Heavy oil exposure suppresses antiviral activities in Japanese flounder Paralichthys olivaceus infected with viral hemorrhagic septicemia virus (VHSV).

A combined treatment of heavy oil (HO) exposure and virus infection induces increased mortality in Japanese flounder (Paralichthys olivaceus). In this study, we addressed how HO exposure affects the immune system, especially antiviral activities, in Japanese flounder. The fish were infected with viral hemorrhagic septicemia virus (VHSV), followed by exposure to HO. We analyzed virus titers in the heart and mRNA expression in the kidney of surviving fish. The virus titers in fish exposed to heavy oil were higher than the threshold for onset. The results suggest that HO exposure may allow the replication of VHSV, leading to higher mortality in the co-treated group. Gene-expression profiling demonstrated that the expression of antiviral-activity-related genes, such as those for interferon and apoptosis induction, were lower in the co-treated group than in the group with VHSV infection only. These results helped explain the high virus titers in fish treated with both stressors. Thus, interferon production in the virus-infected cells and apoptosis induction by natural killer cells worked normally in the VHSV-infected fish without HO exposure, but these antiviral activities were slightly suppressed by HO exposure, possibly leading to extensive viral replication in the host cells and the occurrence of VHS.

Occurrence and Molecular Identification of Microcotyle sebastis Isolated from Fish Farms of the Korean Rockfish, Sebastes schlegelii.

Microcotyle sebastis is a gill monogenean ectoparasite that causes serious problems in the mariculture of the Korean rockfish, Sebastes schlegelii. In this study, we isolated the parasite from fish farms along the coasts of Tongyeong, South Korea in 2016, and characterized its infection, morphology and molecular phylogeny. The prevalence of M. sebastis infection during the study period ranged from 46.7% to 96.7%, and the mean intensity was 2.3 to 31.4 ind./fish, indicating that the fish was constantly exposed to parasitic infections throughout the year. Morphological observations under light and scanning electron microscopes of the M. sebastis isolates in this study showed the typical characteristics of the anterior prohaptor and posterior opisthaptor of monogenean parasites. In phylogenetic trees reconstructed using the nuclear 28S ribosomal RNA gene and the mitochondrial cytochrome c oxidase I gene (cox1), they consistently clustered together with their congeneric species, and showed the closest phylogenetic relationships to M. caudata and M. kasago in the cox1 tree.

Aryl Hydrocarbon Receptor Signaling Is Functional in Immune Cells of Rainbow Trout (Oncorhynchus mykiss).

The arylhydrocarbon receptor (AhR) is an important signaling pathway in the immune system of mammals. In addition to its physiological functions, the receptor mediates the immunotoxic actions of a diverse range of environmental contaminants that bind to and activate the AhR, including planar halogenated aromatic hydrocarbons (PHAHs or dioxin-like compounds) and polynuclear aromatic hydrocarbons (PAHs). AhR-binding xenobiotics are immunotoxic not only to mammals but to teleost fish as well. To date, however, it is unknown if the AhR pathway is active in the immune system of fish and thus may act as molecular initiating event in the immunotoxicity of AhR-binding xenobiotics to fish. The present study aims to examine the presence of functional AhR signaling in immune cells of rainbow trout (Oncorhynchus mykiss). Focus is given to the toxicologically relevant AhR2 clade. By means of RT-qPCR and in situ hybdridization, we show that immune cells of rainbow trout express ahr 2α and ahr 2ß mRNA; this applies for immune cells isolated from the head kidney and from the peripheral blood. Furthermore, we show that in vivo as well as in vitro exposure to the AhR ligand, benzo(a)pyrene (BaP), causes upregulation of the AhR-regulated gene, cytochrome p4501a, in rainbow trout immune cells, and that this induction is inhibited by co-treatment with an AhR antagonist. Taken together, these findings provide evidence that functional AhR signaling exists in the immune cells of the teleost species, rainbow trout.

Host responses of Japanese flounder Paralichthys olivaceus with lymphocystis cell formation.

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease (LCD). In this study, we investigated the mechanisms of lymphocystis cell (LCC) formation from the viewpoint of gene expression changes in the infected fish. LCC occurrence and virus titers in the experimentally infected Japanese flounder, Paralichthys olivaceus were monitored by visual confirmation and real-time PCR, respectively. The gene expression changes in the fish fin were investigated by microarray experiments. LCCs firstly appeared in the fish at 21 days post infection (dpi). LCD incidence increased with time and reached 92.9% at 62 dpi. LCDV genome was firstly detected from dorsal fins at 14 dpi, and the relative amount of the genome gradually-increased until 56 dpi. Since the occurrence of LCC was approximately synchronized with increasing of the virus genome, virus replication might play important roles for LCC formation. The microarray detected a few gene expression changes until 28 dpi. However, the number of expression changed genes dramatically increased between 28 and 42 dpi in which LCCs formation was active. From the microarray data analyses, apoptosis and cell division related genes were down-regulated, whereas cell fusion and collagen related genes were up-regulated at 42 dpi. Together with the observation of morphological changes of LCCs in previous reports, it is suggested that the following steps are involved in LCC formation: the virus infected cells were (1) inhibited apoptotic death and (2) cell division before enlargement, (3) hypertrophied by cell fusion, and (4) surrounded by a hyaline capsule associated with the alteration of collagen fibers.

Gene expression of pro- and anti-apoptotic proteins in rock bream (Oplegnathus fasciatus) infected with megalocytivirus (family Iridoviridae).

Viruses belonging to the genus Megalocytivirus cause diseases in marine fishes primarily in East and Southeast Asian countries. Rock bream iridovirus (RBIV), which is a member of the Megalocytivirus genus, causes severe mass mortalities in rock beam (Oplegnathus fasciatus) in Korea. In this study, we assessed apoptosis-related gene expression patterns in Megalocytivirus-infected rock bream in high mortality and low mortality conditions to determine important apoptosis-related factors, which may affect fish survival/or death. In the high mortality group (100% mortality at 15 dpi), significantly high levels of perforin, granzyme, Fas ligand and caspase 9 expression (5.6-, 10.2-, 13.4- and 4.2-fold, respectively) were observed in the kidney at 8 dpi. Basal expression levels of Fas and caspase 3 were observed at 8 d (1.5-/0.7-fold) and 10 dpi (1.3-/0.6-fold), accompanied by heavy viral loads (8.12 × 10(6)-2.21 × 10(7)/µl). Inhibitor of apoptosis 1 (IAP1) was highly expressed (3.5- to 4.8-fold) at 1 d and 4 dpi; however, IAP1 was reduced when fish died at 8 d and 10 dpi (1.7- to 2.0-fold), which was not significantly different from that of the control group. A similar expression pattern was observed in the low mortality group (18% expected mortality at 30 dpi), which was characterised by a delayed lower magnitude of expression with lower viral loads than the high mortality group. Perforin, granzyme and Fas ligand expression was significantly higher in the low mortality group than in the control group at several sampling points until 30 dpi. Fas and caspases 8, 9 and 3 expression levels showed no statistical significance until 30 dpi. In the low mortality group, significantly higher IAP1 expression compared with the control was observed at 10 d (2.2-fold), 20 d (3.6-fold) and 22 dpi (2.0-fold). In summary, perforin- and granzyme-related apoptosis initiation signals were activated; however, the Fas-induced apoptosis pathway did not efficiently respond. Upregulated IAP1 in RBIV-infected rock bream, which was reported for the first time in this study, exhibited inhibited apoptotic responses in RBIV-infected fish. Although it remains unclear whether apoptosis inhibition aids or impedes fish survival, our data clearly show that the apoptotic response is inhibited in RBIV-infected rock bream.

Development of loop-mediated isothermal amplification method for detection of Kudoa septempunctata (Myxozoa: Multivalvulida) in olive flounder (Paralichthys olivaceus).

A loop-mediated isothermal amplification (LAMP) assay was developed and validated for early, rapid, and sensitive detection of Kudoa septempunctata, a myxosporean parasite found in olive flounder (Paralichthys olivaceus). Recently, several outbreaks associated with ingestion of raw olive flounder muscles harboring mature K. septempunctata spores have been reported, and it is becoming obvious that fresh K. septempunctata spores can cause problems in humans when ingested. Thus, it is necessary to develop reliable detection method of K. septempunctata, to prevent outbreaks and ensure food safety. The LAMP assay has advantages over other molecular detection methods for detecting K. septempunctata in olive flounder muscle, in terms of simplicity, rapidity, and sensitivity. The reaction condition was optimized as 63 °C, 45 min, with three sets of specific primers. The results can be simply confirmed with the naked eye after adding SYBR Green I or by conventional electrophoresis followed by ethidium bromide staining. This LAMP assay did not show any cross-reaction with other kudoid myxosporeans (Kudoa lateolabracis, Kudoa thyrsites) can be found in olive flounder muscles and was validated by testing Kudoa septempunctata spore-spiked samples and field samples. The results showed that the LAMP assay is ten times more sensitive than the conventional polymerase chain reaction in this study and can be applied for early detection for monitoring and epidemiological studies of K. septempunctata in olive flounder aquaculture farms.

Effect of functional electrical stimulation-based mirror therapy using gesture recognition biofeedback on upper extremity function in patients with chronic stroke: A randomized controlled trial.

BACKGROUND: Mirror therapy (MT) is an intervention used for upper extremity rehabilitation in stroke patients and has been studied in various fields. Recently, effective MT methods have been introduced in combination with neuromuscular electrical stimulation or with electromyography (EMG)-triggered biofeedback. The purpose of this study was to investigate the effects of functional electrical stimulation (FES)-based MT incorporating a motion recognition biofeedback device on upper extremity motor recovery to chronic stroke patients. METHODS: Twenty-six chronic stroke patients with onset of more than 6 months were randomly assigned into experimental group (n = 13) and control group (n = 13). Both groups participated in conventional rehabilitation program, while the control group received conventional MT intervention and the experimental group received FES-based MT with motion recognition biofeedback device. All interventions were conducted for 30 min/d, 5 d/wk, for 4 weeks. Upper limb motor recovery, upper limb function, active-range of motion (ROM), and activities of daily living independence were measured before and after the intervention and compared between the 2 groups. RESULTS: The Fugl-Meyer assessment (FMA), manual function test (MFT), K-MBI, and active-ROM (excluding deviation) were significantly improved in both groups (P < .05). Only the experimental group showed significant improvement in upper extremity recovery, ulnar and radial deviation (P < .05). There was a significant difference of change in Brunstrom's recovery level, FMA, MFT, and active-ROM in the experimental group compared to the control group (P < .05). CONCLUSION: FES-based MT using gesture recognition biofeedback is an effective intervention method for improving upper extremity motor recovery and function, active-ROM in patients with chronic stroke. This study suggests that incorporating gesture-recognition biofeedback into FES-based MT can provide additional benefits to patients with chronic stroke.

Serotype distribution and antibiogram of Streptococcus parauberis isolated from fish in South Korea.

Streptococcus parauberis is the dominant etiological agent of streptococcosis, the most devastating bacterial disease in the olive flounder farming industry in South Korea. In this study, the distribution of serotypes, antimicrobial susceptibility, and presence of antimicrobial resistance genes (ARGs) in S. parauberis isolates obtained between 1999 and 2021 was thoroughly investigated to gain insight into the dynamics of their presence and the relationship between serotypes and antimicrobial resistance. Disk diffusion testing of 103 isolates against 10 antimicrobial agents was performed, and epidemiological cut-off values generated through normalized resistance interpretation analysis were used to classify wild-type (WT) and non-wild-type (NWT) populations. Principal component analysis and hierarchical clustering were implemented to achieve an understanding on the relationship between serotypes and antimicrobial resistance patterns. PCR-based serotyping showed that serotype Ia (67.1%) was the most prevalent in South Korea, followed by serotypes Ib/Ic (25.2%) and II (7.7%). The highest proportion of isolates was assigned to NWT against amoxicillin (80.6%), followed by oxytetracycline (77.7%) and erythromycin (48.5%). The time-scale data showed that recently obtained serotypes Ib/Ic and II isolates tended to be categorized as NWT populations resistant to more antibiotics, possibly due to microbial adaptation to antibiotic pressure. ARGs responsible for resistance to oxytetracycline and erythromycin were found only in NWT populations in serotype Ia [tet(S) and erm(B), respectively], and serotype II [tet(M) and mef(J)-msr(I), respectively]. We also found that the mef-msr gene pair in S. parauberis serotype II might be involved in low-level resistance to erythromycin. IMPORTANCE This study presents serotype distribution and antimicrobial susceptibility data along with the antimicrobial resistance genes (ARGs) of Streptococcus parauberis, which is an important bacterial fish pathogen worldwide. In particular, almost all oxytetracycline and erythromycin non-wild-type (NWT) populations harbored tet(S) or tet(M), and erm(B) or mef(J)-msr(I), respectively. Interestingly, these ARGs were distributed in a highly serotype-dependent manner, resulting in a clear correlation between the antibiogram and serotype distribution. Moreover, recent isolates belonging to serotypes Ib/Ic and II tended to be more frequently categorized as NWT against antimicrobials, including amoxicillin and cefalexin compared to old isolates, while a dramatic decrease in erythromycin and clindamycin NWT frequencies was observed in recent serotype Ia isolates, which lacked erm(B). These variations might be attributed to shifts in the antibiotics employed in South Korean aquaculture over time. The overall findings would provide important background knowledge for understanding the epidemiology of S. parauberis infection in aquaculture.

Soft tunic syndrome in the edible ascidian Halocynthia roretzi is caused by a kinetoplastid protist.

An etiological study was conducted to clarify whether the flagellate-like cells found in histological preparations of the tunic of diseased Halocynthia roretzi (Drasche) were the causative agent of soft tunic syndrome in this ascidian. When pieces of softened diseased tunic were incubated overnight in sterile seawater, live flagellated cells, which were actively swimming in the seawater, were observed in 47 out of 61 diseased ascidians (77%), but not in moribund or abnormal individuals with normal tunics (n = 36) nor in healthy animals (n = 19). The flagellate was morphologically very similar to those observed in histological sections of the diseased tunic. By contrast, flagellates were not found in tunic pieces of healthy, moribund, and abnormal individuals that did not exhibit softening of the tunic. Light and electron microscopy revealed that the flagellate has polykinetoplastic mitochondria with discoidal cristae. The cytomorphologies of the flagellate were the same as those of the flagellate-like cells in the diseased tunic. We cultured the flagellate from the softened tunic in vitro and confirmed that the tunics of healthy ascidians, which were immersion-challenged with suspensions of the subcultured flagellates, became softened 17 d after exposure, including the final 12 d in aerated, running seawater. The occurrence of flagellates was also confirmed by incubating pieces of soft tunic from experimentally infected animals in seawater overnight. These results indicate that the flagellate is the causative agent of soft tunic syndrome.

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.

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.

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.

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.

Analysis of genes encoding high-antigenicity polypeptides in three serotypes of Miamiensis avidus.

The ciliate Miamiensis avidus causes scuticociliatosis in Japanese flounder Paralichthys olivaceus. We previously reported three serotypes of this ciliate distinguishable by serotype-specific antigenic polypeptides (serotype I, 30kDa; serotype II, 38kDa; serotype III, 34kDa). In this study, we determined the localization site of the serotype-specific polypeptides in the ciliate and determined the genes encoding the polypeptides, using the isolates IyoI (serotype I), Nakajima (serotype II), and Mie0301 (serotype III). SDS-PAGE and immunoblot analysis of cilia, membrane proteins, and cytoskeletal elements of the ciliates revealed that the polypeptides were abundant in the former two. Scanning electron microscopy of ciliates immobilized by homologous antiserum showed morphological changes in the cilia. These evidences suggested that the polypeptides were ciliary membrane immobilization antigens. The ciliary genes identified showed low identity scores-<51.5% between serotypes. To differentiate the serotypes, we designed serotype-specific PCR primer sets based on the DNA sequences. The PCR-based serotyping results were completely consistent with conventional serotyping methods (immobilization assay and immunoblot analysis). Twenty of 21 isolates were classified as either serotype I or II, and one isolate was undistinguishable. The combination of species-specific PCR previously reported and three serotype-specific PCR could be useful for identifying, serotyping, and surveillance for occurrences of new serotypes of M. avidus.

Miamiensis avidus (Ciliophora: Scuticociliatida) causes systemic infection of olive flounder Paralichthys olivaceus and is a senior synonym of Philasterides dicentrarchi.

The scuticociliate Miamiensis avidus was isolated from olive flounder Paralichthys olivaceus showing typical symptoms of ulceration and hemorrhages in skeletal muscle and fins. In an infection experiment, olive flounder (mean length: 14.9 cm; mean weight: 26.8 g) were immersion challenged with 2.0 x 10(3), 2.0 x 10(4) and 2.0 x 10(5) ciliates ml-1 of the cloned YS1 strain of M. avidus. Cumulative mortalities were 85% in the 2.0 x 10(3) cells ml-1 treatment group and 100% in the other 2 infection groups. Many ciliates, containing red blood cells in the cytoplasm, were observed in the gills, skeletal muscle, skin, fins and brains of infected fish, which showed accompanying hemorrhagic and necrotic lesions. Ciliates were also observed in the lamina propria of the digestive tract, pharynx and cornea. The fixed ciliates were 31.5+/-3.87 pm in length and 18.5+/-3.04 microm in width, and were ovoid and slightly elongated in shape, with a pointed anterior and a rounded posterior, presenting a caudal cilium. Other morphological characteristics were as follows: 13 to 14 somatic kineties, oral ciliature comprising membranelles M1, M2, M3, and paroral membranes PM1 and PM2, contractile vacuole at the posterior end of kinety 2, shortened last somatic kinety and a buccal field to body length ratio of 0.47+/-0.03. In addition, continuous PM1 and PM2, lack of M3 and variable kinetosome numbers in M2 and M3 were frequently observed. Specimens in the current study were compared with previous reports on M. avidus and Philasterides dicentrarchi and confirmed consistently that these 2 taxa are conspecific.

Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae.

We investigated the effect of combined exposure to nodavirus infection and TBT on medaka (Oryzias latipes). Medaka larvae were infected by immersion in medium containing nodavirus at titers of 102.5, 103.5, or 104.5 TCID50/mL. Infected fish then were exposed to TBT at 0, 0.17, 0.52, 1.6, or 4.7µg/L. Of the 12 groups exposed to both stressors, the mortalities of 6 (102.5 TCID50/mL+0.52, 1.6, or 4.7µg/L, 103.5 TCID50/mL+4.7µg/L and 104.5 TCID50/mL+1.6 or 4.7µg/L) were significantly higher than that of each TBT control. Specifically, mortality was 46±5.5% in the group exposed to both 102.5 TCID50/mL virus and 0.52µg/L TBT, which represent the lowest observed effective dose and concentration, respectively, among the 6 groups with increased mortalities. Our results suggest that combined exposure to both stressors suppresses antiviral mechanisms in the fish, thus increasing mortality.

Complete small subunit rRNA gene sequence of the scuticociliate Miamiensis avidus pathogenic to olive flounder Paralichthys olivaceus.

Eight isolates of Miamiensis avidus (scuticociliates) were collected from olive flounder Paralichthys olivaceus with symptoms of severe ulcers and haemorrhages at several culture farms in 1999 and 2003. Cloned strains were produced and the complete small subunit ribosomal RNA gene (SSU rRNA) of each strain was sequenced for classification and phylogenic study. The SSU rRNA is 1759 bp in length and the sequence was deposited in the GenBank under accession number AY550080. All 8 strains exhibited the same sequence, but this sequence did not match any previously deposited scuticociliate SSU rRNA sequence. Phylogenetic analysis placed Miamiensis avidus in a sister lineage to Cohnilembus verminus, Pseudocohnilembus hargisi and P. marinus.

The relationship between gastroesophageal reflux disease and chronic periodontitis.

BACKGROUND/AIMS: The most common cause of chronic periodontitis is poor oral hygiene. Gastroesophageal reflux disease (GERD) enhances the proximal migration of gastric contents and may cause poor oral hygiene. We hypothesized that GERD may increase the risk of chronic periodontitis and investigated this potential relationship. METHODS: A retrospective cross-sectional study was conducted in outpatients between January 1, 2010, and April 30, 2012. GERD was defined as being present based on at least two of the following criteria: etiologic agent(s), identifiable signs and symptoms, and consistent anatomic alterations. A total of 280 patients with chronic periodontitis and 280 controls were analyzed. Information regarding patient demographics and other potential confounding factors for chronic periodontitis were collected through individual medical records. RESULTS: GERD was revealed to be independently associated with an increased incidence of chronic periodontitis (odds ratio [OR], 2.883; 95% confidence interval [CI], 1.775 to 4.682). The other three variables of dental caries (OR, 1.531; 95% CI, 1.042 to 2.249), tobacco use (OR, 2.335; 95% CI, 1.461 to 3.730), and history of medication (calcium channel blocker, cyclosporine, or phenytoin) (OR, 2.114; 95% CI, 1.160 to 3.854) were also determined to be independent risk factors. CONCLUSIONS: The present study supported our hypothesis that GERD can be a risk factor for chronic periodontitis.

Observer variability in gastric neoplasm assessment using the vessel plus surface classification for magnifying endoscopy with narrow band imaging.

BACKGROUND/AIMS: Recent studies have demonstrated that magnifying endoscopy with narrow band imaging (ME-NBI) facilitates differentiation of early gastric cancer from gastric adenoma using vessel plus surface (VS) classification. This study estimated the interobserver and intraobserver agreement of endoscopists using the Yao VS classification system for the gastric mucosal surface. METHODS: We retrospectively reviewed patients who underwent endoscopic submucosal dissection or endoscopic mucosal resection, and selected cases in which preoperative ME-NBI was conducted. Before testing endoscopists, a 20-minute training module was given. Static ME-NBI images (n=47 cases) were presented to seven endoscopists (two experts and five trainees) who were asked to assess the images in 20 seconds using the Yao VS classification system. After 2 weeks, the endoscopists were asked to analyze the images again. The κ statistic was calculated for intraobserver and interobserver variability. RESULTS: The mean κ for intraobserver agreement was 0.69 (experts, 0.74; trainees, 0.64). The mean κ for interobserver agreement was 0.42 (experts, 0.49; trainees, 0.40). CONCLUSIONS: We obtained reliable results as assessed by observer variability, with only brief training on VS classification. The VS classification appears to provide an objective assessment of ME-NBI for trainees who are not familiar with ME-NBI.

Mussel shell impaction in the esophagus.

Mussels are commonly used in cooking around the world. The mussel shell breaks more easily than other shells, and the edge of the broken mussel shell is sharp. Impaction can ultimately cause erosion, perforation and fistula. Aside from these complications, the pain can be very intense. Therefore, it is essential to verify and remove the shell as soon as possible. In this report we describe the process of diagnosing and treating mussel shell impaction in the esophagus. Physicians can overlook this unusual foreign body impaction due to lack of experience. When physicians encounter a patient with severe chest pain after a meal with mussels, mussel shell impaction should be considered when diagnosing and treating the patient.