Hypolipidemic Effect of Rice Bran Oil Extract Tocotrienol in High-Fat Diet-Induced Hyperlipidemia Zebrafish (Danio Rerio) Induced by High-Fat Diet.

In recent years, the potent influence of tocotrienol (T3) on diminishing blood glucose and lipid concentrations in both Mus musculus (rats) and Homo sapiens (humans) has been established. However, the comprehensive exploration of tocotrienol's hypolipidemic impact and the corresponding mechanisms in aquatic species remains inadequate. In this study, we established a zebrafish model of a type 2 diabetes mellitus (T2DM) model through high-fat diet administration to zebrafish. In the T2DM zebrafish, the thickness of ocular vascular walls significantly increased compared to the control group, which was mitigated after treatment with T3. Additionally, our findings demonstrate the regulatory effect of T3 on lipid metabolism, leading to the reduced synthesis and storage of adipose tissue in zebrafish. We validated the expression patterns of genes relevant to these processes using RT-qPCR. In the T2DM model, there was an almost two-fold upregulation in pparγ and cyp7a1 mRNA levels, coupled with a significant downregulation in cpt1a mRNA (p < 0.01) compared to the control group. The ELISA revealed that the protein expression levels of Pparγ and Rxrα exhibited a two-fold elevation in the T2DM group relative to the control. In the T3-treated group, Pparγ and Rxrα protein expression levels consistently exhibited a two-fold decrease compared to the model group. Lipid metabolomics showed that T3 could affect the metabolic pathways of zebrafish lipid regulation, including lipid synthesis and decomposition. We provided experimental evidence that T3 could mitigate lipid accumulation in our zebrafish T2DM model. Elucidating the lipid-lowering effects of T3 could help to minimize the detrimental impacts of overfeeding in aquaculture.

Isolation, identification and characterization of Aeromonas jandaei from diseased Chinese soft-shell turtles.

Aeromonas jandaei is a gram-negative bacterium commonly found in aquatic environments and can induce illnesses in amphibians, reptiles and aquatic animals. In this study, a strain of bacteria was isolated from the diseased Chinese soft-shell turtle (Pelodiscus sinensis), then named strain JDP-FX. This isolate was identified as A. jandaei after analysis of morphological, physiological and biochemical characteristics, as well as 16S rRNA and gyrB gene sequences. Virulence genetic testing further detected temperature-sensitive protease (eprCAI), type III secretion system (TTSS) (ascv), nuclease (nuc), cytotonic enterotoxin (alt) and serine proteinase (ser) in JDP-FX. Compared with healthy Chinese soft-shell turtle, the serum levels of total protein (TP), albumin (ALB) and globulin (GLB) were significantly decreased in the diseased Chinese soft-shell turtle, while, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were significantly increased. Histopathological observations showed that multiple tissues, including intestinal mucosa, liver and kidney, were severely damaged in the diseased Chinese soft-shell turtle. Moreover, the diseased Chinese soft-shell turtle had significant cell degeneration, necrosis, sloughing and interstitial inflammatory cell infiltration. The pathogenicity of JDP-FX was tested via artificial infection. The median lethal dosage (LD50 ) of the strain was 1.05 × 105 colony forming units (CFU/g) per weight of Chinese soft-shell turtle. Drug susceptibility analysis revealed that JDP-FX was susceptible to ceftazidime, minocycline, cefoperazone, ceftriaxone and piperacillin. In addition, JDP-FX was resistant to doxycycline, florfenicol, sulfonamides, gentamicin, ampicillin and neomycin. Therefore, this study may provide guidance for further research into the diagnosis, prevention and treatment of JDP-FX infection.

Isolation and identification of Chryseobacterium indologenes and its pathological changes in Pelodiscus sinensis.

The Chinese revered a species of aquatic reptile known as Pelodiscus sinensis as both an edible and medicinal species. When artificially breeding, many deaths occurred at the farmed P. sinensis, mainly due to excessive breeding density, water contamination, and turtles biting each other secondary to bacterial infections. In this study, an isolate of gram-negative bacteria WH0623 was isolated from the liver and kidney of diseased P. sinensis to trace the potential pathogen of this disease. Based on biochemical characteristics and 16S rRNA gene sequencing analyses, this isolated strain of WH0623 was identified as Chryseobacterium indologenes. The strain's median lethal dose (LD50 ) was 3.3 × 105 colony-forming units (CFU)/g per fish weight tested using artificial infection. Histopathological analysis revealed pathological changes, including cell swelling, hyperaemia, and necrosis in many tissues. Antibiotic susceptibility tests revealed that the bacteria WH0623 was susceptible to doxycycline, sulphonamides, ceftazidime, norfloxacin, and ciprofloxacin. These antibiotics could treat the disease. In conclusion, the pathogen causing the death of farmed P. sinensis was isolated and identified, and a drug-sensitive test was conducted. Our findings contribute to the future diagnosis and treatment of the disease.

Pathological changes of highly pathogenic Bacillus cereus on Pelodiscus sinensis.

An outbreak of a disease with a high mortality rate occurred in a Chinese Softshell Turtle (Pelodiscus sinensis) farm in Hubei Province. This study isolated a highly pathogenic Bacillus cereus strain (Y271) from diseased P. sinensis. Y271 has ß hemolysis, containing both Hemolysin BL (hblA, hblC, and hblD), Non-hemolytic enterotoxin, NHE (nheA, nheB, and nheC), and Enterotoxin FM (entFM) genes. Y271 is highly pathogenic against P. sinensis with an LD50 = 6.80 × 103 CFU/g weight. B. cereus was detected in multiple tissues of the infected P. sinensis. Among them, spleen tissue showed the highest copy number density (1.54 ± 0.12 × 104 copies/mg). Multiple tissues and organs of diseased P. sinensis exhibited significant pathological damage, especially the spleen, liver, kidney, and intestine. It showed obvious tissue structure destruction, lesions, necrosis, red blood cells, and inflammatory cell infiltration. B. cereus proliferating in the spleen, liver, and other tissues was observed. The intestinal microbiota of the diseased P. sinensis was altered, with a greater abundance of Firmicutes, Fusobacterium, and Actinomyces than in the healthy group. Allobaculum, Rothia, Aeromonas, and Clostridium abundance were higher in the diseased group than in the healthy group. The number of unique microbial taxa (472) in the disease group was lower than that of the healthy group (705). Y271 was sensitive to multiple drugs, including florfenicol, enrofloxacin, neomycin, and doxycycline. B. cereus is the etiological agent responsible for the massive death of P. sinensis and reveals its potential risks during P. sinensis cultivation.

The probiotic effects of host-associated Bacillus velezensis in diets for hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂).

This study was to evaluate the potential of a host-associated Bacillus velezensis as a probiotic for hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂). Diets (B0 to B5) containing 0, 0.90 × 108, 0.80 × 109, 0.85 × 1010, 0.90 × 1011, 0.83 × 1012 CFU/kg B. velezensis YFI-E109 were fed to the fish with initial weight (3.07 ± 0.08 g) in a recirculating aquaculture system for six weeks with three replicates, respectively. Probiotic effects were analyzed based on growth, body composition, liver and gut morphology, gut microbiome, and liver metabolome. Analysis of the bacterial genome has shown that the most abundant genes in B. velezensis YFI-E109 were distributed in carbohydrate and amino acid metabolism. Fish in groups B3 and B4 had better growth performance, and higher intestinal amylase (AMS) and lipase (LPS) activities compared with other groups (P < 0.05). Fish in groups B0 and B5 showed significant liver damage, while this status improved in group B3. The liver malondialdehyde (MDA) content in group B3 was lower than that in other groups (P < 0.05). The abundance of Mycoplasma, Ralstonia and Acinetobacter was significantly reduced in B3 and B5 compared to B0. The amino acid and carbohydrate metabolism pathways were enriched in group B3 compared with group B0. In conclusion, dietary B. velezensis YFI-E109 supplementation has the potential to improve growth, liver metabolism, and liver and gut health, and reshape the gut microbiome of hybrid yellow catfish. Excessive B. velezensis YFI-E109 reduced the prebiotic effects. The recommended dietary supplementation of B. velezensis YFI-E109 is 0.31 × 1010 to 0.77 × 1011 CFU/kg for hybrid yellow catfish according to the quadratic regression method by plotting specific growth rate (SGR), feed conversion ratio (FCR), MDA and activities of AMS against dietary B. velezensis YFI-E109 levels.

Transcriptomics in Rare Minnow (Gobiocypris rarus) towards Attenuated and Virulent Grass Carp Reovirus Genotype II Infection.

Grass carp reovirus genotype Ⅱ (GCRV Ⅱ) causes a variety of fish hemorrhagic disease, which seriously affects the sustainable development of grass carp aquaculture in China. Rare minnow (Gobiocypris rarus) is an ideal model fish to study the pathogenesis of GCRV Ⅱ. To investigate the involved molecular responses against the GCRV Ⅱ infection, we performed comparative transcriptomic analysis in the spleen and liver of rare minnow injected with virulent strain DY197 and attenuated strain QJ205. Results showed that the virulent DY197 strain induced more differently expressed genes (DEGs) than the attenuated QJ205 strain, and tissue-specific responses were induced. In the spleen, the attenuated and virulent strains induced different DEGs; the attenuated QJ205 infection activated steroid synthesis pathway that involved in membrane formation; however, virulent DY197 infection activated innate immunity and apoptosis related pathways while suppressing cell proliferation and migration related pathways that are important for damage tissue repair, as well as hemorrhage related pathways. In the liver, the attenuated and virulent strains infection induced similar DEGs; both strains infection activated immunity and apoptosis related pathways but suppressed metabolism-related pathways; virulent DY197 infection especially activated protein digestion and absorption-related pathways and suppressed steroid synthesis pathway. To conclude, virulent strain infection especially induced tissue-specific alterations and caused severe suppression of hemorrhage-related pathways in spleen. Our findings will contribute to better understanding of the interactions between host and GCRV II.

Variations in the Intestinal Microbiota of the Chinese Soft-Shelled Turtle (Trionyx sinensis) between Greenhouse and Pond Aquaculture.

The microbial community structure in aquaculture water plays an important role in the intestinal microbial diversity of aquatic animals. The Chinese soft-shelled turtle (SST) (Trionyx sinensis) is an important aquaculture species of high economic value in the Asia-Pacific region. An intuitive understanding of the microbial diversity and abundances of SST aquaculture is crucial for comprehending these ecosystems. Herein, the evolutionary characteristics of the bacterial communities in the SST and its aquaculture water systems were investigated using Illumina MiSeq sequencing. This experiment sampled nine SSTs from a pond outside a greenhouse and was repeated three times. The sequencing results revealed significant differences in the microflora composition at the phylum and genus levels in both the intestine and aquaculture water of the SSTs in the greenhouse and pond aquaculture environments. A total of 1039 genera belonging to 65 phyla were identified. At the phylum level, the relative abundances of Chloroflexi (24%), Acidobacteria (5%), and Nitrospira (3%) were higher in the greenhouse water than in the pond water. The relative abundances of Bacteroidetes (35%), Actinobacteria (8%), and Cyanobacteria (4%) were higher in the pond water than in the greenhouse water. The intestinal microorganisms in the SSTs experienced significant changes after the SSTs were transferred from a greenhouse culture to a pond culture environment for 28 days. After the SSTs were cultured in the ponds, we observed decreases in the relative abundances of Actinobacteria (39% to 25%), Cyanobacteria (24% to 0.8%), Chlorobacteria (9% to 3%), and Firmicutes (5.5% to 0.8%. However, we observed increases in the relative abundances of Bacteroidetes (2% to 35%) and Acidobacteria (0.3% to 25%). These results showed that the bacterial diversity and richness compositions in the intestinal tract and aquaculture water were the same. However, the relative abundances of bacterial communities varied. The results of this study are of great significance in understanding how the environment affects SST cultures. These data may provide valuable instructions for Chinese soft-shelled turtle aquaculture management.

Development of a droplet digital PCR assay for the sensitive detection of iridovirus in Andrias davidianus.

Chinese giant salamander iridovirus (GSIV) is the first known and causative viral pathogen in Andrias davidianus. Developing a sensitive, accurate and specific assay to detect GSIV in samples is essential to prevent the further spread of the pathogen. In this study, we established a droplet digital PCR (ddPCR) assay that targeted the mcp gene of GSIV, enabling rapid and quantitative detection of the virus. We determined that the optimal annealing temperature, primer concentration and probe concentration were 57.1°C, 50 nM and 500 nM, respectively. We analysed the specificity and sensitivity of the ddPCR assay and found that five common aquatic animal viruses, including Cyprinid herpesvirus 2 (CyHV-2), infectious spleen and kidney necrosis virus (ISKNV), Koi herpesvirus (KHV) and Carp Edema Virus (CEV) displayed negative results based on this GSIV ddPCR assay. The assay can detect GSIV with the lowest detection limit of 3.7 copies per reaction. To evaluate the sensitivity and accuracy of the ddPCR assay, we tested different infected tissue samples with both the ddPCR and TaqMan real-time PCR assays. Our results showed that the ddPCR assay detected GSIV in all samples with 100% positivity, while the TaqMan real-time PCR assay detected GSIV in only 82.1% of samples. The established ddPCR method provided several advantages in detecting GISV, including high sensitivity, high precision and absolute quantification, making it a powerful tool for detection of possible and potential GSIV infection, even in samples with low viral load.

Chinese giant salamander Bcl-w: An inhibitory role in iridovirus-induced mitochondrial apoptosis and virus replication.

B-cell lymphoma-2 (BCL-2) superfamily molecules play crucial roles in mitochondrial apoptosis induced by Chinese giant salamander iridovirus (GSIV). As an anti-apoptotic molecule in the BCL-2 family, the molecular mechanism of Bcl-w during GSIV infection remains unknown. In this study, we characterized for the first time an amphibian Bcl-w from Chinese giant salamander Andrias davidianus (AdBcl-w), and its function and regulatory mechanism during GSIV infection were investigated. AdBcl-w possesses the conserved structural features of Bcl-w and shares 35-54% sequence identities with other Bcl-w. mRNA expression of AdBcl-w was most abundant in liver and muscle. The AdBcl-w mRNA expression was regulated during GSIV infection. Western blotting assays revealed that the level of Bcl-w protein was downregulated markedly as the infection progresses. Confocal microscopy showed that overexpressed AdBcl-w was translocated to the mitochondria after infection with GSIV. Flow cytometry analysis demonstrated that compared with control, the apoptotic progress in cells transfected with AdBcl-w was reduced while that in cells transfected with AdBcl-w siRNA was enhanced. The number of virus major capsid protein gene copies was lower and protein synthesis was reduced in AdBcl-w overexpressing cells. In addition, AdBcl-w could bind directly to the pro-apoptotic molecule AdBak, while this interaction was weakened with GSIV infection. Moreover, p53 level was reduced and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-w overexpressing cells during GSIV infection. These results suggested that AdBcl-w inhibit GSIV replication by regulating the virus induced mitochondrial apoptosis.

Isolation, Identification, and Characterization of Aeromonas veronii from Chinese Soft-Shelled Turtle (Trionyx sinensis).

Aeromonas veronii is widespread in aquatic environments and is capable of infecting various aquatic organisms. A. veronii infection is lethal for Chinese soft-shelled turtles (Trionyx sinensis, CSST). We isolated a gram-negative bacterium from the liver of diseased CSSTs, which was named XC-1908. This isolate was identified as A. veronii based on its morphological and biochemical characteristics, and 16S rRNA gene sequence analysis. A. veronii was pathogenic for CSSTs with an LD50 of 4.17 × 105 CFU/g. The symptoms of CSSTs artificially infected with isolate XC-1908 were consistent with those of the naturally infected CSSTs. The levels of total protein, albumin, and white globule in the serum samples of the diseased turtles were decreased, whereas those of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were elevated. Moreover, the diseased CSSTs exhibited the following histopathological changes: the liver contained numerous melanomacrophage centers, renal glomerulus were edematous, intestinal villi were shed, and in oocytes, the number of vacuoles increased and red-rounded particles were observed. Antibiotic sensitivity tests revealed that the bacterium was sensitive to ceftriaxone, doxycycline, florfenicol, cefradine, and gentamicin, and resistant to sulfanilamide, carbenicillin, benzathine, clindamycin, erythromycin, and streptomycin. This study provides control strategies to prevent outbreaks of A. veronii infection in CSSTs.

Nonstructural protein NS17 of grass carp reovirus Honghu strain promotes virus infection by mediating cell-cell fusion and apoptosis.

Fusion-associated small transmembrane (FAST) proteins can promote cell fusion, alter membrane permeability and trigger apoptosis to promote virus proliferation in orthoreoviruses. However, it is unknown whether FAST proteins perform these functions in aquareoviruses (AqRVs). Non-structural protein 17 (NS17) carried by grass carp reovirus Honghu strain (GCRV-HH196) belongs to the FAST protein family, and we preliminarily explored its relevance to virus infection. NS17 has similar domains to FAST protein NS16 of GCRV-873, comprising a transmembrane domain, a polybasic cluster, a hydrophobic patch and a polyproline motif. It was observed in the cytoplasm and the cell membrane. Overexpression of NS17 enhanced the efficiency of cell-cell fusion induced by GCRV-HH196 and promoted virus replication. Overexpression of NS17 also led to DNA fragmentation and reactive oxygen species (ROS) accumulation, and it triggered apoptosis. The findings illuminate the functions of NS17 in GCRV infection, and provide a reference for the development of novel antiviral strategies.

Characteristics and expression profiles of MHC class â molecules in Carassius auratus.

Major histocompatibility complex class Ⅰ (MHC Ⅰ) molecules play a vital role in adaptive immune systems in vertebrates by presenting antigens to effector T cells. Understanding the expression profiling of MHC Ⅰ molecules in fish is essential for improving our knowledge of the relationship between microbial infection and adaptive immunity. In this study, we conducted a comprehensive analysis of MHC Ⅰ gene characteristics in Carassius auratus, an important freshwater aquaculture fish in China that is susceptible to Cyprinid herpesvirus 2 (CyHV-2) infection. We identified approximately 20 MHC Ⅰ genes discussed, including U, Z, and L lineage genes. However, only U and Z lineage proteins were identified in the kidney of Carassius auratus using high pH reversed-phase chromatography and mass spectrometry. The L lineage proteins were either not expressed or present at an extremely low level in the kidneys of Carassius auratus. We also used targeted proteomics to analyze changes in protein MHC Ⅰ molecules abundance in healthy and CyHV-2-infected Carassius auratus. We observed that five MHC Ⅰ molecules were upregulated, and Caau-UFA was downregulated in the diseased group. This study is the first to reveal the expression of MHC Ⅰ molecules at a large scale in Cyprinids, which enhances our understanding of fish adaptive immune systems.

An Aquareovirus Exploits Membrane-Anchored HSP70 To Promote Viral Entry.

Temperature dependency of viral diseases in ectotherms has been an important scientific issue for decades, while the molecular mechanism behind this phenomenon remains largely mysterious. In this study, deploying infection with grass carp reovirus (GCRV), a double-stranded RNA aquareovirus, as a model system, we demonstrated that the cross talk between HSP70 and outer capsid protein VP7 of GCRV determines temperature-dependent viral entry. Multitranscriptomic analysis identified HSP70 as a key player in the temperature-dependent pathogenesis of GCRV infection. Further biochemical, small interfering RNA (siRNA) knockdown, pharmacological inhibition, and microscopic approaches revealed that the primary plasma membrane-anchored HSP70 interacts with VP7 to facilitate viral entry during the early phase of GCRV infection. Moreover, VP7 functions as a key coordinator protein to interact with multiple housekeeping proteins and regulate receptor gene expression, concomitantly facilitating viral entry. This work illuminates a previously unidentified immune evasion mechanism by which an aquatic virus hijacks heat shock response-related proteins to enhance viral entry, pinpointing targeted preventives and therapeutics for aquatic viral diseases. IMPORTANCE The seasonality of viral diseases in ectotherms is a prevailing phenomenon in the aquatic environment, which causes huge economic losses every year worldwide and hinders sustainable development of the aquaculture industry. Nevertheless, our understanding of the molecular mechanism of how temperature determines the pathogenesis of aquatic viruses remains largely unexplored. In this study, by deploying grass carp reovirus (GCRV) infection as a model system, we demonstrated that temperature-dependent, primarily membrane-localized HSP70 interacts with major outer capsid protein VP7 of GCRV to bridge the virus-host interaction, reshape the host's behaviors, and concomitantly facilitate viral entry. Our work unveils a central role of HSP70 in the temperature-dependent pathogenesis of aquatic viruses and provides a theoretical basis for the formulation of prevention and control strategies for aquatic viral diseases.

Metabolomics in rare minnow (Gobiocypris rarus) after infection by attenuated and virulent grass carp reovirus genotype â ¡.

Grass carp reovirus genotype Ⅱ (GCRV Ⅱ) causes hemorrhagic disease in a variety fish, seriously affecting the aquaculture industry in China. However, the pathogenesis of GCRV Ⅱ is unclear. Rare minnow is an ideal model organism to study the pathogenesis of GCRV Ⅱ. Herein, we applied liquid chromatography-tandem mass spectrometry metabolomics to investigate metabolic responses in the spleen and hepatopancreas of rare minnow injected with virulent GCRV Ⅱ isolate DY197 and attenuated isolate QJ205. Results indicated that marked metabolic changes were identified in both the spleen and hepatopancreas after GCRV Ⅱ infection, and the virulent DY197 strain induced more significantly different metabolites (SDMs) than the attenuated QJ205 strain. Moreover, most SDMs were downregulated in the spleen and tend to be upregulated in hepatopancreas. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that tissue-specific metabolic responses were identified after viruses infection, and the virulent DY197 strain induced more SDMs involved in amino acid metabolism in the spleen, especially the tryptophan metabolism, cysteine and methionine metabolism, which were essential for immune regulation in host; Meanwhile, nucleotide metabolism, protein synthesis and metabolism related pathways were enriched in the hepatopancreas by both virulent and attenuated strains. Our findings revealed the large scale metabolic alterations in rare minnow in response to attenuated and virulent GCRV Ⅱ infection, which will lead to a better understanding of the pathogenesis of viruses and host-pathogens interactions.

Anti-Inflammatory and Antioxidant Properties of Squalene in Copper Sulfate-Induced Inflammation in Zebrafish (Danio rerio).

Long-term or excessive oxidative stress can cause serious damage to fish. Squalene can be added to feed as an antioxidant to improve the body constitution of fish. In this study, the antioxidant activity was detected by 2,2-diphenyl-1-acrylhydrazyl (DPPH) test and fluorescent probe (dichloro-dihydro-fluorescein diacetate). Transgenic Tg (lyz: DsRed2) zebrafish were used to evaluate the effect of squalene on CuSO4-induced inflammatory response. Quantitative real-time reverse transcription polymerase chain reaction was used to examine the expression of immune-related genes. The DPPH assay demonstrated that the highest free radical scavenging exerted by squalene was 32%. The fluorescence intensity of reactive oxygen species (ROS) decreased significantly after 0.7% or 1% squalene treatment, and squalene could exert an antioxidative effect in vivo. The number of migratory neutrophils in vivo was significantly reduced after treatment with different doses of squalene. Moreover, compared with CuSO4 treatment alone, treatment with 1% squalene upregulated the expression of sod by 2.5-foldand gpx4b by 1.3-fold to protect zebrafish larvae against CuSO4-induced oxidative damage. Moreover, treatment with 1% squalene significantly downregulated the expression of tnfa and cox2. This study showed that squalene has potential as an aquafeed additive to provide both anti-inflammatory and antioxidative properties.

Oral Vaccination of Largemouth Bass (Micropterus salmoides) against Largemouth Bass Ranavirus (LMBV) Using Yeast Surface Display Technology.

Largemouth bass ranavirus (LMBV) infects largemouth bass, leading to significant mortality and economic losses. There are no safe and effective drugs against this disease. Oral vaccines that directly target the intestinal mucosal immune system play an important role in resisting pathogens. Herein, the B subunit of Escherichia coli heat-labile enterotoxin (LTB, a mucosal immune adjuvant) and the LMBV main capsid protein (MCP) were expressed using Saccharomyces cerevisiae surface display technology. The yeast-prepared oral vaccines were named EBY100-OMCP and EBY100-LTB-OMCP. The candidate vaccines could resist the acidic intestinal environment. After 7 days of continuous oral immunization, indicators of innate and adaptive immunity were measured on days 1, 7, 14, 21, 28, 35, and 42. High activities of immune enzymes (T-SOD, AKP, ACP, and LZM) in serum and intestinal mucus were detected. IgM in the head kidney was significantly upregulated (EBY100-OMCP group: 3.8-fold; BY100-LTB-OMCP group: 4.3-fold). IgT was upregulated in the intestines (EBY100-OMCP group: 5.6-fold; EBY100-LTB-OMCP group: 6.7-fold). Serum neutralizing antibody titers of the two groups reached 1:85. Oral vaccination protected against LMBV infection. The relative percent survival was 52.1% (EBY100-OMCP) and 66.7% (EBY100-LTB-OMCP). Thus, EBY100-OMCP and EBY100-LTB-OMCP are promising and effective candidate vaccines against LMBV infection.

Anti-inflammatory and antioxidant properties of rice bran oil extract in copper sulfate-induced inflammation in zebrafish (Danio rerio).

Tocotrienols have strong antioxidant properties; however, tocotrienol has not been investigated in detail in aquatic products. In this study, the anti-inflammatory and antioxidant activities of the tocotrienol-rich fraction from rice bran oil and its potential mechanism were verified in a zebrafish CuSO4 inflammation model. The in vitro antioxidant activity was evaluated using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) stable radical method. The copper chelating activity was determined using the pyrocatechol violet method. Intracellular reactive oxygen species in zebrafish were detected using a fluorescent ROS probe. Transgenic Tg (lyz: DsRed2) zebrafish were used for neutrophil transmigration assays. The mRNA expression levels of antioxidant and pro-inflammatory factor genes were measured using quantitative real-time reverse transcription PCR. In the concentration range tested, 100 µg/mL TRF had the highest copper chelating activity (10%). TRF showed DPPH-free radical scavenging ability, which was 53% at 100 µg/mL TRF. TRF effectively repressed ROS generation and inhibited neutrophil migration to the inflamed site. Moreover, TRF upregulated the expression of antioxidant genes sod and gpx4b, inhibited the expression of pro-inflammatory factors tnfa and il8, and suppressed CuSO4-induced inflammation. In conclusion, TRF has significant anti-inflammatory and antioxidant properties, which supports the use of TRF as an aquatic feed additive to improve the anti-inflammatory and antioxidant capacity of aquatic products.

Aflatoxin B1 Induced Oxidative Stress and Gut Microbiota Disorder to Increase the Infection of Cyprinid Herpesvirus 2 in Gibel Carp (Carassius auratus gibelio).

Aflatoxin contamination of food and water is a serious problem worldwide. This study investigated the defensive ability of gibel carp exposed to aflatoxin B1 (AFB1) by challenging it with cyprinid herpesvirus 2 (CyHV-2) infection. The data showed that AFB1 exposure significantly increased the mortality of CyHV-2-infected gibel carp, and enhanced the viral load in the fish liver, kidney, and spleen. The oxidative-antioxidant balance suggested that AFB1 induced severe oxidative stress, including increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the AFB1 exposed group, and the reduced activity of superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase (CAT) in the AFB1 exposed group. Meanwhile, the related expression of nuclear factor erythroid 2-related factor 2 (Nrf2), interferon regulatory factor 3 (IRF3) and the type 1 interferon (IFN1) were noticeably down-regulated, but caspase-1 was up-regulated, after exposure to AFB1, demonstrating that fish are unable to avoid the virus infection. It should be noted that the intestinal microbiota diversity and richness were lower in the AFB1 exposed group, and the composition of intestinal microbiota was affected by AFB1, resulting in the higher abundance of bacteria (such as Aeromonas and Bacteroides) and the lower abundance of potentially beneficial bacteria (such as Cetobacterium and Clostridium) in the AFB1 exposed group. This research provides insight into the possibility that AFB1 may increase the susceptibility of C. gibelio to CyHV-2 infection, and thus amplify the viral outbreak to endanger ecological safety in aquatic environment.

Anti-Inflammatory and Antioxidant Properties of ß-Sitosterol in Copper Sulfate-Induced Inflammation in Zebrafish (Danio rerio).

ß-Sitosterol, which is used extensively in pharmaceuticals, nutraceuticals, and cosmetics, has high nutritional value along with immunomodulatory and anti-inflammatory properties. In this study, we investigated the antioxidant and anti-inflammatory effects of ß-sitosterol in zebrafish and explored the associated molecular mechanisms. In an in vivo antioxidant experiment, zebrafish (Danio rerio) larvae were treated with different concentrations of ß-sitosterol and then exposed to a nonlethal concentration of CuSO4 to induce oxidative stress. Treatment with ß-sitosterol at 70 or 100 µg/mL significantly reduced CuSO4-induced oxidative stress in the zebrafish, demonstrating the strong antioxidant activity of ß-sitosterol. Treatment with ß-sitosterol protected zebrafish larvae against oxidative damage from CuSO4 by upregulating the expressions of sod and gpx4b. In a zebrafish model of inflammation, pretreatment with ß-sitosterol before CuSO4 exposure inhibited neutrophil recruitment and damage to lateral line neuromasts, indicating a potent anti-inflammatory effect derived from reductions in the expressions of il-8 and myd88. The results demonstrate the antioxidative and anti-inflammatory activities of ß-sitosterol and suggest that ß-sitosterol may be useful for the treatment of various inflammatory diseases.

Development and application of a sensitive droplet digital PCR-based method to detect tilapia parvovirus.

Tilapia parvovirus (TiPV) causes severe mortality rates in cultured tilapia, resulting in substantial losses to the fish industry. Droplet digital PCR (ddPCR) is a sensitive, accurate, and absolute quantitation method, plus it does not require a standard curve. Herein we report the development and application of a sensitive ddPCR-based method to rapidly detect and quantify TiPV. Optimal annealing temperature was determined to be 59.3°C, and optimal primer and probe concentrations were 900 nmol/L and 250 nmol/L, respectively. Our ddPCR method was highly specific to TiPV and showed no cross-reactivity with other viruses. Further, the detection limit of ddPCR was 0.07 copies/µl, being lower than that of real-time PCR (qPCR, 4.63 copies/µl). We also investigated the ability of ddPCR to detect TiPV in 50 samples and compared the outcome with qPCR data in terms of sensitivity and accuracy. The results showed that the positive detection rate of ddPCR (32%) was higher than that of qPCR (18%). To conclude, our ddPCR method was effective at detecting TiPV in samples with low viral loads. We believe that its application can facilitate the surveillance of sources and transmission routes of TiPV.