-infected cell cultures for high yield of virus.

Megalocytivirus infection is a major threat in rock bream aquaculture in Korea. To produce a highly concentrated megalocytivirus, primary cells, established cell line and persistently infected cell line were used in this study. Megalocytivirus was inoculated in primary fin cell cultures of red sea bream (Pagrus major), rock bream (Oplegnathus fasciatus), olive flounder (Paralichthys olivaceus) and black sea bream (Acanthopagrus schlegelii) and produced at similar concentrations of 108.99 - 9.88 viral particles/mL in all cultures while produced 107.31 viral particles/mL in grunt fin (GF) cell line. Since only red sea bream fin culture was amenable to subculturing for more than 100 times, it was established into Pagrus major fin (PMF) cell line. A persistently infected PMF cell line (PI-PMF) was obtained by continuous subculturing every 7 days as a batch culture system (PI-PMF-B) after infecting with megalocytivirus. Virus in supernatant of PI-PMF-B was maintained at high concentrations throughout over 50 consecutive subcultures in a relatively narrow range from 108.33 to 108.94 viral particles/mL with high level of CPE. For a more efficient and convenient production, a semi-batch culture system (PI-PMF-S) was developed in which culture media were exchanged at intervals of 3 days without subculturing for more than 50 media exchanges. Despite low virus productivity in a single cell (specific virus productivity, SVP), total cell number was increased in PI-PMF-S, allowing us to efficiently obtain a much higher concentration of virus (108.56 to 109.75 viral particles/mL) than in PMF-B. This is the first study to report detailed new methods for continuous and efficient production of high concentrations of megalocytivivrus with characterization of viral propagation in persistently infected cells.

Development of a high-dose vaccine formulation for prevention of megalocytivirus infection in rock bream (Oplegnathus fasciatus).

A formalin-inactivated red sea bream iridovirus (RSIV) vaccine was prepared using the culture supernatant of a persistently infected Pagrus major fin cell line (PI-PMF) with IVS-1 strain (RSIV subtype II Meglaocytivirus). Rock bream (Oplegnathus fasciatus) were injected with a high-dose, ultracentrifuged megalocytivirus vaccine (Ultra HSCMV, 7.0 × 1010 copies/mL), a high-dose supernatant of cultured megalocytivirus vaccine (HSCMV, 1.0 × 1010 copies/mL), a supernatant of cultured megalocytivirus vaccine (SCMV, 1.0 × 109 copies/mL), and a low-dose of cultured megalocytivirus vaccine (LSCMV, 1.0 × 108 copies/mL). The vaccine efficacies for the various vaccine formulations were determined done following injection challenge with IVS-1 (1.0 × 104 copies/0.1 mL/fish), and the four different vaccines exhibited cumulative mortalities of 10.0 ± 0.0%, 48.3 ± 7.6%, 75.0 ± 5.0%, and 100.0 ± 0.0%, respectively. Additionally, the dose-dependent vaccine efficacy was also confirmed using two different cohabitation methods that included challenges G (general) and I (individual). When squalene + aluminum hydroxide (SqAl) was used as an adjuvant for the HSCMV or SCMV vaccine, cumulative mortalities of 30.0 ± 5.0% and 48.3 ± 7.6%, respectively, were obtained; moreover, these two adjuvants exhibited the highest efficacy in this study. The observed difference in survival post-challenge for the different vaccine concentrations was not reflected in the differences in neutralizing antibody titers. It was found that the water temperature during immune induction plays a less important a role than the water temperature during the challenge test, in which lowering the water temperature from 25 °C to 21 °C during a challenge improved the level of protection from cumulative mortalities from 35% to 10%. This study demonstrated that protection against mortality using inactivated vaccines against RSIVD in rock bream, which are known to be the most susceptible species to RSIV infection, is dependent upon antigen dose and temperature during the challenge.

Influence of temperature shifts on the onset and development of red sea bream iridoviral disease in rock bream Oplegnathus fasciatus.

The effects of various water temperature treatments on the development of red sea bream iridovirus disease (RSIVD) in rock bream Oplegnathus fasciatus challenged with iridovirus Sachun (IVS-1) were determined by measuring the mortality and the viral concentration in the spleen of infected fish. Experimental infections of rock bream with IVS-1 at water temperatures of 18, 21, and 25 degrees C resulted in a cumulative mortality of 100%, but infections at 13 degrees C resulted in 0% mortality, even after 45 d. The disease progressed more rapidly at higher water temperatures; at 25, 21, and 18 degrees C, the mean numbers of days until death were 17, 20, and 30 d, respectively. When the water temperature for fish infected with iridovirus by intramuscular injection was shifted from 13 to 25 degrees C, the cumulative mortality reached 100%, with rapid onset of the disease, independent of the time at which the temperature was shifted, i.e. 7, 14, or 30 d after injection at 13 degrees C. Real-time PCR data revealed that the viral genome copy number in the spleen of rock bream maintained at 13 degrees C increased with time, suggesting the occurrence of viral replication even at 13 degrees C. In the reverse experiment, when the water temperature for fish that were infected at a higher temperature was shifted to 13 degrees C, 3 or 7 d after injection at 25 degrees C, the fish showed 100% cumulative mortality, although the mean number of days until death was higher than that observed for fish maintained at a constant temperature of 25 degrees C. The viral DNA concentration in the spleen of rock bream that had been shifted down to 13 degrees C, 3 or 7 d after injection at 25 degrees C, was not suppressed, but increased and eventually reached levels sufficient to induce mortality at 13 degrees C. However, the level of viral genome copy numbers in the spleen of dead fish at 25 degrees C, regardless of whether those fish were held at a constant temperature of 25 degrees C or shifted up from 13 degrees C, appeared to be greater than the level found in the dead fish shifted down to 13 degrees C after inoculation at 25 degrees C.

Multiplex PCR using YeaD and 16S rRNA gene to identify major pathogens in vibriosis of Litopenaeus vannamei.

The Vibrio species causing major diseases in Litopenaeus vannamei are Vibrio harveyi, Vibrio alginolyticus, and Vibrio parahaemolyticus. For multiplex PCR primers, YeaD was used to detect the three Vibrio species. Bioinformatic analysis such as MultiPLX and primer-BLAST was used to design stable and species-specific multiplex PCR primers. Multiplex PCR results showed clear band patterns with bands at 185 bp for V. alginolyticus, 396 bp for V. harveyi, 805 bp for V. arahaemolyticus, and 596 bp for common Vibrio species. The minimum concentration of DNA was measured by PCR; the value for V. alginolyticus was 0.1 ng, that of V. harveyi was 0.03 ng, and that of V. parahaemolyticus was 0.003 ng. Taken together, YeaD showed stability and specificity in identifying Vibrio species. Our multiplex PCR amplification method is an effective and inexpensive tool for identifying Vibrio species.

Transmission of iridovirus from freshwater ornamental fish (pearl gourami) to marine fish (rock bream).

Freshwater pearl gourami Trichogaster leeri and seawater rock bream Oplegnathus fasciatus infected by the iridoviruses PGIV-SP and IVS-1 were carrying similar numbers of viral particles (2.52 x 10(8) and 2.46 x 10(8) viral genome copies mg(-1) spleen tissue, respectively). The viral genome copy number for both iridoviruses decreased much faster in seawater than in freshwater, reaching a concentration of less than 0.5%, versus 26 to 54% in freshwater, after 4 d of incubation at 25 degrees C. The decrease in copy number altered the infectivity of the viruses, as reflected by the decreased cumulative mortality of rock bream injected intraperitoneally with the incubated iridoviruses. Moreover, uninfected rock bream cohabitated with PGIV-SP-challenged rock bream showed 100% cumulative mortality; a similar experiment using IVS-1 had the same result, implying the potential for iridoviral transmission from freshwater ornamental fish to marine fish even in a marine environment. Of 58 outwardly healthy marine fish groups collected from various markets, 2 rock bream groups and 1 sea perch group Lateolabrax sp. tested positive for PGIV-SP by 2-step polymerase chain reaction (PCR). Thus, PGIV-SP from freshwater ornamental fish may have crossed both environmental and species barriers to infect marine fish such as rock bream.

Outbreaks and risks of infectious spleen and kidney necrosis virus disease in freshwater ornamental fishes.

We examined the distribution of iridoviruses in 10 freshwater ornamental fish species hatched in Korea and imported from other Asian countries using both 1-step and 2-step polymerase chain reation (PCR). None of the 10 fish species analyzed were free of iridovirus as shown by 2-step PCR positive results, and 3 species yielded 1-step PCR positive results with associated mortality. Cloned PCR amplicons of the adenosine triphosphatase (ATPase) and major capsid protein (MCP) genes in genomic DNA of iridovirus showed the same nucleotide sequences as that of infectious spleen and kidney necrosis virus (ISKNV) isolated from the mandarinfish Siniperca chuatsi. These results indicate the presence of ISKNV disease in various ornamental fish as new host species and that the disease is widespread throughout different Asian countries including Korea, Singapore and China. Such infections were either clinical with associated mortality (and 1-step PCR positive) or asymptomatic in fish that were externally healthy (and only positive in 2-step PCR). Molecular analyses of the K2 region performed on iridovirus samples isolated from freshwater ornamental fishes revealed deletion/insertion of repetitive sequences of various lengths (42 to 339 bp), depending on the ISKNV isolates, without substitutions. Experimental infection of pearl gourami Trichogaster leeri and silver gourami T. microlepis with a tissue homogenate of pearl gourami infected by ISKNV induced 70 and 20% cumulative mortalities in the pearl and silver gourami, respectively.

Prevalence of tet(B) and tet(M) genes among tetracycline-resistant Vibrio spp. in the aquatic environments of Korea.

Of 24 tetracycline(Tc)-resistant Vibrio spp. isolated from different marine sources in Korea between 1993 and 2003, 23 were identified as carrying both tet(B) and tet(M), while 1 strain carried tet(B) only. In conjugation experiments, 3 strains appeared to be able to transfer both tet(B) and tet(M) to the recipient. Both discriminatory PCR and sequence analysis showed that tet(M) genes of Vibrio spp. appear to be a single allele containing a specific region of tet(M) in Tn1545. However, erm(B) and aphA3, known to be linked to Tn1545-like genes, were not detected in Tc-resistant Vibrio spp., even in 9 strains resistant to erythromycin. In analysis to examine the relative position of tet(B) and tet(M), it was shown that tet(M) was present at the 3'-end of the insertion sequence IS10 of Tn10 carrying tet(B). At the junctional region between Tn10 and tet(M), we found a 14 bp sequence of unknown function and the deletion of regulatory sequences reported to be needed for tet(M) expression in conjugative transposons. This is the first report of the simultaneous presence of tet(B) and tet(M), and of the tet(M) gene being linked to the 3'-end of Tn10 in Tc-resistant Vibrio spp. in Korea.

Molecular cloning and characterization of Cathepsin B from a scuticociliate, Uronema marinum.

A cDNA encoding cathepsin B was cloned from the scuticociliate, Uronema marinum, which invades the olive flounder, Paralichthys olivaceus, leading to high mortalities in culturing fish. The full-length scuticociliate cathepsin B (ScCtB) gene contains an open reading frame of 1053 base pairs encoding 350 amino acids. A homology search revealed that ScCtB shares sequence identity with several piscine cathepsin Bs (48%-45%). The protein of ScCtB from U. marinum extracts was purified 12.8-fold by a one step purification process using a DEAE-Sephagel high performance liquid chromatography (HPLC) column. It had a molecular mass of 30 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, which was consistent with predicting molecular mass of mature protein (29.2 kDa) of ScCtB. The protease activity of the ScCtB enzyme was demonstrated by electrophoresis in a gelatin-acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate, Z-arginyl-arginyl-7-amido-4-methylcoumarin (Z-Arg-Arg-AMC). The optimum pH for the protease activity was 5.5. Typical of cysteine proteases, the enzyme was inhibited by trans-epoxysuccinyl-L-leucyl-amido(4-guanidino)butane (E-64) and leupeptin.

Molecular cloning of the gyrA gene and characterization of its mutation in clinical isolates of quinolone-resistant Edwardsiella tarda.

Knowing the entire sequence of the gene encoding the DNA gyrase Subunit A (gyrA) of Edwardsiella tarda could be very useful for confirming the role of gyrA in quinolone resistance. Degenerate primers for the amplification of gyrA were designed from consensus nucleotide sequences of gyrA from 9 different Gram-negative bacteria, including Escherichia coli. With these primers, DNA segments of the predicted size were amplified from the genomic DNA of E. tarda and then the flanking sequences were determined by cassette ligation-mediated polymerase chain reaction. The nucleotide sequence of gyrA was highly homologous to those of other bacterial species, in both the whole open-reading frame and the quinolone-resistance-determining region (QRDR). The 2637-bp gyrA gene encodes a protein of 878 amino acids, preceded by a putative promoter, ribosome binding site and inverted repeated sequences for cruciform structures of DNA. However, the nucleotide sequence of the flanking region did not show any homologies with those of other bacterial DNA gyrase Subunit B genes (gyrB) and suggested the gyrase genes, gyrA and gyrB, are non-continuous on the chromosome of E. tarda. All of the 12 quinolone-resistant isolates examined have an alteration within the QRDR, Ser83 --> Arg, suggesting that, in E. tarda, resistance to quinolones is primarily related to alterations in gyrA. Transformation with the full sequence of E. tarda gyrA bearing the Ser83 --> Arg mutation was able to complement the sequence of the gyrA temperature-sensitive mutation in the E. coli KNK453 strain and to induce increased resistance to quinolone antibiotics at 42 degrees C.

CpG motif in synthetic ODN primes respiratory burst of olive flounder Paralichthys olivaceus phagocytes and enhances protection against Edwardsiella tarda.

Effects of synthetic cytidine-phosphate-guanosine (CpG) oligodeoxynucleotide (ODN) on respiratory burst activity of olive flounder (Paralichthys olivaceus) head-kidney phagocytes and on protection against lethal infection with Edwardsiella tarda were investigated. Phagocytes precultured with a CpG ODN showed significantly higher chemiluminescence (CL) responses than phagocytes precultured with guanosine-phosphate-cytidine (GpC) ODN or culture medium alone (control) at all concentrations. Supernatants produced from leucocytes, which were pulsed with CpG ODN, induced significantly higher respiratory burst activity than supernatants produced by GpC ODN or culture medium alone. In an in vivo experiment, respiratory burst activities of the head kidney phagocytes in the groups injected either 0.25 or 0.5 microg fish(-1) of CpG ODN were significantly higher than those in the groups injected with GpC ODN or HBSS (control) at 3, 5 and 7 d after injection. The groups of fish injected with 0.25 or 0.5 microg of CpG ODN showed higher survival rates (83.3%) than groups treated with GpC ODN (33.3%) and a control group (8.3%) after challenge with E. tarda. The present in vitro and in vivo experiments have demonstrated the ability of synthetic CpG ODN to increase phagocyte respiratory burst activity and disease resistance in olive flounder.