Spleen Of Fish Research Articles

Toxicity of Low-Level Multiple-Mycotoxin Mixture in Nile Tilapia (Oreochromis niloticus) Is Prevented with Organically Modified Clinoptilolite Feed Additive

Organically modified clinoptilolite (member of the zeolite family of minerals; MinazelPlus®) feed additive, with an average weight of 30 ± 2 g, was used to prevent mycotoxicosis in Nile tilapia (Oreochromis niloticus) through its supplementation for 42 days to the diet contaminated with multiple mycotoxins: aflatoxin B1 (40 µg/kg), fumonisin B1 and B2 (600 µg/kg), zearalenone (50 µg/kg), and deoxynivalenol (150 µg/kg). The fish were divided randomly into four experimental groups (basal diet control—C; fed 2 g/kg MinazelPlus®—MZ; fed multiple mycotoxins—MT; and fed a combination of MinazelPlus® and multiple mycotoxins—MZ + MT). Each group consisted of triplicate aquarium setups, with six fish in each replicate. Sampling was performed in weeks 2, 4, and 6. The lymphocyte count was significantly higher in the MZ group compared with the MT group and the MT + MZ group in week 6. An overall decrease in the neutrophil count was observed in the experimental groups. Histopathological analysis was performed in weeks 2 and 6, revealing significant changes in the liver, intestines, kidney, and spleen of fish from the MT group, while the MT + MZ and MZ groups were similar to the control. The addition of 2 g/kg MinazelPlus® has the ability to prevent and reduce the adverse effects of chronic exposure to low concentrations of multiple mycotoxins in juvenile Nile tilapia.

The Impact of Acute Ammonia Nitrogen Stress on Serum Biochemical Indicators and Spleen Gene Expression in Juvenile Yellowfin Tuna (Thunnus albacares).

The presence of ammonia nitrogen in water has a significant impact on the serum and spleen of fish, potentially leading to changes in substances such as proteins in the serum while also causing damage to the immune function of the spleen. To investigate the effects of ammonia nitrogen (NH3-N) stress on juvenile yellowfin tuna (Thunnus albacares), this study established three NH3-N concentrations, 0, 5, and 10 mg/L, denoted as L0, L1, and L2, respectively. Serum and spleen samples were collected at 6, 24, and 36 h. The effects of different NH3-N concentrations and exposure times on the physiological status of juvenile fish were investigated by analyzing serum biochemical indices and splenic gene expression. The results indicate that in the L1 group, the serum high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), complement 3 (C3) and complement 4 (C4) levels, and acid phosphatase (ACP) activity showed a trend of initially increasing and then decreasing. In the L2 group, the serum low-density lipoprotein cholesterol (LDL-C), HDL-C, and C4 levels and ACP activity also displayed an initially rising and then declining trend, while TG, C3, and creatinine (CRE) levels and alkaline phosphatase (AKP) activity showed an upward trend. In the L1 group, glutathione peroxidase 1b (GPX1b), interleukin 10 (IL-10), interleukin 6 receptor (IL-6r), and tumor necrosis factor alpha (TNF-α) gene expression levels in the spleen exhibited a trend of first increasing and then decreasing. In the L2 group, IL-10, IL-6r, tumor necrosis factor beta (TNF-β), caspase 2 (casp2), and caspase 9 (casp9) gene expression levels in the spleen also showed an initial increase followed by a decrease. When NH3-N levels are below 5 mg/L, it is recommended to limit stress exposure to no more than 36 h for the juvenile fish. For concentrations ranging from 5 to 10 mg/L, stress should be strictly controlled to within 24 h. Exposure to high NH3-N levels may affect biochemical indicators such as serum lipid metabolism, immunity, and metabolism in juvenile fish, and may damage the expression of antioxidant, immune gene, and apoptosis factors in the spleen. This study aims to deepen our understanding of the effects of NH3-N on juvenile tuna, with the goal of establishing effective water quality monitoring and management strategies. This will ensure the quality of aquaculture water, reduce the harm caused by NH3-N to juvenile yellowfin tuna, and enhance aquaculture efficiency and product quality.

Marine macroalgae Chaetomorpha aerea as a dietary supplement: Optimizing immunity and resistance to Edwardsiella tarda in tilapia (Oreochromis mossambicus)

The intensification of aquaculture has led to a rise in fish infections, necessitating the search for alternative antibiotics. In this context, our study investigated the effects of dietary supplementation with Chaetomorpha aerea, a filamentous green algae, on the immune health and resistance to infections in tilapia (Oreochromis mossambicus). Diets containing varying concentrations of C. aerea (0, 1, 2, 5, and 10 g/kg) were prepared and administered to the fish for 30 days, followed by a challenge with Edwardsiella tarda to evaluate survival rates. The results were significant. The diet containing 5 g/kg of C. aerea (group T3) brought about substantial improvements in hematological parameters, including increases in red blood cell count (RBC), hematocrit (Hct), and hemoglobin (Hb). The T3 group exhibited a robust immune response, with higher lysozyme and ceruloplasmin activity in immunological assays. LBP gene expression was significantly elevated in the spleen and thymus of fish in the T3 group, which correlated with higher survival after bacterial challenge compared to the control group. Principal Component Analysis (PCA) and cluster analysis confirmed that the 5 g/kg concentration stood out for maximizing immunological benefits without compromising the overall health of the fish. These findings highlight the robust immune response in the T3 group, a key finding of our study. We conclude that supplementation with C. aerea represents a promising and sustainable alternative in the formulation of diets for tilapia, contributing to improved health and resistance to diseases. Future studies are recommended to explore its application in other species and development stages, in addition to evaluating other health biomarkers.

Identification of a novel circovirus associated with turbot (Scophthalmus maximus) acute hemorrhage disease

Turbot (Scophthalmus maximus) is an economically important farming fish in China. However, an emerging disease named turbot acute hemorrhage disease (TAHD) has been affecting turbot farms since November 2019. TAHD is characterized by severe bleeding in the fish fins and may lead to significant mortality in one or two weeks, with accumulated mortality exceeding 90 %. The TAHD spread rapidly across the major turbot farming regions in China and resulted in significant economic losses. Virome sequencing was utilized in the diseased fish to discover a novel turbot circovirus (TCV). The TCV particle is ∼30 nm in size and is located in the fish spleen and kidney. The TCV genome is 1774 bp long, with three open reading frames (ORFs) encoding the replication protein (Rep), capsid protein (Cap), and ORF3 of unknown function. The identity of the TCV Rep sequence was <53 % compared to the reported circovirus sequences. Phylogenetic analysis of the Rep and Cap protein sequences revealed that TCV is a novel circovirus. The polymerase chain reaction detection method was used to analyze the clinical TAHD samples from 2019, which were all TCV-positive. Various cell lines, including turbot kidney, epithelioma papilloma cyprinid, Chinook salmon embryo, and spotted halibut kidney, were used for TCV isolation. However, no cytopathic effect or replication was observed. The diseased fish tissue homogenate filtrate was used for experimental infection. As a result, all of the infected turbot showed signs of natural infection, with increasing numbers of cap gene copies post-infection. Based on these results, it was hypothesized that the novel circovirus TCV is closely associated with TAHD. Our research also indicated that the circovirus represents a significant threat to fish and more attention should be paid to it in aquaculture.

Inactivated LMBV vaccine with nano‑aluminum adjuvant provided immune protection in largemouth bass (Micropterus Salmoides)

Largemouth bass ranavirus (LMBV) has caused severe economic losses in largemouth bass (Micropterus salmoides) aquaculture. Vaccination is acknowledged as an efficient strategy to prevent viral diseases in fish. In this study, an inactivated virus vaccine with nano‑aluminum adjuvant was successfully developed to protect largemouth bass effectively against LMBV. LMBV was inactivated by formalin and mixed with nano‑aluminum adjuvant under moderate agitation. The nano‑aluminum hydroxide adjuvant showed a cuboidal structure under the scanning transmission electron microscope (STEM), scanning electron microscope (SEM), and transmission electron microscope (TEM), and LMBV antigen-adsorbing rate was 39.49 % superior to those of micro‑aluminum hydroxide and IMS1312 adjuvants. The results from both external and internal damage assessments indicated that the nano‑aluminum vaccine had good safety for Micropterus salmoides. The relative percentage of survival was 80 % with a booster immunization in largemouth bass. The expression levels of immune-related genes such as TNF-α, IFN, and IL-8 were significantly up-regulated in the spleen of fish immunized by nano‑aluminum adjuvant vaccine compared to those in inactivated LMBV and control groups. The neutralizing antibody in nano‑aluminum adjuvant vaccine group showed higher abundance and lasted for a longer period. Lymphocytes from the head kidney of largemouth bass in the nano‑aluminum adjuvant vaccine group showed stronger proliferation activity in vitro post-stimulation with ConA. Moreover, stronger migration of the dendritic cells was induced by the nano‑aluminum adjuvant vaccine. Collectively, our findings suggested that the inactivated LMBV vaccine with nano‑aluminum adjuvant could provide efficient protection for largemouth bass against LMBV infection. The nano‑aluminum adjuvant has potential application with inactivated viral vaccines in fish.

Molecular characterization and gene expression of pattern recognition receptors in brown-marbled grouper (Epinephelus fuscoguttatus) fingerlings responding to vibriosis infection

The pathogen recognition system involves receptors and genes that play a crucial role in activating innate immune response in brown-marbled grouper (Epinephelus fuscoguttatus) as a control agent against various infections including vibriosis. Here, we report the molecular cloning of partial open reading frames, sequences characterization, and expression profiles of Pattern Recognition Receptors (PRRs) in brown-marbled grouper. The PRRs, namely pglyrp5, tlr5, ctlD, and ctlE in brown-marbled grouper, possess conserved domains and showed shared evolutionary relationships with other fishes, humans, mammals, birds, reptilians, amphibians, and insects. In infection experiments, up to 50% mortality was found in brown-marbled grouper fingerlings infected with Vibrio alginolyticus compared to 27% mortality infected Vibrio parahaemolyticus and 100% survival of control groups. It is also demonstrated that all four PRRs had higher expression in samples infected with V. alginolyticus compared to V. parahaemolyticus. This PRRs gene expression analysis revealed that all four PRRs expressed rapidly at 4-h post-inoculation even though the Vibrio count was only detected earliest at 12-h post-inoculation in samples. The highest expression recorded was from V. alginolyticus inoculated fish spleen with up to 73-fold change for pglyrp5 gene, followed by 14 to 38-fold expression for the same treatment in spleen, head kidney, and blood samples for other PRRs, namely tlr5, ctlD, and ctlE genes. Meanwhile less than a 10% increase in expression of all four genes was detected in spleen, head kidney, and blood samples inoculated with V. parahaemolyticus. These findings indicated that pglyrp5, tlr5, ctlD, and ctlE play important roles in the early immune response to vibriosis infected, brown-marbled grouper fingerlings.

Ficus hirta Vahl. alleviate LPS induced apoptosis via down-regulating of MiR-411 in orange-spotted grouper (Epinephelus coioides) spleen cell

Ficus hirta Vahl. (FhV) has been shown to have antimicrobial and antiviral efficacy. To further ascertain the pharmacological properties of FhV., and to search for alternatives to antibiotics. An in vitro experiment was carried out to evaluate what influence FhV. would have on LPS-induced apoptosis. In this study, Fas, an apoptosis receptor, was cloned, which included a 5′-UTR of 39 bp, an ORF of 951 bp, a protein of 316 amino acids, and a 3′-UTR of 845 bp. EcFas was most strongly expressed in the spleen tissue of orange-spotted groupers. In addition, the apoptosis of fish spleen cells induced by LPS was concentration-dependent. Interestingly, appropriate concentrations of FhV. alleviated LPS-induced apoptosis. Inhibition of miR-411 further decreased the inhibitory effect of Fas on apoptosis, which reduced Bcl-2 expression and mitochondrial membrane potential, enhanced the protein expression of Bax and Fas. More importantly, the FhV. could activate miR-411 to improve this effect. In addition, luciferase reporter assays showed that miR-411 binds to Fas 3'-UTR to inhibit Fas expression. These findings provide evidence that FhV. alleviates LPS-induced apoptosis by activating miR-411 to inhibit Fas expression and, therefore, provided possible strategies for bacterial infections in fish.

Микроэлементный состав крови русского осетра

Establishing quantitative levels of chemical elements in the blood plays a leading role in studying the mi-croelement status of the body. The purpose of the work was to study the microelement composition of the blood and spleen of the Russian sturgeon Acipenser gueldenstaedtii (Brandt, 1833). The objects of the study were females and males of Russian sturgeon of different ages. The determination of metals was carried out using atomic absorption spectrometry. In each sample, iron, copper, manganese, zinc, nickel, cobalt, chromium, lead, and cadmium were determined. Specific features of the accumulation of chemical elements in the blood and spleen of Russian sturgeon have been revealed. The content of iron and zinc in the blood and spleen of fish is in the greatest quantities. At the same time, the concentration of zinc in the blood of fish, as well as the concentration of iron, depends on the systematic position of the fish, and falls within the limits of the concentrations of chemical elements according to A. P. Vinogradov: the content of iron and zinc in the blood of Teleostei is generally higher than in the blood of Elasmobranchii. Russian sturgeon belongs to the subclass Chondrostei and has characteristics of cartilaginous and bony fish, perhaps this can explain the amount of iron and zinc found in the blood. A positive correlation was revealed between iron and copper in the blood of female and male Russian sturgeon. At the same time, no gender differences were found in the content of chemical elements in the blood of female and male Russian sturgeon, with the exception of nickel, the concentration of which is higher in the blood of males. It has been shown that as fish age, the concentration of nickel, manganese, lead and copper in the blood increases.

Metabolomics analysis of splenectomy of Nile tilapia (Oreochromis niloticus) reveals the spleen involved in regulating liver lipids and amino acids metabolism

The liver is essential for nutritional balance in fish, and liver damage in farmed fish often arises from factors like overfeeding, causing various health issues. Clinical observations indicate that liver diseases frequently involve spleen dysfunction, and there is evidence to suggest that the spleen has a significant impact on liver function. However, there has been no discussion on the role of the spleen in maintaining liver health in fish. To understand the role of fish spleen in liver metabolism, this study selected Nile tilapia (Oreochromis niloticus) as the experimental material and first established a tilapia splenectomy model. Various biochemical parameters of the liver and serum were measured, and the liver metabolism characteristics of the splenectomy group (SP group) and the sham operation group (SO group) were analyzed using metabolomics. After splenectomy, biochemical parameters of the liver and serum showed abnormalities, including significant increases in total cholesterol (T-CHO) and serum total bile acid (TBA) levels, alongside a significant decrease in liver TBA levels, suggesting impaired metabolic function and cholesterol deposition in the liver. Metabolomics analysis showed that metabolites such as lipids, lipid-like molecules, and organic acids and derivatives were differentially regulated between the SO and SP group. KEGG analysis showed that differential metabolites were enriched in lipid metabolism and amino acid metabolism. The metabolic pathway analysis of differential metabolites showed that after splenectomy, the low-activity urea cycle in the liver may accelerate lipid synthesis, while low concentrations of aromatic amino acids and taurine may inhibit lipid catabolism. These results indicate that after splenectomy, the liver metabolic capacity is impaired, which causes abnormal lipid metabolism by interfering with amino acid metabolism, making splenectomy tilapia liver at risk of liver disease, including cholesterol deposition, hepatic steatosis and nonalcoholic fatty liver disease (NAFLD). Our results show that the spleen is involved in regulating liver lipid and amino acid metabolism, and the spleen may interfere with lipid metabolism by regulating liver amino acid metabolism. Our data can provide support for further research on liver and spleen functions and the immune-nutrient metabolism mechanisms in fish, as well as new ideas for healthy fish farming.

Autophagy, apoptosis, and inflammation response of Sebastiscus marmoratus to different concentrations of hydroxychloroquine

Hydroxychloroquine (HCQ) is an important public health therapeutic agent widely used in the prevention and treatment of malaria and autoimmune diseases, with some antiviral effects, as well as a common autophagy inhibitor. Its autophagy-inhibiting effect attracts great research interest in mammals but is still little studied in fish. We even have no idea about the effects of HCQ on different tissues of fish and what concentrations should be used for safety studies. This study investigated the effect of different concentration of HCQ treatments on the survival, tissue structure, and expressions of genes related to autophagy, apoptosis, and inflammation in the gill, spleen, testis, and ovary of Sebastiscus marmoratus. The results showed that the higher HCQ concentration (77.40 and 154.80 mg/mL) led to mass mortality within an hour. The half-lethal concentration (LC50, 24 h) of HCQ for S. marmoratus was approximately 48.95 mg/mL. When exposed to 30.96 mg/mL HCQ for 24 hours, autophagy was blocked as revealed by electron microscopy from gill, spleen and testis of fish. The expressions of autophagy-related genes (LC3/Cx43), apoptosis-related genes (Cas3/p53), and inflammation-related genes (TNF-α/IL8) exhibited tissue-specific and dose-dependent responses. The gonads showed preferential expression of all these genes and were found to be sensitive and regular after HCQ treatment. For example, at a concentration of 30.96 mg/mL, the testis demonstrated a regularity that suggests it is an ideal candidate tissue for studying the role of HCQ or autophagy. This study systematically revealed the response of S. marmoratus to different concentrations of HCQ and provided optional assay concentrations for key tissues, serving as an important reference for the future studies on HCQ and autophagy in S. marmoratus. Furthermore, the potential crosstalk between autophagy, apoptosis, and inflammatory pathways initially identified in this study could be helpful for the future research on autophagy regulation in marine fish.

Isolation and complete genome sequence analysis of Vibrio rotiferianus strain TO-01 from Trachinotus ovatus to reveal its pathogenicity and drug resistance

Vibrio rotiferianus is a pathogenic bacterium that is widely distributed in the ocean and can cause diseases in a variety of marine animals, including fish, shrimp and shellfish. In this study, we isolated and identified V. rotiferianus strain TO-01 from diseased Trachinotus ovatus. Infected fish exhibited a range of pathological symptoms, including lethargy in swimming, blackened body coloration, skin ulceration and erratic side swimming. Tissue section examination revealed pronounced damage to the liver, spleen and gills of the infected fish. An analysis of immune factor expression revealed a significant increase in the expression of inflammatory factors, including interleukin (IL)-1, IL-2, IL-6 receptor and IL-21, in the spleen and kidney of fish infected with TO-01 at 6 h post-infection. To gain a more in-depth comprehension of the pathogenicity and drug resistance of V. rotiferianus, we conducted whole genome sequencing. The total genome length of V. rotiferianus was 4734,075 bp, and 74 virulence factors were predicted within the genome. Most of these virulence genes were associated with bacterial adhesion and flagellar motility. A gene associated with group sensing, cqsA, and a gene associated with hemolysin, tlh, were also identified. The prediction of drug resistance genes was in agreement with the results of drug sensitivity assays, both of which indicated that TO-01 has significant resistance to β-lactams. The findings of this study significantly enhance our understanding of the pathogenic mechanism of V. rotiferianus and give crucial insights into disease prevention and control in the management of T. ovatus.

Lipidomics and transcriptomics analysis revealed the role of the spleen of Nile tilapia (Oreochromis niloticus) in lipid metabolism

In fish, the development of the spleen is not as perfect as that of higher vertebrates, but it has the same functions that the spleen should have. Up to date, research on fish spleen is mostly focused on its immune function, and the roles and mechanisms of spleen in the regulation of fish metabolism are still unclear. For this purpose, we established a spleen-deficient model of Nile tilapia (Oreochromis niloticus) by splenectomy and integrated lipidomics and transcriptomics to study the changes and mechanisms of lipid metabolism in splenectomy tilapia so as to understand the effect of spleen on lipid metabolism in tilapia. We found that splenectomy can lead to lipid deposition in the liver of tilapia, significant changes in liver and serum lipid mass spectrometry, abnormal lipid metabolism, decreased anti-oxidative stress ability, and being more prone to lipid toxicity and fatty liver. Combined with the results of transcriptomics, we found that splenectomy resulted in down-regulation of differentially expressed genes in “cellular iron ion homeostasis,” “extracellular region,” “small molecule binding,” and “TGF-beta signaling pathway,” and iron metabolism-related genes such as tfa, bmp2b, bmp6, hamp, and slc40a1, etc. were closely related to differential lipid changes. Therefore, it is speculated that the abnormal lipid metabolism in tilapia after splenectomy is closely related to iron metabolism, and its molecular mechanism may be related to the hepcidin regulatory pathway—BMP-SMAD signaling pathway. This study can provide a new understanding of the relationship and mechanism between immune and nutritional metabolism in fish and also provide new ideas for the study of fish hepatic diseases.

Enrofloxacin, Effective Treatment of Pseudomonas aeruginosa and Enterococcus faecalis Infection in Oreochromis niloticus.

Enrofloxacin is a broad-spectrum synthetic antimicrobial drug widely used in veterinary medicine. The present study aimed to determine the effective enrofloxacin dose for treating Pseudomonas aeruginosa and Enterococcus faecalis infection in Oreochromis niloticus. P. aeruginosa and E. faecalis isolates were verified using selective differential media and biochemically using the Vitek 2 test. Bacterial isolates were virulent for O. niloticus with LD50 equal to 2.03 × 106 and 2.22 × 107 CFU fish-1 for P. aeruginosa and E. faecalis, respectively. Infected fish suffered from decreased feed intake followed by off-food, tail erosion, darkening of the external body surface, exophthalmia, ascites, and loss of escape reflex. Internally, congested hemorrhagic hepatopancreas with engorged distended gall bladder were dominant. The posterior kidney was congested with enlarged spleen, and empty elementary tract. Pathologically, severe degenerative changes were dominant in the hepatopancreas, posterior kidney, spleen, stomach, and gills of infected fish. Antimicrobial sensitivity test indicated the high susceptibility of P. aeruginosa and E. faecalis to enrofloxacin with MIC estimated at 1 and 0.0625 µg/mL, respectively. Enrofloxacin effectively protected O. niloticus against E. faecalis and P. aeruginosa infection when used with medicated feed at doses of 10 and 20 mg kg-1 body weight.

Ferroptosis and iron mineralization involved in the death and survival of orange‐spotted groupers challenged with Pseudomonas plecoglossicida

AbstractPseudomonas plecoglossicida (P. plecoglossicida) is a pathogen in aquaculture that causes considerable economic loss. According to artificial infection experiments, the fish were classified into control group, moribund group, and survival group. Compared to the control group, both the moribund group and the survival group of fish had fewer red blood cells (RBCs) and lower oxygen saturation (SaO2). Furthermore, the fish in the survival group has more RBCs and SaO2 compared to the moribund group. The concentrations of total iron, ferrous iron, ferric iron, and mineralized iron in the fish spleen of the moribund and survival groups were lower compared to those of the control group. Additionally, the concentrations of these iron components in the fish spleen of the survival group were higher than those of the moribund group. The results demonstrated that iron mineralization is involved in the survival of fish challenged with P. plecoglossicida. Compared to the control and survival groups, the fish spleen had several distinguishing features in the moribund group, including less reduced glutathione (GSH), higher mitochondrial complex V activity, more lipid peroxidation, and reactive oxygen species, as well as reduced glutathione peroxidase 4 (gpx4) expression. Moreover, there were intact cell membranes, a normal nucleus size, no chromatin concentration, and disappearance of cristae in the mitochondria of the spleens of the moribund group. The characteristics of spleen cells in the moribund group were consistent with ferroptosis, suggesting that ferroptosis was involved in the death of fish challenged with P. plecoglossicida.

Acanthopanax senticosus cultures fermented by Lactobacillus rhamnosus enhanced immune response through improvement of antioxidant activity and inflammation in crucian carp (Carassius auratus)

Lactic acid bacteria (LAB) have been recognized as safe microorganism that improve micro-flora disturbances and enhance immune response. A well-know traditional herbal medicine, Acanthopanax senticosus (As) was extensively utilized in aquaculture to improve growth performance and disease resistance. Particularly, the septicemia, skin wound and gastroenteritis caused by Aeromonas hydrophila threaten the health of aquatic animals and human. However, the effects of probiotic fermented with A. senticosus product on the immune regulation and pathogen prevention in fish remain unclear. Here, the aim of the present study was to elucidate whether the A. senticosus fermentation by Lactobacillus rhamnosus improve immune barrier function. The crucian carp were fed with basal diet supplemented with L. rhamnosus fermented A. senticosus cultures at 2 %, 4 %, 6 % and 8 % bacterial inoculum for 8 weeks. After trials, the weight gain rate (WGR), specific growth rate (SGR) were significantly increased, especially in LGG-6 group. The results confirmed that the level of the CAT, GSH-PX, SOD, lysozyme, and MDA was enhanced in fish received with probiotic fermented product. Moreover, the L. rhamnosus fermented A. senticosus cultures could trigger innate and adaptive immunity, including the up-regulation of the C3, C4, and IgM concentration. The results of qRT-PCR revealed that stronger mRNA transcription of IL-1β, IL-10, IFN-γ, TNF-α, and MyD88 genes in the liver, spleen, kidney, intestine and gills tissues of fish treated with probiotic fermented with A. senticosus product. After infected with A. hydrophila, the survival rate of the LGG-2 (40 %), LGG-4 (50 %), LGG-6 (60 %), LGG-8 (50 %) groups was higher than the control group. Meanwhile, the pathological damage of the liver, spleen, head-kidney, and intestine tissues of probiotic fermentation-fed fish could be alleviated after pathogen infection. Therefore, the present work indicated that L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy to protecting fish from pathogenic bacteria infection.

Effects of Acipenserid herpesvirus 2 on the outcome of a Streptococcus iniae co-infection in white sturgeon (Acipenser transmontanus)

Acipenserid herpesvirus 2 (AciHV-2) is a large double-stranded DNA virus in the family Alloherpesviridae that causes catastrophic outbreaks in young naive white sturgeon (Acipenser transmontanus) populations, with mortalities of up to 80%. Survivors of these infections are suspected to remain latently infected. The gram-positive zoonotic bacterium Streptococcus iniae is another important sturgeon pathogen that causes severe myositis and up to 50% mortality during natural outbreaks. Throughout the last decade, co-infections of AciHV-2 and S. iniae have been reported in cultured white sturgeon in California resulting in severe presentations of piscine streptococcosis. This phenomenon of herpesvirus and streptococcus co-infection appears to span multiple taxa since in humans, it is recognized that a Human herpesvirus 3 infection (VZV) is a negative prognostic indicator for pediatric Invasive Group A Streptococcal infections (IGASI). While a decrease in humoral immunity caused by VZV has been hypothesized as a potentially important factor in IGASI cases, no natural animal model exists to study this process. Moreover, no studies have investigated these reported co-infections in white sturgeon. Therefore, the goal of this study was to investigate the effects of a recent AciHV-2 infection on the outcome of a subsequent S. iniae challenge in white sturgeon fingerlings. When fish were infected with 108 colony forming units (CFU) of S. iniae intramuscularly (IM), a statistically significant decrease in survival of 41% was detected in the co-infection group compared to the S. iniae group (p-value &amp;lt; 0.001). This difference was not observed when fish were infected with 106 CFU of S. iniae IM. At this lower infection dose, however, a statistically significant downregulation of tnfα was observed in the spleen of fish in the co-infection group compared to the S. iniae group (p-value = 0.0098). Analysis of serum from survivors revealed a statistically significant reduction in anti-S. iniae serum IgM and serum serotransferrin in fish from the co-infection group compared to the S. iniae group (p-value = 0.0134 and p-value = 0.0183, respectively). Further studies are indicated to determine what interactions lead to the decreased production of pathogen-specific IgM, serotransferrin, and TNFα in the host.

Ecotoxicological responses of juvenile Sparus aurata to BDE-99 and BPA exposure: A multi-biomarker approach integrating immune, endocrine and oxidative endpoints

Pentabromodiphenyl ether (BDE-99) and bisphenol A (BPA) are synthetic organic compounds present in several daily use products. Due to their physicochemical properties, they are ubiquitously present in aquatic ecosystems and considered highly persistent. Recent evidence has confirmed that both emerging compounds are toxic to humans and terrestrial mammals eliciting a wide range of detrimental effects at endocrine and immune levels. However, the ecotoxicological responses that they can trigger in vertebrate marine species have not yet been established. Hence, this study aimed to investigate the ecotoxicological responses of juvenile Sparus aurata upon chronic (28 days) dietary exposure to BDE-99 and BPA (alone and combined) following an integrated multi-biomarker approach that combined fitness indicators (Fulton's K and splenosomatic indexes) with endocrine [cortisol, 17β-estradiol (E2), 11-ketotestosterone (11-KT) concentrations] and immune (peroxidase and antiprotease activities) endpoints in fish plasma, and oxidative stress [superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) activities, and lipid peroxidation (LPO)] endpoints in the fish spleen. The mixture of BDE-99 and BPA yielded the highest IBR index value in both plasma and spleen biomarkers, therefore, suggesting that the effects of these compounds are more severe when they act together. Endocrine biomarkers were the most responsive in the three contaminated treatments. Fitness indicators were not affected by the individual nor the interactive effects of BDE-99 and BPA. These findings highlight the relevance of accounting for the interactive effects of emerging chemical contaminants and integrating responses associated with distinct biological pathways when investigating their impacts on marine life, as such a multi-biomarker approach provides a broader, more realistic and adequate perspective of challenges faced by fish in a contaminated environment.

Evaluation of immunogenicity and protective efficacy of the outer membrane porin F (OprF) against Pseudomonas plecoglossicida in large yellow croaker (Larimichthys crocea)

Large yellow croaker (Larimichthys crocea) farming dominates the marine aquaculture industry in China. However, the epidemic outbreaks of visceral white nodules disease (VWND), caused by bacterial pathogen Pseudomonas plecoglossicida, have emerged as a significant concern within the large yellow croaker industry. Although vaccination is considered to be an effective method for preventing and controlling P. plecoglossicida infection, there is currently no commercially available vaccine targeting this bacterium. In the present study, the outer membrane porin F (OprF) of P. plecoglossicida was characterized and revealed a high sequence similarity with that of other Pseudomonas species. The recombinant OprF protein (rOprF) produced in Escherichia coli was then evaluated for its immunogenicity and protective role against P. plecoglossicida in large yellow croaker. The rOprF was identified to have immunogenicity by Western blot using large yellow croaker anti-P. plecoglossicida sera. Additionally, the indirect immunofluorescence assay (IIFA) provided evidence indicating the surface exposure of OprF in P. plecoglossicida. Fish vaccinated twice via intraperitoneal (IP) injection with the purified rOprF combined with commercial adjuvant ISA 763A VG exhibited a relative percent survival (RPS) of 70.60% after challenge with virulent P. plecoglossicida strain through immersion. The administration of rOprF resulted in a notable increase in specific serum antibody levels and serum lysozyme activity compared to the control groups. The immune-related genes in the spleen and head kidney of rOprF-vaccinated fish were remarkably upregulated compared with the PBS-vaccinated sham group after the P. plecoglossicida challenge. In summary, the findings of this study suggest that rOprF exhibits considerable potential in inducing a robust immune response, making it a viable candidate for vaccination against P. plecoglossicida infection in large yellow croaker.

Role of the Pseudomonas plecoglossicida fliL gene in immune response of infected hybrid groupers (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).

Pseudomonas plecoglossicida, a gram-negative bacterium, is the main pathogen of visceral white-point disease in marine fish, responsible for substantial economic losses in the aquaculture industry. The FliL protein, involved in torque production of the bacterial flagella motor, is essential for the pathogenicity of a variety of bacteria. In the current study, the fliL gene deletion strain (ΔfliL), fliL gene complement strain (C-ΔfliL), and wild-type strain (NZBD9) were compared to explore the influence of the fliL gene on P. plecoglossicida pathogenicity and its role in host immune response. Results showed that fliL gene deletion increased the survival rate (50%) and reduced white spot disease progression in the hybrid groupers. Moreover, compared to the NZBD9 strain, the ΔfliL strain was consistently associated with lower bacterial loads in the grouper spleen, head kidney, liver, and intestine, coupled with reduced tissue damage. Transcriptomic analysis identified 2 238 differentially expressed genes (DEGs) in the spleens of fish infected with the ΔfliL strain compared to the NZBD9 strain. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the DEGs were significantly enriched in seven immune system-associated pathways and three signaling molecule and interaction pathways. Upon infection with the ΔfliL strain, the toll-like receptor (TLR) signaling pathway was activated in the hybrid groupers, leading to the activation of transcription factors (NF-κB and AP1) and cytokines. The expression levels of proinflammatory cytokine-related genes IL-1β, IL-12B, and IL-6 and chemokine-related genes CXCL9, CXCL10, and CCL4 were significantly up-regulated. In conclusion, the fliL gene markedly influenced the pathogenicity of P. plecoglossicida infection in the hybrid groupers. Notably, deletion of fliL gene in P. plecoglossicida induced a robust immune response in the groupers, promoting defense against and elimination of pathogens via an inflammatory response involving multiple cytokines.

Vibrio cholerae O1 Inhabit Intestines and Spleens of Fish in Aquaculture Ponds.

Vibrio cholerae is the causative agent of cholera, an acute diarrheal disease that spreads locally and globally in epidemics and pandemics. Although it was discovered that fish harbor V. cholerae strains in their intestines, most investigations revealed non-toxic V. cholerae serogroups in fish. Due to the rarity of toxigenic V. cholerae serogroups, it is difficult to cultivate these strains from environmental samples. Hence, here we aimed to uncover evidence of the occurrence of toxigenic V. cholerae in the intestines and spleens of various fish species. By using molecular detection tools, we show that V. cholerae O1 and strains positive for the cholera toxin inhabit both healthy and diseased fish intestines and spleens, suggesting that fish may serve as intermediate vectors of toxigenic V. cholerae. No significant differences were found between the abundance of toxigenic V. cholerae (either O1 or cholera toxin positive strains) in the healthy and the diseased fish intestines or spleens. In conclusion, a variety of fish species may serve as potential vectors and reservoirs of toxigenic V. cholerae as they form a link between the other reservoirs of V. cholerae (chironomids, copepods, and waterbirds). Similarly, they may aid in the spread of this bacterium between water bodies.