Red sea bream iridovirus (RSIV) is a highly contagious viral infection that affects various fish species and poses a significant threat to the global aquaculture industry. Thus, accurate and timely diagnosis is paramount for sustainable management of fish health. This study rigorously evaluated the diagnostic efficacy of various polymerase chain reaction (PCR) assays, focusing on those recommended by the World Organization for Animal Health (WOAH) and the assays newly proposed by WOAH's Aquatic Animals Health Standards Commission. Specifically, this study assessed conventional PCR, nested PCR, modified 1-F/1-R, and real-time PCR assays using a 95% limit of detection (LoD95%), as well as diagnostic sensitivity (DSe) and specificity (DSp) tests across different RSIV severity grades (G0-G4). In previous studies, the LoD95% for the 1-F/1-R and 4-F/4-R conventional assays were 225.81 and 328.7 copies/reaction, respectively. The modified 1-F/1-R exhibited a lower LoD95% of 51.32 copies/reaction. Notably, the nested PCR had an LoD95% of 11.23 copies/reaction, and the real-time PCR assay had an LoD95% of 12.02 copies/reaction. The DSe varied across RSIV severity grades, especially in the lower G0-G2 grades. The nested PCR and modified 1-F/1-R assays displayed the highest DSe, making them particularly useful for early-stage screening and detection of asymptomatic carriers. In addition, the PCR assays did not cross-react with any other aquatic pathogens except RSIV. Our findings significantly advanced the diagnostic capabilities of RSIVD by suggesting that nested PCR and modified 1-F/1-R assays are particularly promising for early detection. We propose their inclusion in future WOAH guidelines for a more comprehensive diagnostic framework.
Fish Diseases , Iridovirus , Sea Bream , Virus Diseases , Animals , Iridovirus/genetics , Real-Time Polymerase Chain Reaction/veterinary
Surgical techniques are gaining attention for treating physical diseases in aquaculture and aquarium fish. Sturgeon is a suitable species for surgical experiments due to its industrial significance. Maintaining homeostasis is crucial during surgical procedures, and the liver plays a major role in immune regulation. High temperature is suggested to improve physiological activity and wound healing. This study investigated differences in hepatectomy sturgeons' tolerance and histopathological responses of internal organs. Moreover, this study investigated the effects of high temperatures on wound healing and hematopoietic recovery in fish undergoing surgical procedures. The liver condition was found to play a pivotal role in the analysis, and cortisol levels were affected by anesthesia. The results showed that high temperature facilitated hematopoietic recovery and wound healing, but excessive induction of physiological activity caused damage. Managing high temperatures and liver conditions induced a remarkable improvement in wound healing. However, anesthesia itself can be a significant stressor for fish, and wound healing requires a greater amount of energy. Further research is needed to understand the stress factors caused by surgical procedures and anesthesia and to promote animal welfare in fishery products.
Aquaculture, a crucial sector of the global food industry, faces a myriad of issues due to parasitic invasions. One such parasite, Microcotyle sebastis, which afflicts Korean rockfish in South Korea, has a significant economic impact. The impending danger of resistance to traditional anthelmintics necessitates the exploration of new antiparasitic candidates. Although the efficacy of salinomycin against aquatic parasites such as ciliates and sporozoans is known, its influence on monogeneans has yet to be studied. Therefore, this study investigated the efficacy and safety of salinomycin for the treatment of M. sebastis infections, presenting the first exploration of salinomycin's therapeutic potential against monogeneans. In vitro examinations revealed a minimum effective concentration of salinomycin of 5 mg/kg, which led to necrosis of the haptor upon dislodging from the gill filaments. The one-time oral administration of the drug at concentrations of 5 mg/kg and 10 mg/kg showed a significant dose-dependent reduction in parasite counts, with no apparent behavioral side effects in Korean rockfish. Biochemical analyses monitored the liver, heart, and kidney enzymes, specifically aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatine kinase-myocardial band (CK-MB). At both 20 °C and 13 °C, no significant differences were observed in the levels of AST and ALT. However, at 20 °C, alterations in BUN levels were evident on Day 14, a deviation not observed at 13 °C. The CK-MB analysis revealed elevated enzyme levels at both temperatures when compared to the control group, reflecting the similar changes observed in terrestrial animals administered salinomycin. The biochemical data suggest that the oral administration of salinomycin is potentially more favorable at 13 °C than at 20 °C. Although our findings warrant further comprehensive studies, including on the long-term and potential effects on nontarget species and water quality, they also suggest that salinomycin could be considered as an alternative or adjunctive treatment if resistance to the currently used praziquantel against M. sebastis is confirmed.
This study detected two potential pathogens, Vibro parahaemolyticus, which causes acute hepatopancreatic necrosis disease (AHPND), and white spot syndrome virus (WSSV), in fishing bait in South Korea. However, their infectious nature was not confirmed, possibly due to the degradation caused by freezing/thawing or prolonged storage under frozen conditions. While infectivity was not confirmed in this study, there is still a significant risk of exposure to these aquatic products. Furthermore, fishing bait and feed should be handled with caution as they are directly exposed to water, increasing the risk of disease transmission. In Australia, cases of WSSV infection caused by imported shrimp intended for human consumption have occurred, highlighting the need for preventive measures. While freezing/thawing is a method for inactivating pathogens, there are still regulatory and realistic issues to be addressed.
Parasitic infections pose significant challenges in aquaculture, and the increasing resistance to conventional anthelmintics necessitates the exploration of alternative treatments. Levamisole hydrochloride (HCl) has demonstrated efficacy against monogenean infections in various fish species; however, research focused on Microcotyle sebastis infections in Korean rockfish (Sebastes schlegelii) remains limited. Therefore, this study aimed to evaluate the efficacy of levamisole HCl against M. sebastis infections in Korean rockfish with the goal of optimizing anthelmintic usage in aquaculture. In this study, we first assessed the susceptibility of M. sebastis to levamisole HCl in vitro. Subsequently, in vivo evaluations were conducted to assess the drug's efficacy, safety, and to identify optimal administration methods. In vitro experiments revealed concentration-dependent sensitivity of M. sebastis to levamisole HCl, with a minimum effective concentration (MEC) of 100 mg/L. In vivo experiments employed oral administration, intraperitoneal injection, and immersion treatments based on the MEC. Oral administration proved to be a safe method, yielding efficacy rates of 27.3% and 41.6% for 100 mg/kg and 200 mg/kg doses, respectively, in contrast to the immersion and injection methods, which induced symptoms of abnormal swimming, vomiting, and death. Biochemical analyses conducted to assess the safety of levamisole HCl revealed a transient, statistically significant elevation in the levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) on day three post-administration at 20 °C. Following this, no substantial differences were observed. However, at 13 °C, the enzyme levels remained relatively consistent, emphasizing the role of water temperature conditions in influencing the action of levamisole HCl. Our research findings substantiate the efficacy of levamisole HCl against M. sebastis in Korean rockfish, underscoring its potential for safe oral administration. These results provide valuable insights for developing parasite control strategies involving levamisole HCl in Korean rockfish populations while minimizing adverse impacts on fish health and the environment. However, this study bears limitations due to its controlled setting and narrow focus. Future research should expand on these findings by testing levamisole HCl in diverse environments, exploring different administration protocols, and examining wider temperature ranges.
In recent years, studies have highlighted the significant impact of probiotic treatment on the central nervous system (brain) and stress regulation through the microbiota-gut-brain axis, yet there have been limited knowledge on this axis in fish. Therefore, this study aimed to enhance the current understanding of the mechanisms underlying probiotic effects on neurotransmission and stress alleviation in fish through transcriptomic profiling. In this study, olive flounders (Paralichthys olivaceus) were subjected to two trial setups: a 1-month lab-scale trial and a 6-month field-scale trial, with and without the probiotic strain Lactococcus lactis WFLU12. RNA-Seq analysis was performed using liver samples collected from fish at one-month post-feeding (mpf) in both trials. Additionally, fish growth was monitored monthly, and serological parameters were measured at one mpf in the field-scale experiment. The results of the lab-scale trial showed that probiotic administration significantly upregulated genes related to neurotransmission, such as htr3a, mao, ddc, ntsr1, and gfra2. These findings highlight the impact of probiotics on modulating neurotransmission via the microbiota-gut-brain axis. In the field-scale experiment, fish growth was significantly promoted and the sera levels of AST, LDH, and cortisol were significantly higher in the control group compared to the probiotics group. Furthermore, genes involved in stress responses (e.g. hsp70, hsp90B1, hspE1, prdx1, and gss) and transcriptional regulators (e.g. fos, dusp1, and dusp2) exhibited significant upregulation in the control group compared to the probiotics group, indicating that probiotic administration can alleviate stress levels in fish. Overall, this study provides valuable insights into the mechanisms underlying the beneficial effects of probiotics in fish, specifically regarding their impact on neurotransmission and stress alleviation.
Flounder , Probiotics , Animals , Transcriptome , Probiotics/pharmacology , Gene Expression Profiling/veterinary , Synaptic Transmission
In this study, the starry flounder L1 cell adhesion molecule (L1CAM) sequence was obtained using next-generation sequencing, and the integrity of the sequence was verified by cloning and sequencing. First, the amino acid sequence was predicted using the cDNA sequence, and the gene was then identified through multiple sequence alignment analysis with related sequences and phylogenetic analysis. Thus, homogeneity was confirmed. The expression level of PsL1CAM (Platichthys stellatus L1CAM) mRNA in healthy starry flounder was detected in all tissues used in the experiment, and tissue- and gene-specific expression levels were confirmed. In addition, as a result of mRNA expression analysis after artificial infection with viral hemorrhagic septicemia virus (VHSV) and Streptococcus parauberis PH0710, significant expression changes and characteristics were confirmed following infection with VHSV and S. parauberis PH0710. After artificial infection with VHSV, the expression level of PsL1CAM mRNA was significantly upregulated in almost all major tissues of the starry flounder, whereas it was significantly downregulated in mucosal-associated lymphoid tissues, such as the gills and intestine. Infection with S. parauberis PH0710 significantly upregulated the expression of PsL1CAM mRNA in almost all major tissues of the starry flounder, whereas it was significantly downregulated in the heart after infection. Our results indicate that PsL1CAM may be involved in the host immune response to starry flounders.
Infestations of parasites, particularly those caused by copepods and monogeneans, are a major hindrance to aquaculture and have a big negative economic impact. Sebastes schlegelii, a farmed Korean rockfish, is particularly prone to copepods and monogeneans. This study comprehended how parasitic copepods and rockfish from a farm in Tongyeong-si, Gyeongsangnam-do, Korea, are related to the monogenean trematode Microcotyle sebastis. Our research revealed that monogeneans predominated, with a rate of 98.4% and an average infection intensity of 7 per infected fish. With an average infection intensity of 4 per sick fish, the infection rate for parasitic copepods was equally considerable, coming in at 91.7%. The high co-infection rate of both parasite families, at 90.8%, is noteworthy. Furthermore, Lepeophtheirus elegans and Peniculus truncatus, 2 parasitic copepod species, were discovered to have M. sebastis eggs attached to their bodies. This demonstrates a connection between parasitic copepod infestations in farmed Korean rockfish and the monogenean trematode M. sebastis. These discoveries highlight the necessity for creating more potent parasite control methods for the aquaculture sector and shed light on the intricate relationships between various parasite species.
Copepoda , Parasites , Perciformes , Trematoda , Animals , Fisheries , Republic of Korea/epidemiology
Red sea bream iridovirus (RSIV) causes significant economic losses in aquaculture. Here, we analyzed the pathogenicity, viral shedding, and transmission dynamics of RSIV in rock bream (Oplegnathus fasciatus) by employing immersion infection and cohabitation challenge models. Rock bream challenged by immersion exposure exhibited 100% mortality within 35 days post RSIV exposure, indicating that the viral shedding in seawater peaked after mortality. At 25 °C, a positive correlation between the viral loads within infected rock bream and virus shedding into the seawater was observed. Specific RSIV lesions were observed in the spleen and kidney of the infected rock bream, and the viral load in the spleen had the highest correlation with the histopathological grade. A cohabitation challenge mimicking the natural transmission conditions was performed to assess the virus transmission and determine the pathogenicity and viral load. The RSIV-infected rock breams (donors) were cohabited with uninfected rock bream, red sea bream (Pagrus major), and flathead grey mullet (Mugil cephalus) (recipients) at both 25 °C and 15 °C. In the cohabitation challenge group maintained at 15 °C, no mortality was observed across all experimental groups. However, RSIV was detected in both seawater and the recipient fish. Our results provide preliminary data for further epidemiological analyses and aid in the development of preventive measures and management of RSIVD in aquaculture.
Red sea bream iridovirus (RSIV) causes significant economic losses in the aquaculture industry. We analyzed the pathogenicity of RSIV in flathead grey mullets (Mugil cephalus), the correlation of histopathological lesions, and interspecies horizontal transmission, through immersion infection and cohabitation challenges. Flathead grey mullets, which were challenged by immersion infection, exhibited mortality at 14 and 24 days after RSIV exposure. Viral shedding in seawater peaked 2-3 days before or after the observed mortality. Specific lesions of RSIV were observed in the spleen and kidney, and the correlation between histopathological grade and viral load was the highest in the spleen. In a cohabitation challenge, flathead grey mullets were the donors, and healthy rock bream, red sea bream, and flathead grey mullets were the recipients. Viral shedding in seawater was the highest in flathead grey mullet and rock bream at 25 °C, with 106.0 RSIV copies L/g at 14 dpi. No mortality was observed in any group challenged at 15 °C, and no RSIV was detected in seawater after 30 dpi. The virus shed from RSIV-infected flathead grey mullets caused horizontal transmission through seawater. These findings suggest that rapid decision-making is warranted when managing disease in fish farms.
Red sea bream iridovirus (RSIV) is an important aquatic virus that causes high mortality in marine fish. RSIV infection mainly spreads through horizontal transmission via seawater, and its early detection could help prevent disease outbreaks. Although quantitative PCR (qPCR) is a sensitive and rapid method for detecting RSIV, it cannot differentiate between infectious and inactive viruses. Here, we aimed to develop a viability qPCR assay based on propidium monoazide (PMAxx), which is a photoactive dye that penetrates damaged viral particles and binds to viral DNA to prevent qPCR amplification, to distinguish between infectious and inactive viruses effectively. Our results demonstrated that PMAxx at 75 µM effectively inhibited the amplification of heat-inactivated RSIV in viability qPCR, allowing the discrimination of inactive and infectious RSIV. Furthermore, the PMAxx-based viability qPCR assay selectively detected the infectious RSIV in seawater more efficiently than the conventional qPCR and cell culture methods. The reported viability qPCR method will help prevent the overestimation of red sea bream iridoviral disease caused by RSIV. Furthermore, this non-invasive method will aid in establishing a disease prediction system and in epidemiological analysis using seawater.
Fish Diseases , Iridovirus , Sea Bream , Animals , Iridovirus/genetics , Sea Bream/genetics , Propidium , Polymerase Chain Reaction
An 8-week feeding trial was conducted to evaluate the effects of various dietary levels of garlic juice processing waste (GJPW) on the growth, feed utilization, digestive and antioxidant enzyme activity, growth- and antioxidant-related gene expression, and resistance to Streptococcus iniae infection of juvenile black rockfish (Sebastes schlegelii). A total of 450 juvenile rockfish were randomly distributed into 30 L rectangular tanks (30 fish per tank). Five experimental diets were prepared in triplicate. The fish were fed experimental diets supplemented with GJPW at concentrations of 0 (GJPW0, control), 2.5 (GJPW2.5), 5 (GJPW5), 7.5 (GJPW7.5), and 10 g kg-1 (GJPW10) diet. All of the GJPW-supplemented treatments (2.5, 5, 7.5, and 10 g kg-1) significantly enhanced weight gain (WG), specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio (PER), and digestive enzyme activity (amylase, trypsin, and lipase). A decreasing trend was seen in plasma aspartate aminotransferase (ALT), alanine aminotransferase (AST), and glucose (GLU) content with increasing dietary levels of GJPW. In contrast, plasma lysozyme and antioxidant enzyme activities were significantly increased with increasing dietary GJPW levels. Furthermore, GJPW administration significantly upregulated the expression of insulin-like growth factor-1 (IGF-1), superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) in the liver of rockfish. A challenge test with S. iniae showed significantly higher resistance in the GJPW-supplemented treatments than in the control. In short, dietary supplementation GJPW enhanced growth performance and antioxidant response in juvenile black rockfish, with suitable effects in fish fed with 2.5 g kg-1 GJPW for 8 weeks.
Red sea bream iridoviral disease (RSIVD) causes serious economic losses in the aquaculture industry. In this paper, we evaluated RSIV kinetics in rock bream under various rearing water temperatures and different RSIV inoculation concentrations. High viral copy numbers (approximately 103.7-106.7 RSIV genome copies/L/g) were observed during the period of active fish mortality after RSIV infection at all concentrations in the tanks (25 °C and 20 °C). In the group injected with 104 RSIV genome copies/fish, RSIV was not detected at 21-30 days post-infection (dpi) in the rearing seawater. In rock bream infected at 15 °C and subjected to increasing water temperature (1 °C/d until 25 °C) 3 days later, the virus replication rate and number of viral copies shed into the rearing seawater increased. With the decrease in temperature (1 °C/d) from 25 to 15 °C after the infection, the virus replicated rapidly and was released at high loads on the initial 3-5 dpi, whereas the number of viral copies in the fish and seawater decreased after 14 dpi. These results indicate that the number of viral copies shed into the rearing seawater varies depending on the RSIV infection level in rock bream.
Interleukin-1 beta (IL-1ß) is transcribed by monocytes, macrophages, and dendritic cells in response to activation of toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) or cytokine signalling and causes a rapid inflammatory response to infection. IL-8, also known as chemokine C-X-C motif ligand (CXCL)-8, is regulated by IL-1ß and affects the chemotaxis of macrophages and neutrophils upon pathogen infection. In healthy red sea bream, rsbIL-1ß is most highly distributed in the liver, and rsbIL-8 is most highly distributed in the head kidney. In response to RSIV infection, rsbIL-1ß and rsbIL-8 mRNA are significantly upregulated in the kidney and spleen. This may be because the primary infection targets of RSIV are the kidney and spleen. In the gills, both genes were significantly upregulated at 7 days after RSIV infection and may be accompanied by a cytokine storm. In the liver, both genes were significantly downregulated at most observation points, which may be because the immune cells such as macrophages and dendritic cells expressing rsbIL-1ß or rsbIL-8 migrated to other tissues because the degree of RSIV infection was relatively low. Using a GFP fusion protein, it was confirmed that rsbIL-1ß and rsbIL-8 were localized to the cytoplasm of Pagrus major fin (PMF) cells. RsbIL-1ß overexpression induced the expression of interferon gamma (IFN-γ), myxovirus-resistance protein (Mx) 1, IL-8, IL-10, TNF-α, and MyD88, while rsbIL-8 overexpression induced the expression of IFN-γ, Mx1, rsbIL-1ß and TNF-α. In addition, overexpression of both genes significantly reduced the genome copies of RSIV and significantly reduced the viral titers. Therefore, rsbIL-1ß and rsbIL-8 in red sea bream play an antiviral role against RSIV through their normal signalling.
DNA Virus Infections , Fish Diseases , Iridoviridae , Iridovirus , Perciformes , Sea Bream , Animals , Antiviral Agents , Interferon-gamma , Interleukin-10 , Interleukin-1beta/genetics , Interleukin-8 , Iridoviridae/physiology , Ligands , Myeloid Differentiation Factor 88 , Pathogen-Associated Molecular Pattern Molecules , Perciformes/genetics , RNA, Messenger , Tumor Necrosis Factor-alpha
Septin is an evolutionarily conserved family of GTP-binding proteins. Septins are known to be involved in a variety of cellular processes, including cell division, chromosome separation, cell polarity, motility, membrane dynamics, exocytosis, apoptosis, phagocytosis, DNA damage responses, and other immune responses. In this study, the sequences of the septin gene family of starry flounder were obtained using NGS sequencing, and the integrity of the sequences was verified through cloning and sequencing. At first, the amino acid sequence was annotated using the cDNA sequence, and then, the gene sequence was verified through multiple sequence alignment and phylogenetic analyses using the related conserved sequences. The septin gene family was classified into three subgroups based on the phylogenetic analysis. High conservation within the domain and homology between the genes reported in different species were confirmed. The expression level of septin gene family mRNA in each tissue of healthy starry flounder was evaluated to confirm the tissue- and gene-specific expression levels. Additionally, as a result of the analysis of mRNA expression after simulated pathogen infection, significant expression changes and characteristics were confirmed upon infection with bacteria (Streptococcus parauberis PH0710) and virus (VHSV). Based on the current results and that of previous studies, to confirm the immunological function, Septin 2, 3, and 8 were produced as recombinant proteins based on the amino acid sequences, and their role in phagocytosis was further investigated. The results of this study indicate that septin gene family plays a complex and crucial role in the host immune response to pathogens of starry flounder.
Flounder , Animals , Flounder/genetics , Phylogeny , RNA, Messenger , Septins/genetics , Sequence Alignment
