Surface protrusions on nervous necrosis virus (NNV) particles play crucial roles in NNV antigenicity and infectivity. Pocket structures at the apex of surface protrusions are most likely responsible for initiating NNV infection by functioning as cell receptor binding sites. To create a scientifically robust model, we developed mouse monoclonal antibodies (MAbs) that recognize common epitopes on pocket structures shared among serologically distinct NNVs. We obtained 108 hybridoma clones that produced NNV-specific MAbs by immunizing eight mice using highly purified red-spotted grouper NNV (RGNNV) with and without physicochemical treatments. Finally, 13 hybridoma clones that produced MAbs which recognized different epitopes on RGNNV and/or striped jack NNV (SJNNV) were obtained. NNV-neutralizing activity was not observed in any of the MAbs. We believe that insufficient amounts of NNV used for immunization might be one of the factors responsible for preferential suppression of the generation of NNV-neutralizing antibodies.

Interferon regulatory factors (IRFs) exhibit antiviral and antibacterial properties. In this study, we cloned the IRF8 genes of red sea bream (Pagrus major) and analyzed their expression following red sea bream iridovirus (RSIV) infection. Multi-sequence analysis revealed conserved features, such as the IRF-association domain, nuclear localization signal, and DNA-binding domain. Phylogenetic analysis demonstrated that PmIRF8 clustered with IRF8 from other fish species. Quantitative real-time PCR was employed to examine IRF8 expression in normal tissues, revealing high expression levels in the gill (26.7-fold), spleen (25.7-fold) and brain (22.6-fold). This finding aligns with previous research, which has established that IRF8 is associated with immune cell development and is highly expressed in lymphomyeloid tissues. Additionally, temporal expression of PmIRF8 following RSIV infection demonstrated an initial low expression in the gills, followed by an upregulation at 36 hours post-infection (hpi). A similar expression pattern was observed in the spleen. The liver showed variable expression, while the kidney exhibited an increase at 6 hpi, followed by another upregulation at 3 days post-infection. These results suggest that PmIRF8 plays a role in regulation and suppression during the immune response. These findings are corroborated by previous studies on other fish species. Overall, this investigation presents the characterization of IRF genes in red sea bream and provides foundational data for comparative analysis of IRF genes.

Nervous necrosis virus (NNV) has caused significant economic losses in Korea's aquafarms due to mass mortality in sevenband groupers during the summer season. However, the mechanisms of NNV infection are not well understood. To investigate these mechanisms and search for antiviral proteins, we have established a differentially expressed genes (DEGs) from naive sevenband grouper and NNV-infected ones. Among the highly expressed proteins was Cathepsin H, a lysosomal cysteine proteinase that plays a role in lysosomal protein breakdown. The open reading frame (ORF) of Cathepsin H (SG.CH) was 987 nucleotides long that codes for a protein of 328 amino acids (approximately 20 kDa molecular weight). The gene showed a high degree of identity (98%) with the Cathpesin H from the giant grouper (Epinephelus lanceolatus). When the gene expression profile was analyzed in various tissues, the highest expression was found in the kidney. While following the NNV infection, the expression profile of the Cathepsin H gene displayed a significant upregulation in brain at 48 hours after infection. These experimental results can help understand the mechanism of NNV infection.

White spot syndrome virus (WSSV) is a viral disease that causes significant economic losses in the global shrimp industry, primarily through horizontal transmission among aquatic animals. The virus can also be spread by shellfish, which can concentrate and release the virus through filter-feeding activity. The potential for virus transmission depends on whether the virus can survive in the environment for a while without being digested. From the artificial degradation analysis in the environmental water, the number of WSSV genome copies decreased more rapidly in seawater than in freshwater, and at 18℃ compared to 23℃. Moreover, WSSV had a lower digestion rate with shellfish digestive enzyme compared to the reduction rate in seawater, indicating that WSSV may persist in the digestive tracts of shellfish for longer, increasing the chance of transmission. These findings conclude that shellfish play a significant role in the transmission of WSSV and further research is needed to determine their specific contribution to the virus spread in aquatic environments.

Viral haemorrhagic septicemia virus (VHSV) is mainly transmitted horizontally by water-borne transmission. To prevent the spread of infectious VHSV in the aquatic environment, the water treatment is needed in aquaculture system. Although several studies have investigated to reduction of VHSV titer using the water treatment system, comparing of viral titer reduction in seawater by using different types of disinfection system has not been studied yet. Here, we determined the disinfectant effect of VHSV in seawater by using 4 types (UV radiation, plasma-AOP, electrolysis, ozonation) disinfection system. Moreover, cumulative mortality and detection rate of VHSV in olive flounder (Paralichthys olivaceus) after cohabitation challenges was also investigated viral reduction by disinfection system (UV radiation or plasma-AOP) or by untreated system. VHSV copy numbers were decreased from 10^5.54 copies/mL to 10^2.17±0.53 copies/mL, 10^2.31±0.22 copies/mL, 10^2.44±0.27 copies/mL, and 10^3.14±0.11 copies/mL by the UV radiation, plasma-AOP, electrolysis, ozonation, respectively. In cohabitation challenge, the cumulative mortality and VHSV detection rate of olive flounder by the plasma-AOP treatment is lower than that by the UV radiation treatment and positive control. These results suggest that the plasma-AOP treatment system can be useful for efficient inactivation of VHSV.

In this study, we studied the use of 10 commercial disinfectant candidates in aquaculture field conditions for fish pathogenic viruses; Marine birnavirus, Infectious pancreatic necrosis virus, Hirame rhabdovirus. Sodium hypochlorite inactivated more than 99% under the treatment conditions of 23 ppm for 20 minutes, 150 ppm for chlorine dioxide, 40-80 ppm for povidone-iodine, 50 ppm for glutaraldehyde, and 150 ppm for didecyl dimethyl ammonium chloride. The virus inactivation ratio exceeded 99% under the treatment conditions of hydrogen peroxide at 1,050 ppm for 60 minutes and Formalin at 500 ppm or more, and the virus inactivation concentration of hydrogen peroxide and formalin was too high. These indicate that its disinfection effect was insignificant. Quaternary ammonium compounds were not expected to inactivate viruses even at 225 ppm of active ingredient, dichlorobenzen at 2,250 ppm, and copper sulfate at 3,000 ppm, so it was showed that it could not be used as a disinfectant. As a result of this study, sodium hypochlorite, chlorine dioxide, povidone-iodine, glutaraldehyde, and didecyl dimethyl ammonium chloride were confirmed to have antiviral effects.

The current study aimed to identify the primary cause of mortality in a commercial Oreochromis niloticus (O. niloticus) farm. Furthermore, the efficacy of poly-β-hydroxybutyrate (PHB) as a feed additive to prevent mortality was investigated after in-vitro testing. Also, a histopathological examination was carried out. The samples of naturally diseased O. niloticus showed swellings and hemorrhages on the body surface. Moreover, the post-mortem examination revealed black fluids with an awful odor, a congested liver, and intestinal gasses. Concerning diagnosis of the main cause of mortality in diseased O. niloticus, , there were eight isolates might be Edwardsiella tarda based on the biochemical identification results. Also, the identification was confirmed by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The resulting spectra of two previously identified E. tarda strains were then compared to those found in the Bruker database and the two strains were identified as E. tarda at the species level. For detection of pathogenicity of identified strains, the virulence E. tarda hemolysin (ETHA) gene were detected at band 1078 bp in the eight identified strains. Regarding the in-vitro antimicrobial activity of PHB against E. tarda, the antibacterial activity of blood and tissues had been calculated using the Agar and well diffusion procedures Prior to and after the challenge,. Lower bacterial counts and a larger inhibition zone were signs of the tested materials' concentration-dependent antibacterial activity. For in-vivo evaluation of PHB in dietary-fed O. niloticus, PHB was effective in preventing Edwardsiellosis, with the lowest mortality rates in the group fed 10 g PHB/Kg feed after being injected with E. tarda. In comparison, the group that received a 5 g PHB/Kg feed after receiving an injection of E. tarda saw 16.6% mortalities and a 66.7 percent relative survival rate. The control positive group had 50% mortality. The results of the biochemical testes showed that O. niloticus's kidney and liver functions were unaffected by dietary PHB supplementation. Protein, albumin, globulin, urea, creatinine, aspartate transaminase levels were identical to those of the control negative group. According to the current study, the groups fed high and low PHB concentrations had considerably higher levels of immunoglobulin M, complement 5, and interleukin-1 than the control group. The number of total viable bacteria was lower in the gut of PHB-fed groups than in control. Compared to mild histological alterations in those fed with a lower dosage and severe histopathological abnormalities in the control group, the greater dose of PHB effectively prevented Edwardsiellosis in O. niloticus with decreased mortality and no histopathological changes.

Nanotechnology seeks to improve material effectiveness to have a greater impact on medicine. The current study was performed to determine how effective dietary propolis (PR) and propolis nanoparticles (PR-NPs) were at improving immune and anti-oxidant status, lowering cortisol levels as stress indicators, and preventing Pseudomonas aeruginosa infection in O. niloticus. Histopathology was carried out. Fish (N = 516, 25 ± 2g) were fed basal diets supplemented with 1 gm PR/kg, 2 gm PR/kg, 1 gm PR-NPs/kg, 2 gm PR-NPs/kg feed and control diet for 28 days. When compared to the control, interlukin-1, IgM, complement 5, and levels of lysozyme were dramatically higher in PR and PR-NPs fed-groups. Antioxidant enzymes and glutathione levels were higher in fish fed PR and PR-NPs, but malondialdehyde levels were lower. Cortisol levels decreased in feeding groups compared to the control. When compared to O. niloticus group fed 1 gm PR/kg and the positive control, propolis nanoparticles successfully prevented P. aeruginosa infection and the mortality rate was zero. When compared to the negative control, the positive control group's histopathological findings revealed severe histopathological changes. In low and high groups fed PR-NPS, normal structures were observed as well as high concentrations of PR after being injected with P. aeruginosa. The group that was fed low concentrations of PR after being injected with P. aeruginosa showed only minor histopathological changes. Conclusion: Dietary supplementation of PR or PR-NPs may have a beneficial effect on aquaculture, with PR-NPs having a superior effect.

이전에 Edwardsiella tarda로 분류되었던 많은 어류 병원세균들이 다양한 연구에서 E. piscicida 또는 E. anguillarum으로 재분류되었다. 본 연구에서는 국내에서 1996년부터 2010년까지 양식 넙치와 뱀장어의 감염어에서 분리하여 E. tarda로 동정하였던 17균주의 재분류를 실시하였다. 이를 위해 species-specific primers를 이용한 특정 PCR amplicon 검출과 gyrB 염기서열에 기반한 계통발생학적 분석을 수행하였다. 또한 숙주에 따른 분리주 특성의 차이를 알아보기 위하여 항균제 감수성 패턴과 random amplified polymorphic DNA (RAPD) PCR의 amplicons에 대한 염기서열을 분석하였다. E. piscicida 특이 프라이머를 사용한 PCR 결과 17개의 분리주만 amplicon을 형성하였고, E. tarda 프라이머를 사용했을 때는 E. tarda type strain (ATCC 15947)에서만 amplicon이 나타났다. 17개 분리주의 gyrB 염기 서열에 따른 계통수에서 모든 분리주는 E. piscicida의 참조 균주들과 함께 E. tarda type strain 과는 분리되는 clade를 형성하였으며 넙치와 뱀장어에서 분리된 균들은 각각의 진화 그룹으로 구분되었다. 항균제 감수성 시험 결과 넙치에서 분리된 균들은 doxycycline, oxytetracycline, tetracycline에서 non-wild type (NWT)이 나타났고 oxolinic acid, flumequine에 대해서는 뱀장어에서 분리된 균에서만 NWT이 나타났다. E. piscicida 분리주들의 RAPD 패턴에서 차이나는 3개 amplicons의 뉴클레오티드 서열을 분석한 결과 Edwardsiella phage GF-2, E. piscicida MS-18-199의 플라스미드 pEPMS-18199 및 E. anguilllarum ET080813의 플라스미드 1에서 일치하는 영역이 확인되었다. 이러한 결과에 근거하여, 본 연구의 17 분리주는 모두 E. piscicida로 분류될 수 있으며, 분리주의 특성은 숙주의존적인 차이를 보이는 것으로 판단되었다.

Spleen, liver and intestinal mucus of Anguilla japonica cultured in a domestic recirculating aquaculture system were sampled to isolate and identify culturable bacteria. Based on 16s rRNA sequence analysis, the most frequently isolated bacterium was Lactococcus lactis. To determine the antibiotic susceptibility of L. lactis, the minimum inhibitory concentration (MIC) test was conducted using five isolates (A1, A21, A65, A125 and A130) from Anguilla japonica and three isolates (KCTC2013, KCTC3769 and KCTC3899) from Korean Collection for Type Culture. Differences of MICs between A. japonica-derived isolates and KCTC isolates were observed in Tetracycline, Oxytetracycline, Doxycyclin and Erythromycin. MICs obtained for five L. lactis isolates from A. japonica were >64 ㎍/mL for Tetracycline, >256 ㎍/mL for Oxytetracycline, 16-32 ㎍/mL for Doxycyclin, and > 64㎍/mL for Erythromycin. On the other hand, KCTC isolates showed the MICs of 0.25 ㎍/mL, 0.25 ㎍/mL, 0.12 ㎍/mL and 0.03-0.06 ㎍/mL for respective antibiotics. Furthermore, it was confirmed that genomic DNA and plasmid DNA isolated from A. japonica-derived L. lactis isolates have antibiotic resistance genes and transfer origin. These findings suggest that A. japonica-derived L. lactis isolates may have the potential to mediate the transfer of various antibiotic resistance genes. Therefore, it is advisable to use probiotics along with the feed or use them as postbiotics rather than dispersing them directly into the water in aquaculture system.

Trichodinid ciliates were found from the skin scrapings of culture starry flounder (Platichthys stellatus) obtained from a local fisheries market in Gangneung. Skin scrapings were prepared and stained by either Giemsa solution or silver nitrate impregnation, for morphological observation. PCR analysis was also conducted to make molecular identification and phylogenetic analysis. Morphological parameters obtained from our specimens were the most similar with Trichodina hokkaidoensis of barfin flounder from Japan and also olive flounder from Korea. The partial 18S rDNA sequences of our specimen (n=7) also showed 100.0% similarity with those of barfin flounder and olive flounder. Several trichodinids are known to have a broad host specificity and this is thought to be the case for T. hokkaidoensis. Starry flounder is a strong candidate for land-based aquaculture production, as a target species for prospective aquaculture diversification and therefore pathogenic potentials of this ciliate should be considered.

Scuticociliatosis can occur from juveniles to adult olive flounder (Paralichthys olivaceus) and the infection usually causes mass mortality. There is no clear way to treat it once the parasite has invaded the fish's internal organs. Therefore, periodic monitoring of scuticociliatosis for surveillance and prompt response is a crucial part of reducing damage to olive flounder farms. In this study, we conducted scuticociliatosis monitoring of olive flounder farms in jeju island to investigate the detection rate of the causative agents (Miamiensis avidus and Uronema marinum), and determine the phylogenetic classification of the detected pathogens. Disease monitoring was conducted on a total of 3,282 olive flounder and the detection rate was highest in August (15.83%) during the investigation period. The sequences of all 4 strains obtained from olive flounder farms in 4 different regions, were 100% homologous with SSU rRNA gene of M. avidus deposited in GenBank.

The internal ribosome entry site (IRES) elements from various RNA viruses are widely used to express proteins in bicistronic or multicistronic ways in mammalian cells. However, research on the application of IRESs in fish cells has been poorly conducted. In this study, to evaluate the availability of various viral IRESs in fish cells based on a bicistronic vector system, the translation activity of IRESs from viruses of invertebrates (aphid lethal paralysis virus ALPV; cricket paralysis virus, CrPV; and Plautia stali intestine virus, PSIV), of mammals (encephalomyocarditis virus, EMCV), and of fish (infectious pancreatic necrosis virus, IPNV; marine birnavirus, MABV; and snakehead retrovirus, SnRV) was analyzed in various fish cell lines originated from salmonid (chinook salmon embryonic cells, CHSE-214), cyprinid (Epithelioma papulosum cyprinid, EPC), and flatfish (Hirame natural embryonic cells, HINAE). Translation mediated by EMCV IRES was shown in all cell lines, but the activity was weak in CHSE-214 and HINAE cells while high activity was shown in EPC cells, suggesting that although EMCV IRES can be broadly used in various fish cells, the translational activity can be varied according to different cell lines. PSIV IRES showed moderate activity in EPC cells and low activity in CHSE-214 and HINAE cells. CrPV IRES showed weak activity in EPC and HINAE cells and no activity in CHSE-214 cells. In the case of ALPV IRES, green fluorescence was observed only in EPC cells but the activity was very weak. These results suggest that dicistroviral IRESs analyzed in this study would not be the best option for protein expression in various fish cells. The translation activity of SnRV IRES in fish cells was first verified in EPC cells, though the activity was very weak. The 5'UTR of MABV segment A showed high translation activities in all examined fish cell lines, suggesting that IRES of MABV can be used to develop multicistronic expression systems or cap-independent RNA-based translation systems in a wide range of fish cells.

본 연구에서는 넙치(Paralichthys olivaceus, average weight 230.27 g; average length 27.99 cm)에서 amprolium hydrochloride의 주사 투여에 따른 급성독성을 평가하기 위해 50, 100, 150, 200, 400, 600 mg/kg 용량으로 설정하고 단회주사투여한 결과 LD₅₀ 값은 148.18 mg/kg이었다. 또한 약물을 주사투여한 후 1, 3, 7, 14, 28일째에 혈액학적 분석을 실시한 결과 GOT 수치는 80 mg/kg 용량구간에서만 대조구에 비해 1일차 및 3일차까지 최고수치를 나타내었으며, BUN 수치는 1일과 3일차 80 mg/kg 용량구간과 7일차에 40 및 80 mg/kg 구간에서 대조구에 비해 유의적인 증가를 나타내었다. 추가적으로 조직학적 변화를 확인한 결과 7, 14, 28일차, 20, 40, 80 mg/kg 용량 구간에서 신장 조직에서 melanomacrophage center와 부종이 관찰되었다. 결과적으로 amprolium hydrochloride 주사 투여는 넙치에서 독성을 나타내기 때문에 넙치의 생체 내 실험에서 여러가지 악영향을 미치는 것으로 보아 상당한 독성이 있는 것으로 판단된다.

Florfenicol (FF)은 chloramphenicol(CM)계의 항생제로 CM의 대표적 부작용인 재생불량성 빈혈을 유발하지 않는 장점을 활용하기 위해 개발되었다. FF은 세계적으로 안전하게 사용중인 동물용의약품 중 하나로 우리나라도 어류에게 임상적으로의 사용이 허가된 항생제이다. 우리나라의 FF의 용법, 용량 및 휴약기간은 일본과 설정이 동일하다 하지만 최대잔류허용기준(maximum residue limit, MRL)은 0.2 mg/kg과 8.0 mg/kg으로 상이하게 설정되어있다. 하지만 이를 뒷받침할 국내 기준에 맞는 잔류량 특성 연구가 미비한 실정이다. 본 연구는 식품안전을 목적으로 휴약기간을 설정하기 위하여 농림축산검역본부에서 고시한 동물용의약품 잔류성 시험지침에 따라 시험을 진행하였으며, 양식 뱀장어인 Anguilla japonica에서 FF의 잔류 양상을 분석하였다. FF을 5일간 반복투여 방법으로 승인된 임상 용량인 10 mg/kg과 2배인 20 mg/kg까지 투여하였으며, 잔류경향을 평가한 결과 이전에 설정되어있던 7일의 휴약기간을 약 60일로 연장할 필요가 있는 것으로 밝혀졌다. 이 연구의 결과는 이전에 7일이었던 휴약기간을 최근(2022년) 60일로 연장하는데 근거자료로 활용된 바 있다.

Infectious diseases are a major cause of death at the fish farm. Therefore, various disinfectants are used in aquaculture to sterilize pathogens. In this study, the disinfectant efficacy of triple salt was evaluated against Salmonella typhimurium and important aqua pathogens. In the case of bacteria under the conditions of distilled water (DW), hard water (HW), and organic matter (OM), S. typhimurium was inactivated with 0.56, 0.5 and 100 parts per thousands (ppt), V. parahaemolyticus (AHPND) was inactivated with 0.17, 0.13 and 20 ppt and V. anguillarum was inactivated with 0.25, 0.17 and 33.3 ppt. Also, in the case of the virus under the conditions DW, HW and OM, VHSV was inactivated with 0.1, 0.1 and 0.5 ppt and SVCV was inactivated with 0.5, 0.5 and 1 ppt. In addition, this study investigated the disinfection effect of triple salt against WSSV by bioassay using Litopenaeus vannamei. Investigating the antiviral effect of disinfectants under DW, HW, and OM conditions, WSSV was inactivated with 0.05, 0.05, 0.8 ppt. The results from this study showed that the triple salt was a highly effective disinfectant against aqua pathogens. Therefore, we can provide basic data for establishing the standards for use of triple salt in aquaculture.

Viral hemorrhagic septicemia virus (VHSV) is the etiological agent of viral hemorrhagic septicemia (VHS), which is one of the most serious viral diseases affecting cultured olive flounder (Paralichthys olivaceus) in Korea. Mortality due to VHSV infection in olive flounder appears in the cold-water season at 8 to 15℃. Among factors contributing to the onset of VHS epizootics in olive flounder, the water temperature plays essential role in regulation of VHS outbreaks. In order to estimate the VHSV infectivity and distribution, we performed cell culture and qRT-PCR in flounder target organ after VHSV infection. VHSV titration and distribution were similar between spleen and kidney. The different differential expression kinetics of four immune-related genes in spleen and kidney were observed in this study. Perforin was immediately up-regulated and then continuously expressed within 48h in both of spleen and kidney. Mx gene showed the highest immune-related gene expression in this study. IL-8 and ISG-15 were expressed in spleen. It was peaked at 15h and then gradually decreased, whereas these were not relatively expressed in kidney. Our study showed that the immune response in host was immediately activated, further study needs to interpret the interaction between host and pathogen for their survival.

In March 2022, Leather carp (Cyprinus carpio nudus) raised in a pond continued to die with skin ulcers, and the diseased fish were taken to our Laboratory for diagnosis. Severe skin lesions such as petechial hemorrhage, abrasions and perforation were the main gross signs of the diseased fish. Histopathological examination revealed a cyst of Dermocystidium-like organisms in the subcutaneous layer. In addition, secondary or cross infection by different kinds of protistan parasites and bacterial organisms was suspected.

In this study, dead electric eel (Electrophorus electricus) imported from Peru was investigated with histopathological analysis. On the third day after import, the electric eel began to exhibit clinical symptoms including severe ulceration, exposed muscles with hemorrhage in head and body lesions. Aeromonas hydrophila and A. veronii were detected from skin lesions, whereas A. hydrophila and Plesiomonas shigelloides were detected from internal organs. Histopathological analysis revealed epithelial desquamation in ulcer, epithelial and goblet cell hyperplasia, and lymphocyte infiltration in epidermis. In dermis, macrophage infiltration and colonization of A. hydrophila or A. veronii were seen. The activation and infiltration of macrophage associated with bacterial septicemia were observed in kidney and spleen. It is presumed to be mortality of electric eel due to septicemia caused by opportunistic infectious bacteria after transport stress.