• Journal of fish pathology
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• v.26 no.1
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• pp.31-38
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• 2013

The edible ascidian, Halocynthia roretzi is a commercially important fisheries resource in Korea. However, there have been outbreaks of mass mortality due to soft tunic syndrome. It was discovered recently that the cause of death is infection by a protozoan parasite Azumiobodo hoyamushi. Alamar blue assay and microscopic counting were used to estimate anti-protozoal effects of 20 drugs having different action mechanisms. Through comparison of alamar blue assay and microscopic counting, 6 drugs were found to be potential in protozoan-killing effects: amphotericin B, formalin, hydrogen peroxide, bithionol, benzalkonium chloride, bronopol (24hr-$EC_{50}{\leq}20{\mu}g/ml$). The preliminary data can be used as a basis to develop anti-protozoal agents against A. hoyamushi.

• Parasites, Hosts and Diseases
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• v.52 no.3
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• pp.305-310
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• 2014

Ascidian soft tunic syndrome (AsSTS) caused by Azumiobodo hoyamushi (A. hoyamushi) is a serious aquaculture problem that results in mass mortality of ascidians. Accordingly, the early and accurate detection of A. hoyamushi would contribute substantially to disease management and prevention of transmission. Recently, the loop-mediated isothermal amplification (LAMP) method was adopted for clinical diagnosis of a range of infectious diseases. Here, the authors describe a rapid and efficient LAMP-based method targeting the 18S rDNA gene for detection of A. hoyamushi using ascidian DNA for the diagnosis of AsSTS. A. hoyamushi LAMP assay amplified the DNA of 0.01 parasites per reaction and detected A. hoyamushi in 10 ng of ascidian DNA. To validate A. hoyamushi 18S rDNA LAMP assays, AsSTS-suspected and non-diseased ascidians were examined by microscopy, PCR, and by using the LAMP assay. When PCR was used as a gold standard, the LAMP assay showed good agreement in terms of sensitivity, positive predictive value (PPV), and negative predictive value (NPV). In the present study, a LAMP assay based on directly heat-treated samples was found to be as efficient as DNA extraction using a commercial kit for detecting A. hoyamushi. Taken together, this study shows the devised A. hoyamushi LAMP assay could be used to diagnose AsSTS in a straightforward, sensitive, and specific manner, that it could be used for forecasting, surveillance, and quarantine of AsSTS.

• Journal of fish pathology
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• v.28 no.3
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• pp.117-123
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• 2015

Economic loss by soft tunic syndrome of edible ascidian, Halocynthia roretzi has become a serious problem. Recently, it has discovered that the cause of this syndrome is infection by a protozoan parasite Azumiobodo hoyamushi. However, only a few studies have been conducted to control this parasitic disease. In a previous research, non-specific disinfectants have been found to be effective in controling the causative parasite. In an attempt to eradicate this causative parasite, organic acids were tested in this study to evaluate their in vitro and in vivo efficacy. In vitro tests showed that 8 different organic acids used in this study were moderately or highly effective with protozoan-killing effects ($EC_{50}=153{\sim}275{\mu}g/ml$). Despite weak in vivo penetration of organic acids into the tunic tissues, treatment with high concentration reduced the mortality of ascidian caused by infection the parasite, indicating that we might be able to develop a disinfection method using environmentally-friendly organic acids.

• Fisheries and Aquatic Sciences
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• v.19 no.1
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• pp.1.1-1.6
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• 2016

Although soft tunic syndrome (STS) in the ascidian is a serious disease, helpful measures have yet not been established. It was examined in this study by applying aniti-parasitic drugs to eradicate the causative protozoa Azumiobodo hoyamushi from infected ascidians. Formalin was synergistic in killing parasites in vitro when co-treated with hydrogen peroxide ($H_2O_2$) or bronopol, but not with chloramine-T or povidone-iodine (PVP-I), when tested with in vitro parasite culture. The synergistic effects did not change when $formalin-H_2O_2$ (or bronopol) ratios were changed. It was found that treatment periods less than 60 min achieved a sub-maximal efficacy. Increasing drug concentration while keeping 30 min period improved anti-parasitic effects. Anti-parasitic effects of $formalin(F)+H_2O_2$(H) were also assessed in an in vivo STS model infected with cultured parasites. It was observed that combined 50 (40F + 10H) and 100 (80F +20H) ppm were effective in partially preventing STS-caused mortality. In horizontally transmitted artificial STS model, significant prevention of ascidian mortality was also observed after 50 ppm. Marked reduction of living parasites were noted after drug treatments in vivo. The results provide a highly useful basis to develop a preventive or treatment measure against the currently uncontrollable STS in the ascidian.

• Fisheries and Aquatic Sciences
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• v.20 no.7
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• pp.12.1-12.7
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• 2017

Soft tunic syndrome (STS) is a protozoal disease caused by Azumiobodo hoyamushi in the edible ascidian Halocynthia roretzi. Previous studies have proven that combined formalin-hydrogen peroxide ($H_2O_2$) bath is effective in reducing STS progress and mortality. To secure target animal safety for field applications, toxicity of the treatment needs to be evaluated. Healthy ascidians were bathed for 1 week, 1 h a day at various bathing concentrations. Bathing with 5- and 10-fold optimum concentration caused 100% mortality of ascidians, whereas mortality by 0.5- to 2.0-fold solutions was not different from that of control. Of the oxidative damage parameters, MDA levels did not change after 0.5- and 1.0-fold bathing. However, free radical scavenging ability and reducing power were significantly decreased even with the lower-than-optimal 0.5-fold concentration. Glycogen content tended to increase with 1-fold bathing without statistical significance. All changes induced by the 2-fold bathing were completely or partially restored to control levels 48 h post-bathing. Free amino acid analysis revealed a concentration-dependent decline in aspartic acid and cysteine levels. In contrast, alanine and valine levels increased after the 2-fold bath treatment. These data indicate that the currently established effective disinfectant regimen against the parasitic pathogen is generally safe, and the biochemical changes observed are transient, lasting approximately 48 h at most. Low levels of formalin and $H_2O_2$ were detectable 1 h post-bathing; however, the compounds were completely undetectable after 48 h of bathing. Formalin-$H_2O_2$ bathing is effective against STS; however, reasonable care is required in the treatment to avoid unwanted toxicity. Drug residues do not present a concern for consumer safety.