Genome based quantification of VHSV in multiple organs of infected olive flounder (Paralichthys olivaceus) using real-time PCR.

BACKGROUND: Viral hemorrhagic septicemia (VHS) is a serious viral disease that infects the olive flounder in South Korea. The Korean aquaculture industry experienced an economic loss caused by the high infectivity and mortality. OBJECTIVE: This study aimed to evaluate the infection density of VHSV in various organs of the olive flounder including spleen, liver, kidney, stomach, esophagus, intestine, gill, muscle, heart, and brain. Olive flounders were collected from a local fish farm and injected subcutaneously with 106 PFU/fish. METHODS: Each 15 fish were sampled at 0, 3, and 7 days post challenge (dpc), respectively, to perform quantitative analysis of VHSV using SYBR-green based real-time PCR in various tissues including spleen, liver, head-kidney, body-kidney, muscle, esophagus, stomach, intestine, gill, and brain. RESULTS: Organs infected with VHSV were obtained after 3 and 7 days. Each organs were examined for viral infection using real-time PCR. The data obtained from this experiment revealed copy numbers higher than 10 copies per 100 ng cDNA in the spleen (15.26 ± 3.11 copies/100 ng of cDNA), muscle (11.24 ± 2.25 copies), and gill (14.23 ± 6.26 copies), but lower in liver, head-kidney, body-kidney, esophagus, brain and stomach. CONCLUSION: The present study, together with previous data, demonstrated that the gill, spleen, and muscle are the major target organs of VHSV in olive flounder. Therefore, central monitoring of spleen, gill and muscle should be considered and might be necessary if anti-VHSV treatment is to be successful in infected olive flounder.

Co-Expression Network Analysis of Spleen Transcriptome in Rock Bream (Oplegnathus fasciatus) Naturally Infected with Rock Bream Iridovirus (RBIV).

Rock bream iridovirus (RBIV) is a notorious agent that causes high mortality in aquaculture of rock bream (Oplegnathus fasciatus). Despite severity of this virus, no transcriptomic studies on RBIV-infected rock bream that can provide fundamental information on protective mechanism against the virus have been reported so far. This study aimed to investigate physiological mechanisms between host and RBIV through transcriptomic changes in the spleen based on RNA-seq. Depending on infection intensity and sampling time point, fish were divided into five groups: uninfected healthy fish at week 0 as control (0C), heavy infected fish at week 0 (0H), heavy mixed RBIV and bacterial infected fish at week 0 (0MH), uninfected healthy fish at week 3 (3C), and light infected fish at week 3 (3L). We explored clusters from 35,861 genes with Fragments Per Kilo-base of exon per Million mapped fragments (FPKM) values of 0.01 or more through signed co-expression network analysis using WGCNA package. Nine of 22 modules were highly correlated with viral infection (|gene significance (GS) vs. module membership (MM) |> 0.5, p-value < 0.05). Expression patterns in selected modules were divided into two: heavy infected (0H and 0MH) and control and light-infected groups (0C, 3C, and 3L). In functional analysis, genes in two positive modules (5448 unigenes) were enriched in cell cycle, DNA replication, transcription, and translation, and increased glycolysis activity. Seven negative modules (3517 unigenes) built in this study showed significant decreases in the expression of genes in lymphocyte-mediated immune system, antigen presentation, and platelet activation, whereas there was significant increased expression of endogenous apoptosis-related genes. These changes lead to RBIV proliferation and failure of host defense, and suggests the importance of blood cells such as thrombocytes and B cells in rock bream in RBIV infection. Interestingly, a hub gene, pre-mRNA processing factor 19 (PRPF19) showing high connectivity (kME), and expression of this gene using qRT-PCR was increased in rock bream blood cells shortly after RBIV was added. It might be a potential biomarker for diagnosis and vaccine studies in rock bream against RBIV. This transcriptome approach and our findings provide new insight into the understanding of global rock bream-RBIV interactions including immune and pathogenesis mechanisms.

Genome based quantification of Miamiensis avidus in multiple organs of infected olive flounder (Paralichthys olivaceus) by real-time PCR.

INTRODUCTION: Miamiensis avidus is the major parasitic pathogen affecting the olive flounder, Paralichthys olivaceus. Recent epidemiological studies have shown that M. avidus infections are becoming increasingly severe and frequent in the olive flounder farming industry. OBJECTIVES: This study aimed to evaluate the infection density of M. avidus in various organs of the olive flounder including spleen, liver, kidney, stomach, esophagus, intestine, gill, muscle, heart, and brain. Olive flounders were collected from a local fish farm. METHODS: Each fish was injected subcutaneously with 2.75 × 103 CFU M. avidus/ fish. Organs infected with M. avidus were obtained after 7 and 25 days. Each organ was examined for parasitic infection using real-time PCR. The primers were designed according to the sequences of 28 s in M. avidus, which was used as a target gene. RESULTS: Each organ was examined for parasitic infection using real-time PCR. The primers were designed according to the sequences of 28 s in M. avidus, which was used as a target gene. The levels of 28 s rRNA were used to calculate quantitative gene copy number. Real-time PCR of brain (60.58 ± 38.41), heart (64.03 ± 62.40), muscle (6.10 ± 3.12), gill (5.06 ± 4.56), intestine (2.38 ± 1.69), esophagus (4.22 ± 3.72), stomach (3.25 ± 2.68), kidney (0.81 ± 0.15), liver (0.63 ± 0.15), and spleen (11.18 ± 4.08) was performed at 3 days post-infection. At 7 days post-infection, heart (754.15 ± 160.85), brain (247.90 ± 62.91), spleen (38.81 ± 17.52), liver (7.47 ± 4.54), kidney (10.90 ± 3.41), stomach (19.50 ± 8.86), esophagus (39.37 ± 14.10), intestine (17.54 ± 12.63), gill (38.27 ± 20.20), and muscle (33.62 ± 15.07) were measured. CONCLUSION: The present study, together with previous data, demonstrated that the gill, intestine, and brain are the major target organs of M. avidus in olive flounder. However, this does not mean that tiny amounts of DNA extracted from those tissues of fish during the early stages of infection can guarantee successful detection and/or quantification of M. avidus. Our data suggest that the brain might be the best organ for detection in the early stage.

Dietary Probiotic Effect of Lactococcus lactis WFLU12 on Low-Molecular-Weight Metabolites and Growth of Olive Flounder (Paralichythys olivaceus).

The use of probiotics is considered an attractive biocontrol method. It is effective in growth promotion in aquaculture. However, the mode of action of probiotics in fish in terms of growth promotion remains unclear. The objective of the present study was to investigate growth promotion effect of dietary administration of host-derived probiotics, Lactococcus lactis WFLU12, on olive flounder compared to control group fed with basal diet by analyzing their intestinal and serum metabolome using capillary electrophoresis mass spectrometry with time-of flight (CE-TOFMS). Results of CE-TOFMS revealed that 53 out of 200 metabolites from intestinal luminal metabolome and 5 out of 171 metabolites from serum metabolome, respectively, were present in significantly higher concentrations in the probiotic-fed group than those in the control group. Concentrations of metabolites such as citrulline, tricarboxylic acid cycle (TCA) intermediates, short chain fatty acids, vitamins, and taurine were significantly higher in the probiotic-fed group than those in the control group. The probiotic strain WFLU12 also possesses genes encoding enzymes to help produce these metabolites. Therefore, it is highly likely that these increased metabolites linked to growth promotion in olive flounder are due to supplementation of the probiotic strain. To the best of our knowledge, this is the first study to show that dietary probiotics can greatly influence metabolome in fish. Findings of the present study may reveal important implications for maximizing the efficiency of using dietary additives to optimize fish health and growth.

Complete Genome Sequence of Multiple-Antibiotic-Resistant Streptococcus parauberis Strain SPOF3K, Isolated from Diseased Olive Flounder (Paralichthys olivaceus).

Here, we report the complete genome sequence of multiple-antibiotic-resistant Streptococcus parauberis strain SPOF3K, isolated from the kidney of a diseased olive flounder in South Korea in 2013. Sequencing using a PacBio platform yielded a circular chromosome of 2,128,740 bp and a plasmid of 23,538 bp, harboring 2,123 and 24 protein-coding genes, respectively.

A Comparison of Genotypic and Phenotypic Methods for Analyzing the Susceptibility to Sulfamethoxazole and Trimethoprim in Edwardsiella piscicida.

In a study of 39 isolates of Edwardsiella piscicida made from Korean aquaculture sites, sul genes were detected in 16 isolates and dfr genes in 19. Ten isolates were shown to contain both sul and dfr genes. MIC and disc diffusion zones assays were performed to measure the phenotypic susceptibilities of the 39 isolates. Normalized resistance interpretation was applied to these data to categorize isolates as either fully susceptible or as manifesting reduced susceptibility. The standard CLSI protocols specify the use of a mixture of sulfamethoxazole/trimethoprim (20:1) in both MIC and disc diffusion tests. Using the CLSI MIC protocol, 100% of the isolates containing dfr genes, but only 75% of the isolates containing sul genes, were categorized as manifesting reduced susceptibility. Using the CLSI disc diffusion protocol, only 58% of the isolates containing dfr genes and 69% of those containing sul genes were categorized as manifesting reduced susceptibility. When the single agent trimethoprim was substituted for the combined mixture in both the MIC and disc diffusion protocols, 100% of the dfr-positive isolates were categorized as NWT. When the single-agent sulfamethoxazole was substituted, the analysis of the MIC characterized 100% and the disc zone data 94% of the sul-positive isolates as manifesting reduced susceptibility. It is argued that the use of trimethoprim and sulfamethoxazole as single agents in phenotypic susceptibility tests would provide more meaningful data than the currently recommended use of these two agents combined.