Draft genome sequence of multiple antibiotic-resistant Vibrio harveyi isolated from diseased olive flounder (Paralichthys olivaceus) farmed in South Korea.

Vibrio harveyi strain 22FBVib0145 was isolated from a diseased olive flounder farmed in Jeju, Korea. Here, we report the draft genome sequence of this strain. It is 6,238,277 bp in length with a G + C content of 44.8%.

Characterization and Pathogenicity of Flavobacterium psychrophilum Isolated from Rainbow Trout (Oncorhynchus mykiss) in Korea.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease in salmonids and rainbow trout fry syndrome. This pathogen has attained a global presence and can spread both horizontally and vertically. However, it was not documented in Korea before September 2018. In this study, the objectives were to characterize Flavobacterium psychrophilum strain FPRT1, isolated from diseased rainbow trout genotypically and phenotypically. We also conducted various investigations to better understand its impact and assess potential control measures. We acquired fifty rainbow trout (approximately 70 g in weight) and transferred them to a laboratory aquarium. During the initial acclimation period, we observed mortality and examined affected fish for clinical signs. We isolated the bacterium from the spleen of infected rainbow trout using tryptone yeast extract salts agar supplemented with glucose, naming this FPRT1. Antibiotic susceptibility testing was carried out, and from the result, we selected enrofloxacin to administer to the trout orally to reduce mortality. To evaluate pathogenicity, we exposed the trout to FPRT1 at different water temperatures (8, 15, and 22 °C). Genomic analysis was conducted to identify the serotype and relatedness of FPRT1 to European strains. Affected fish displayed clinical signs, such as ulcerative lesions in the mandible, anemia with pale gills, exophthalmia, and increased mucus secretion. Internal symptoms included pale liver and enlarged spleen. FPRT1 was susceptible to erythromycin, enrofloxacin, florfenicol, oxytetracycline, and gentamicin, but resistant to oxolinic acid and sulfamethoxazole/trimethoprim. Oral administration of enrofloxacin resulted in a decrease in mortality from 28% to 6%. Pathogenicity tests revealed varying mortality rates due to FPRT1 at different temperatures. The highest rates were observed at 8 °C (ranging from 43% to 100%) for both intraperitoneal and intramuscular injections, and lower rates occurred at 22 °C (ranging from 0% to 30%), with intramuscular injections displaying higher susceptibility. Genomic analysis identified FPRT1 as serotype 2 and indicated its close genetic relationship with European strains based on the core genome and dispensable genome. The substantial genomic similarity between our strain and European strains suggests the possibility of bacterial spread through the importation of fertilized eggs from Europe. In conclusion, this study highlights the introduction of the previously undocumented pathogen (F. psychrophilum) into Korean rainbow trout populations. The detection of this pathogen and its pathogenicity assessment is not only important for understanding its impact on local aquaculture but also for establishing surveillance and control measures to prevent further transmission and outbreaks in the region.

Mechanisms underlying probiotic effects on neurotransmission and stress resilience in fish via transcriptomic profiling.

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.

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.

Development of a rapid and sensitive real-time diagnostic assay to detect and quantify Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome.

The oomycete Aphanomyces invadans causes epizootic ulcerative syndrome (EUS), a World Organization for Animal Health (WOAH)-listed disease that has seriously impacted a wide range of fish worldwide. Currently, only three conventional polymerase chain reaction (PCR) assays are recommended for the detection of A. invadans. The robust quantitative PCR (qPCR) assay has recently become more important due to its highly accurate nature and the applicability of qPCR-based environmental DNA (eDNA) detection in the monitoring of pathogens in aquatic environments. Therefore, in this study, we developed a novel TaqMan probe-based qPCR method to sensitively and quantitatively detect A. invadans. The assay limit of detection was determined using 10-fold serial dilutions of linearized A. invadans plasmid. Assay sensitivity was assessed in the presence of interfering substances and compared to three WOAH-listed primers using the mycelia and zoospores of A. invadans with and without fish muscle tissue. The assay specificity was also theoretically and experimentally assessed against other oomycetes, fish muscle tissue, and water samples. The assay's repeatability and reproducibility were determined. In this study, the limit of detection of the developed assay was 7.24 copies of A. invadans genomic DNA per reaction (95% confidence interval (CI): 2.75 to 19.05 copies/reaction). The assay showed the same sensitivity in the presence of other substances. Compared to the WOAH-recommended PCR assays, this assay had 10-times higher sensitivity for all tested samples. There were no cross-reactions with other closely related oomycetes, fish muscle, or water samples, indicating that the assay was highly specific for A. invadans. The repeatability and reproducibility tests showed little variation, ranging from 0.1-0.9% and 0.04-1.1%, respectively, indicating the high consistency, repeatability, and reliability of the developed assay. This highly rapid, sensitive, specific, and consistent EUS qPCR assay would be of importance in transboundary disease management and the monitoring of pathogens in aquatic environments.

Molecular mechanisms underlying the vulnerability of Pacific abalone (Haliotis discus hannai) to Vibrio harveyi infection at higher water temperature.

Climate change is one of the most important threats to farmed abalone worldwide. Although abalone is more susceptible to vibriosis at higher water temperatures, the molecular mode of action underlying this has not been fully elucidated. Therefore, this study aimed to address the high susceptibility of Halitotis discus hannai to V. harveyi infection using abalone hemocytes exposed to low and high temperatures. Abalone hemocytes were divided into four groups, 20C, 20 V, 25C, and 25 V, depending on co-culture with (V)/without (C) V. harveyi (MOI = 12.8) and incubation temperature (20 °C or 25 °C). After 3 h of incubation, hemocyte viability and phagocytic activity were measured, and RNA sequencing was performed using Illumina Novaseq. The expression of several virulence-related genes in V. harveyi was analyzed using real-time PCR. The viability of hemocytes was significantly decreased in the 25 V group compared to cells in the other groups, whereas phagocytic activity at 25 °C was significantly higher than at 20 °C. Although a number of immune-associated genes were commonly upregulated in abalone hemocyte exposed to V. harveyi, regardless of temperature, pathways and genes regarding pro-inflammatory responses (interleukin-17 and tumor necrosis factor) and apoptosis were significantly overexpressed in the 25 V group compared to the 25C group. Notably, in the apoptosis pathway, genes encoding executor caspases (casp3 and casp7) and pro-apoptotic factor, bax were significantly up-regulated only in the 25 V group, while the apoptosis inhibitor, bcl2L1 was significantly up-regulated only in the 20 V group compared to the control group at the respective temperatures. The co-culture of V. harveyi with abalone hemocytes at 25 °C up-regulated several virulence-related genes involved in quorum sensing (luxS), antioxidant activity (katA, katB, and sodC), motility (flgI), and adherence/invasion (ompU) compared to those at 20 °C. Therefore, our results showed that H. discus hannai hemocytes exposed to V. harveyi at 25 °C were highly stressed by vigorously activated inflammatory responses and that the bacterial pathogen overexpressed several virulence-related genes at the high temperature tested. The transcriptomic profile of both abalone hemocytes and V. harveyi in the present study provide insight into differential host-pathogen interactions depending on the temperature conditions and the molecular backgrounds related to increased abalone vulnerability upon global warming.

Complete Genome Sequences of Streptococcus parauberis Serotype Ia, Ib/Ic, and II Isolates from Diseased Olive Flounder in South Korea.

We report the complete genome sequences of three isolates of Streptococcus parauberis, representing serotypes Ia, Ib/Ic, and II, which were isolated from diseased olive flounder (Paralichthys olivaceus) in South Korea.

Complete Genomic Sequences of Two Aeromonas hydrophila Isolates Derived from Diseased Fish in South Korea.

Aeromonas hydrophila is a Gram-negative pathogen that is associated with motile aeromonad septicemia in various fish species. Here, we report the complete genomic sequences of two A. hydrophila isolates derived from diseased fish in South Korea.

Downregulated pol-miR-140-3p induces the expression of the kinesin family member 5A against Streptococcus parauberis infection in olive flounder.

MicroRNAs (miRNAs) are small non-coding RNAs that participate in various biological and cellular processes by regulating target gene expression. miRNAs are also known to play vital roles in the pathogenesis of various diseases, including infections, as well as the disease progression and defense responses. In this study, we examined the expression levels of pol-miR-140-3p and its target gene, kinesin family member 5A (KIF5A), in association with the Streptococcus parauberis (S. parauberis) infection, a major bacterial pathogen that causes streptococcosis in olive flounder (Paralichthys olivaceus). KIF5A is a heavy chain isoform of kinesin-1, which is known to be brain-specific, and this study is the first examination of KIF5A expression related to the regulation of miRNA in olive flounder (named PoKIF5A). There were significant differences in expression levels between infected and healthy olive flounder as the expression of pol-miR-140-3p in the infected fish was lower than that in the control, while the expression of PoKIF5A was higher in the infected fish than in the healthy controls. These contradictory results suggest that downregulated pol-miR-140-3p induces the expression of PoKIF5A against S. parauberis infection in olive flounder.

Whole-Genome Sequences of Three Edwardsiella piscicida Isolates from Diseased Fish in South Korea.

Edwardsiella piscicida is a Gram-negative pathogen that is associated with edwardsiellosis in aquaculture systems worldwide. Here, we report the whole-genome sequences of three E. piscicida isolates derived from cultured fish in South Korea.

Dual-Organ Transcriptomic Analysis of Rainbow Trout Infected With Ichthyophthirius multifiliis Through Co-Expression and Machine Learning.

Ichthyophthirius multifiliis is a major pathogen that causes a high mortality rate in trout farms. However, systemic responses to the pathogen and its interactions with multiple organs during the course of infection have not been well described. In this study, dual-organ transcriptomic responses in the liver and head kidney and hemato-serological indexes were profiled under I. multifiliis infection and recovery to investigate systemic immuno-physiological characteristics. Several strategies for massive transcriptomic interpretation, such as differentially expressed genes (DEGs), Poisson linear discriminant (PLDA), and weighted gene co-expression network analysis (WGCNA) models were used to investigate the featured genes/pathways while minimizing the disadvantages of individual methods. During the course of infection, 6,097 and 2,931 DEGs were identified in the head kidney and liver, respectively. Markers of protein processing in the endoplasmic reticulum, oxidative phosphorylation, and the proteasome were highly expressed. Likewise, simultaneous ferroptosis and cellular reconstruction was observed, which is strongly linked to multiple organ dysfunction. In contrast, pathways relevant to cellular replication were up-regulated in only the head kidney, while endocytosis- and phagosome-related pathways were notably expressed in the liver. Moreover, interestingly, most immune-relevant pathways (e.g., leukocyte trans-endothelial migration, Fc gamma R-mediated phagocytosis) were highly activated in the liver, but the same pathways in the head kidney were down-regulated. These conflicting results from different organs suggest that interpretation of co-expression among organs is crucial for profiling of systemic responses during infection. The dual-organ transcriptomics approaches presented in this study will greatly contribute to our understanding of multi-organ interactions under I. multifiliis infection from a broader perspective.

Transcriptome analysis unveils survival strategies of Streptococcus parauberis against fish serum.

Streptococcus parauberis is an important bacterial fish pathogen that causes streptococcosis in a variety of fish species including the olive flounder. Despite its importance in the aquaculture industry, little is known about the survival strategy of S. parauberis in the host. Therefore, the objective of this study was to produce genome-wide transcriptome data and identify key factors for the survival of S. parauberis SPOF3K in its host. To this end, S. parauberis SPOF3K was incubated in olive flounder serum and nutrient-enriched media as a control. Although S. parauberis SPOF3K proliferated in both culture conditions, the transcriptomic patterns of the two groups were very different. Interestingly, the expression levels of genes responsible for the replication of an S. parauberis plasmid in the presence of olive flounder serum were higher than those in the absence of olive flounder serum, indicating that this plasmid may play an important role in the survival and proliferation of S. parauberis in the host. Several ATP-binding cassette transporters known to transport organic substrates (e.g., biotin and osmoprotectants) that are vital for bacterial survival in the host were significantly up-regulated in S. parauberis cultured in serum. In addition, groEL, dnaK operon, and members of the clp protease family, which are known to play important roles in response to various stressors, were up-regulated in S. parauberis incubated in serum, thus limiting damage and facilitating cellular recovery. Moreover, important virulence factors including the hyaluronic acid capsule (has operon), sortase A (srtA), C5a peptidase (scp), and peptidoglycan O-acetyltransferase (oatA) were significantly upregulated in S. paraubers in serum. These results indicate that S. paraubers can resist and evade the humoral immune responses of fish. The transcriptomic data obtained in this study provide a better understanding of the mode of action of S. parauberis in fish.

Differential proteome profile of gill and spleen in three pathogen-infected Paralichthys olivaceus.

BACKGROUND: Olive flounder (Paralichthys olivaceus) is one of the major cultured fish species in Asia including Korea. However, the mass mortality of olive flounder caused by various pathogens leads to huge economic loss. The pathogens that lead to fish mortality include parasites, bacteria, and viruses that can cause various kinds of diseases. OBJECTIVE: The purpose of this study was to investigate the protein expression patterns in the gills and spleens of olive flounder after artificial infection. We hypothesized that proteomics levels in gills and spleen may be differentially expressed depending on infectious agents. METHODS: To investigate the expression pattern of proteins in gills and spleens, olive flounders were experimentally infected with VHSV (virus), S. parauberis (bacteria), or M. avidus (pathogenic ciliate). Proteins were extracted from the gills and spleens of infected olive flounder. We used 2-DE analysis with LC-MS/MS to investigate proteome changes in infected olive flounders. RESULTS: The results of the LC-MS/MS analyses showed different protein expression profiles depending on pathogenic sources and target organs. Proteins related to cytoskeletal structure like keratin, calmodulin and actin were mostly expressed in the infected gills. Proteins involved in the metabolism pathway like glycolysis were expressed mainly in the spleens. The protein profiles of S. parauberis and VHSV infection groups had many similarities, but the profile of the M. avidus infection group was greatly different in the gill and spleen. CONCLUSION: Our results indicate that measures according to the characteristics of each pathogen are necessary for disease prevention and treatment of farmed fish.

Complete Genome Analysis of Flavobacterium psychrophilum Strain FPRT1, Isolated from Diseased Rainbow Trout (Oncorhynchus mykiss) in South Korea.

Here, we report the complete genome sequence of Flavobacterium psychrophilum FPRT1, isolated from the spleen and kidney of diseased rainbow trout (Oncorhynchus mykiss). Whole-genome sequencing was performed using the PacBio RS II platform, which yielded a circular chromosome of 2,795,347 bp harboring 2,895 protein-coding genes.

Complete Genome Sequence of Aeromonas salmonicida subsp. masoucida Strain BR19001YR, Isolated from Diseased Korean Rockfish (Sebastes schlegelii).

We report the complete genome sequence of the virulent Aeromonas salmonicida subsp. masoucida strain BR19001YR, isolated from diseased black rockfish (Sebastes schlegelii). Sequencing of the circular chromosome and three plasmids using the PacBio and Illumina platforms yielded 4,982,192 bp with a 58.24% G+C content.

Multi-Omics Analysis Provides Novel Insight into Immuno-Physiological Pathways and Development of Thermal Resistance in Rainbow Trout Exposed to Acute Thermal Stress.

In recent years, poikilothermic animals such as fish have increasingly been exposed to stressful high-temperature environments due to global warming. However, systemic changes in fish under thermal stress are not fully understood yet at both the transcriptome and proteome level. Therefore, the objective of this study was to investigate the immuno-physiological responses of fish under extreme thermal stress through integrated multi-omics analysis. Trout were exposed to acute thermal stress by raising water temperature from 15 to 25 °C within 30 min. Head-kidney and plasma samples were collected and used for RNA sequencing and two-dimensional gel electrophoresis. Gene enrichment analysis was performed: differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were identified to interpret the multi-omics results and identify the relevant biological processes through pathway analysis. Thousands of DEGs and 49 DEPs were identified in fish exposed to thermal stress. Most of these genes and proteins were highly linked to DNA replication, protein processing in the endoplasmic reticulum, cell signaling and structure, glycolysis activation, complement-associated hemolysis, processing of released free hemoglobin, and thrombosis and hypertension/vasoconstriction. Notably, we found that immune disorders mediated by the complement system may trigger hemolysis in thermally stressed fish, which could have serious consequences such as ferroptosis and thrombosis. However, antagonistic activities that decrease cell-free hemoglobin, heme, and iron might be involved in alleviating the side effects of thermally induced immuno-physiological disorders. These factors may represent the major thermal resistance traits that allow fish to overcome extreme thermal stress. Our findings, based on integration of multi-omics data from transcriptomics and proteomics analyses, provide novel insight into the pathogenesis of acute thermal stress and temperature-linked epizootics.

Overfeeding-Induced Obesity Could Cause Potential Immuno-Physiological Disorders in Rainbow Trout (Oncorhynchus mykiss).

Although over-nutrition from overfeeding-induced obesity is known to be highly associated with metabolic and immunological disorders in humans, little is known about overfeeding-induced obesity in fish farming. The purpose of this study was to investigate changes in immuno-physiological parameters, to better understand the potential risk of overfeeding-induced obesity in fish. Commercial feed was provided to fish in the overfed group until they refuse to eat, but fish in the control group was fed with the feed at 1% bodyweight per day. The hemato-serological, histological, and immunological changes were observed at weeks 2 and 8. Rainbow trout leukocytes were co-incubated with oxidized low-density lipoprotein (OxLDL), and the phagocytes engulfing the OxLDL and the presence of apoptotic cells were evaluated. The body weight, body mass index (BMI), and hepatosomatic index (HSI) index were significantly higher in the overfed group, and high lipid accumulation and fatty changes were also observed in their livers, indicating that the feeding regime used in this study led to overfeeding-induced obesity. Likewise, much higher numbers of and larger vacuoles were observed in overfed fish macrophages, showing unclear boundaries between the cytoplasm and extracellular space. In the overfed group, the expression of IL-10, HSP70, TLR2, and CD36 was significantly higher, and lymphocyte apoptosis was more evident, indicating that overfeeding-induced obese fish might have immunologic disorders. This was the first study to demonstrate that overfeeding-induced obesity could cause an immune-physiological imbalance in rainbow trout, making them more vulnerable to infectious diseases and various stressful conditions. This study will contribute to improvements in fish nutrition, feeding practices, fish nutrition, and disease prevention in the aquaculture industry.