Fish Immunity Research Articles

Neutrophils and aquatic pathogens.

IntroductionNeutrophilic granulocytes are short‐lived cells continuously circulating in the vascular system of vertebrates. They play a basic and decisive role in the innate immune defence of the hosts against all types of pathogenic microorganisms.MethodsBased on a literature study, the functions of neutrophils and cells with similar functions are described. The study places special emphasis on organisms in the aquatic environment and the pathogens occurring in that particular environment.ResultsThe evolutionary origin of this specific cell type is not clear, but its most basic traits (recognition of foreign elements, extracellular trap release, phagocytosis and elimination of ingested material) are found in phagocytes in members of evolutionary ancient invertebrate groups spanning from amoebae, sponges, sea‐anemones, mollusks (snails and mussels), arthropods (crustaceans and insects) to echinoderms (sea stars and sea urchins). Their functions as innate immune sentinels and effector cells in these groups are well described. Neutrophilic granulocytes with elongated and lobed nuclei (possibly allowing cell movements through narrow extracellular spaces and leaving space for phagosomes) occur in vertebrates including fish, amphibians, reptiles, birds and mammals although the morphology of the nucleus, stainability of cytoplasmic granula, and the antimicrobial armament vary among groups. Following the pathogen invasion of a fish host, the neutrophils migrates from the vascular system into the infection focus. They apply their PRRs (including TLRs) to recognize the invader as non‐self, produce netosis by casting extracellular chromatin containing traps in the microenvironment. These nets assist the immobilization of invading microbes and prevents their further spread. The cells attach to and engulf the microbes by phagocytosis, whereafter they eliminate the pathogen in phagolysosomes equipped with a range of killing mechanisms and attract, by release of chemokines, additional immune cells (monocytes, macrophages and lymphocytes) to the site of invasion. Their role in innate immunity of fish hosts towards aquatic pathogens has been elucidated by in vivo and in vitro studies. Neutrophils interact with virus (e.g. IPNV and VHSV), bacteria (e.g. Aeromonas, Vibrio, Edwardsiella, Mycobacterium and Renibacterium) and parasites, including monogeneans (Gyrodactylus), cestodes (Diphyllobothrium), trematodes (Diplostomum) and ciliates (Ichthyophthirius and Philasterides). Despite the decisive function of neutrophils in innate immunity and early protection, the excessive production of ROS, RNS and NETs may lead to pathological disturbances in the host, which are exacerbated if the pathogens evolve immune evasion mechanisms.ConclusionNeutrophils in aquatic organisms play a central role in innate immunity but may serve as a toll and a support in acquired protection. The strong impact of the cellular reactions not only on pathogen but also on host tissues emphasizes that an optimal immune reaction is balanced, involves targeted and specific effector mechanisms, which leaves a minimum of collateral damage in host organs.

Structural characteristics and mucosal immune response of the interbranchial lymphoid tissue in the gills of flounder (Paralichthys olivaceus)

A specialized lymphoepithelial tissue termed the interbranchial lymphoid tissue (ILT) is recently identified in several fish species. However, the structural variation and mucosal immune functions of the ILT remain largely unknown. In this study, the anti-Zap-70 MAb was firstly determined to specifically recognize ZAP-70 protein, and CD4-1+, CD4-2+ and CD8β+ T-cells, but not IgM+ B cells, in peripheral blood leucocytes of flounder (Paralichthys olivaceus). Then we found that aggregates of Zap-70+ cells were located in the epithelium covering the bottom of the interbranchial cleft and along the afferent and efferent edges of the filaments in a cross view, where a meshwork of epithelial cells containing diffused lymphoid cells was exhibited, confirming these structures as the ILT; In a sagittal view, Zap-70+ cells were situated at the base of the filaments (here named as proximal ILT, pILT) and in the interlamellar epithelium (named as distal ILT, dILT). Also, a few IgM+ B cells were distributed at these sites. The lymphoepithelium within pILT and dILT was very thin with a low number of Zap-70+ cells in premetamorphosis and postclimax larvae of flounder, and got thicker containing much more Zap-70+ cells in juvenile and adult individuals. The aggregates of CD4-1+/Zap-70+, CD4-2+/Zap-70+, and CD8β+/Zap-70+ T-cell subsets were identified in the ILT. Post bath vaccination with inactivated Edwardsiella tarda and then intraperitoneal injection of EdU, the amounts of EdU+ and Zap-70+ cells obviously increased at 3 d and 7 d, and co-localization of EdU+/Zap-70+ cells identified the presence of proliferative T cells; meanwhile, MHC class II-expressing cells were increased. These findings indicated that the ILT in gills of flounder was an important site for the induction of local T cell-mediated immunity, which would lead to a better understanding of mucosal immunity and defense mechanisms of teleost fish.

Immunoproteomic identification of OmpA with potential stimulation of serum‐specific antibody in Nile tilapia ( Oreochromis niloticus ) and its ability to protect against Edwardsiella ictaluri infection

Edwardsiella ictaluri is a Gram-negative bacterium that has been reported to be disease causing in several economic fish including Nile tilapia. Here, immunoproteomic identification found that outer membrane protein A (OmpA) could react with serum from fish naturally infected with E. ictaluri. The expression and production of recombinant OmpA protein could be successfully conducted in Escherichia coli strain BL21(DE3) with the molecular mass of 38 kDa after inclusion bodies were successfully solubilized and refolded. Immunization of fish with the recombinant OmpA protein showed that the recombinant proteins could induce the production of specific antibodies in serum of immunized fish (p < 0.05) at 1-week postimmunization (7 dpi) and reached the highest antibody titre at 3 weeks postimmunization (21 dpi). The serum-specific antibody from fish immunized with the recombinant OmpA exhibited bacterial agglutination property. Furthermore, the partial protection of Nile tilapia was observed in OmpA immunized fish with the relative per cent survival (RPS) of 39.81%. In summary, this study explored the immunoproteomic method as a starting point to investigate OmpA of E. ictaluri and might be considered as a candidate protein for recombinant vaccine development in Nile tilapia against this bacterial infection.

The use of the probiotic «Subtilis-C» in the breeding of the Nile tilapia (Oreochromis niloticus)

With intensive cultivation technologies, fish are exposed to stress factors associated with intensive breeding. They reduce the immunity of cultivated objects. In addition, the aquatic environment is a good substrate for the development of microorganisms, including pathogenic ones, which aggravates the situation. Therefore, increasing the immunity of fish is an urgent task of their breeding. One of the ways to enhance the immune resistance of the body is the use of immunomodulators. The purpose of this work was to study the effect of the probiotic “Subtilis-S” on the productivity and hematological status of fish. At the beginning of the experiment, the tilapia females (7 heads per aquarium) were separated from the males. Males were kept individually. The fish of the experimental groups received the probiotic “Subtilis-S” as a feed additive for two months at the rate of 1 g/kg of feed. A month after the start of the experiment, the males were hooked up to the females (one male in each aquarium). After gestation, the larva was taken from the females and planted in separate containers. During the experiment, fertilization of eggs occurred in 80% of females in the experimental group and 50% in the control. The results of fish appraisal showed that the probiotic “Subtilis-S” had no effect on the growth and mass accumulation of tilapia: no significant differences in the main zootechnical parameters were found in the fish of the experimental and control groups. In the leukogram of tilapia treated with “Subtilis-S”, the proportion of lymphocytes increased due to a decrease in immature forms of the myeloid series; for rod-shaped neutrophils, the differences are significant. Which probably indicates the activation of a specific link of the immune defense. In the blood of experienced tilapia, in contrast to the control, there is a small percentage of eosinophils and basophils, that is, there is an increase in the nonspecific link of cellular immunity. The use of the probiotic “Subtilis-S” as a feed additive enhances the immunity of tilapia and increases the survival of fish in aquaculture, as well as increases their fertility.

Oral Vaccination With Recombinant Pichia pastoris Expressing Iridovirus Major Capsid Protein Elicits Protective Immunity in Largemouth Bass (Micropterus salmoides).

Largemouth bass iridovirus (LMBV) can cause high mortality and lead to heavy economic loss in the cultivation of largemouth bass, but there was no effective treatment. Here, the present study constructed a recombinant Pichia pastoris expressing LMBV major capsid protein (MCPD). The recombinant GS115-pW317-MCPD was then used to immunize largemouth bass via oral administration, and mucosal immune response mediated by immunoglobulins (Igs) was measured after oral immunization. Serum antibody levels were measured by ELISA, neutralizing antibody titers were determined by serum neutralization test (SNT), antigen presentation-related gene expressions were detected by RT-PCR, and the histopathological characteristics of immunized fish were assessed after challenging with 0.1 ml 107.19 TCID50/ml LMBV. The relative percentage survival (RPS) was also determined. Our results showed that the serum antibody titers of immunized fish were significantly higher than that of control groups (P < 0.05). IgT and IgM expressions in gut were increased significantly after vaccination with GS115-pW317-MCPD; however, much stronger response in gut was observed as compared with gill. The expression levels of major histocompatibility complex (MHC) II, CD8, and T-cell receptor (TCR) were significantly elevated in GS115-pW317-MCPD group (P < 0.05), while CD4 and MHC I transcription levels remained unchanged after oral immunization (P > 0.05). The RPS of fish orally immunized with 1.0 × 108 CFU/g GS115-pW317-MCPD was reached up to 41.6% after challenge with 0.1 ml 109.46 TCID50/ml LMBV. Moreover, orally immunizing with GS115-pW317-MCPD can relieve the pathological damage caused by LMBV. Therefore, GS115-pW317-MCPD showed a promising potential against LMBV.

Exon-Intron Circular RNA circRNF217 Promotes Innate Immunity and Antibacterial Activity in Teleost Fish by Reducing miR-130-3p Function.

Circular RNA (circRNA) is produced by splicing head to tail and is widely distributed in multicellular organisms, and circRNA reportedly can participate in various cell biological processes. In this study, we discovered a novel exon-intron circRNA derived from probable E3 ubiquitin-protein ligase RNF217 (RNF217) gene, namely, circRNF217, which was related to the antibacterial responses in teleost fish. Results indicated that circRNF217 played essential roles in host antibacterial immunity and inhibited the Vibrio anguillarum invasion into cells. Our study also found a microRNA miR-130-3p, which could inhibit antibacterial immune response and promote V. anguillarum invasion into cells by targeting NOD1. Moreover, we also found that the antibacterial effect inhibited by miR-130-3p could be reversed with circRNF217. In mechanism, our data revealed that circRNF217 was a competing endogenous RNA of NOD1 by sponging miR-130-3p, leading to activation of the NF-κB pathway and then enhancing the innate antibacterial responses. In addition, we also found that circRNF217 can promote the antiviral response caused by Siniperca chuatsi rhabdovirus through targeting NOD1. Our study provides new insights for understanding the impact of circRNA on host-pathogen interactions and formulating fish disease prevention to resist the severely harmful V. anguillarum infection.

Molecular cloning, expression analysis of the IgT gene and detection of IgT+ B cells in the half-smooth tongue sole (Cynoglossus semilaevis)

IgT is a specific Ig isotype in teleosts, which plays extremely important roles in the mucosal immunity of fish. In the present study, the membrane-bound and secretory IgT of the half-smooth tongue sole (Cynoglossus semilaevis) were identified for the first time. The V-D-J-C structure of two forms of csIgT are translated by the same Cτ gene, and the secretory tail and transmembrane domain were encoded through alternative splicing at the 3′ end of the Cτ4. The CH regions of csIgT had high similarity with that of other flatfish (P. olivaceus and S. maximus). In healthy C. semilaevis, sIgT and mIgT were mainly expressed in mucus related tissues such as skin, intestine and gill. The transcript levels of sIgT and mIgT mRNA showed a significant induction in the immune-related tissues upon Vibrio Harveyi infection. A polyclonal rabbit anti-csIgT was successfully prepared using the csIgT heavy chain recombinant protein. Using this antibody, we detected the native IgT with the molecular mass at 220 kDa in skin total protein under non-reducing SDS-PAGE condition. Immunofluorescence analysis indicated that IgT+ B lymphocytes were intensively located in the skin, gill, intestine, and head kidney of C. semilaevis. These results suggest that IgT may participate in the immune response of C. semilaevis, which will facilitate the investigations of the immunoglobulins of marine fish.

The mechanism of immune related signal pathway Egr2-FasL-Fas in transcription regulation and methylated modification of Paralichthys olivaceus under acute hypoxia stress

Apoptosis genes Egr2, Fas and FasL are related to immune responses. However, the mechanism of these genes inducing apoptosis in fish are still not very clear. An acute hypoxia treatment (1.73 ± 0.06 mg/L) for 24 h was carried out on Japanese flounder (Paralichthys olivaceus). The increasingly dense apoptotic signals at 3 h, 6 h, 12 h by TUNEL in skeletal muscle indicated that hypoxia could quickly affect muscle growth and development. Furthermore, we concluded that the Egr2-FasL-Fas signal pathway, which was located at the upstream of apoptotic executor protein caspases, was related to the apoptosis by quantitative real-time PCR, protein concentration detection in ELISA and double gene in situ hybridization methods. The mechanism of the pathway was researched in transcription regulation and epigenetic modification by dual-luciferase reporter assay and bisulfite modified method, respectively. Egr2, as a transcription factor, could up-regulate the expression of FasL gene. And its binding site was mainly between −479 to −1 of FasL gene promoter. The 5th CpG dinucleotides (−514) methylation levels in FasL gene were significantly affected by hypoxia, and they were negatively correlated with its expressions. These suggested that the −514 site may be a very important site to regulate the FasL gene expression. Above results, we concluded that hypoxia activated the immune related signal pathway Egr2-FasL-Fas to induced skeletal muscle apoptosis to affect growth and development of Japanese flounder. The study revealed the mechanism of hypoxia induced apoptosis, which could provide a reference for fish immunity and aquaculture management.

The effects of blunt snout bream (Megalobrama amblycephala) IL-6 trans-signaling on immunity and iron metabolism via JAK/STAT3 pathway

Interleukin-6 (IL-6) is a pleiotropic inflammatory cytokine, which plays a dual role in mammalian inflammation through both classical signaling (IL-6 binds to IL-6 receptor/IL-6R) and trans-signaling (IL-6 binds to soluble IL-6R). However, the function of IL-6, especially the regulatory mechanism of IL-6 trans-signaling in immunity and iron metabolism remains largely unclear in teleost. Here, L8824 cells (Ctenopharyngodon idella hepatic cells) were stimulated with blunt snout bream (Megalobrama amblycephala) IL-6 combination with sIL-6R protein (rmaIL-6+rmasIL-6R/maIL-6 trans-signaling) or STAT3 inhibitor (c188-9), and RNA-sequencing, global transcriptional analyses. The enrichment analysis of GO and KEGG showed that maIL-6 trans-signaling is mainly involved in stress and inflammation response, and the activation of STAT3 is mainly related to cell proliferation, apoptosis and immune regulation. Furthermore, after treated L8824 cells with JAK2 inhibitors, it was found that the induction of IL-6 trans-signaling on the selected immune-related genes could be inhibited. These results implied that in early stage after rmaIL-6+rmasIL-6R treatment, the maIL-6 trans-signaling played an important role in the immune regulation through the JAK2/STAT3 pathway. By extending the rmaIL-6+rmasIL-6R treatment time, it was found that maIL-6 trans-signaling could promote the expression of iron metabolism related genes (ft, tf, tfr1, hamp and fpn1) in L8824 cells, indicating that maIL-6 trans-signaling may be involved in iron metabolism in the non-acute immune phase. Finally, after treated L8824 cells with JAK2 and STAT3 inhibitors, it was found that only tf and fpn1 were regulated by maIL-6 trans-signaling through the JAK2/STAT3 pathway. These findings provided novel insights into IL-6 trans-signaling regulatory mechanism in teleost, enriching our knowledge of fish immunity and iron metabolism.

Immune gene expression and protective effects in goldfish (Carassius auratus L.) immunized with formalin-inactivated cyprinid herpesvirus-2 (CyHV-2) vaccine

The goldfish hematopoietic necrosis viral disease (GHNVD) has led to worldwide economic losses in goldfish aquaculture. The present study has focused on the development of an inactivated vaccine for the cyprinid herpesvirus (CyHV-2) and to check the immunogenicity of the vaccine in the host. The fantail goldfish fin (FtGF) cell line was used in the propagation of the CyHV-2 and the viral titer obtained were of 107.8 TCID50/ml. Followed by the virus was inactivated using 0.1% formalin for 2 days. Various concentrations of formalin-inactivated CyHV-2 (1%, 0.7%, 0.5%, 0.3% and 0.1%) were studied in the FtGF cell line. Morphological changes were observed in the FtGF cell line in all other concentrations of formalin except 0.1% formalin-inactivated CyHV-2 vaccine. The goldfishes were intraperitoneally injected with 300 μl of vaccine and various immune gene responses were studied for a period of 30 days. The gene expression of the adaptive markers CD8, CD4, IFN-ϒ, the cytokines (IL-10, IL-12) was studied in kidney and spleen tissues. Formalin-inactivated CyHV-2 vaccine showed a significant up-regulation of the genes CD8 and IFN-ϒ by the 6th hr post-vaccination onwards. The experimental fish were challenged intraperitoneally with CyHV-2 virus of concentration 107.8 TCID50/ml after 30 days of post-vaccination. A significant difference in cumulative mortality rate was observed for the vaccinated fishes from the unvaccinated fishes. The relative percent survival for formalin immunized fish was 74.03%. Our results have proven that the formalin-inactivated vaccines were efficient and it resulted in triggering the immune gene expression in goldfish. The development and further enhanced studies for this vaccine will lead to a promising low-cost commercial vaccine for CyHV-2 viral infection.

Atlantic Salmon (Salmo salar) bacterial and viral innate immune responses are not impaired by florfenicol or tetracycline administration

Antibiotics are used to treat bacterial infections in fish aquaculture, and these drugs can interact with immune cells/the immune system and potentially leave fish vulnerable to viral, fungal, parasitic, or other bacterial infections. However, the effects of antibiotics on fish immunity have largely been overlooked by the aquaculture industry. We tested, at 12 and 20 °C, whether tetracycline and florfenicol (the most commonly used antibiotics in commercial aquaculture), affected the Atlantic salmon's capacity to respond to bacterial or viral stimulation. Atlantic salmon were acclimated to 12 or 20 °C and fed with tetracycline or florfenicol (100 and 10 mg kg of body weight−1 day−1, respectively) medicated feed for 15 or 10 days, respectively. Thereafter, we evaluated their immune function prior to, and after, an intraperitoneal injection of Forte Micro (containing inactivated cultures of Aeromonas salmonicida, Vibrio anguillarum, Vibrio ordalii and Vibrio salmonicida) or the viral mimic polyriboinosinic polyribocytidylic acid (pIC). We measured the transcript expression levels of 8 anti-bacterial and 8 anti-viral putative biomarker genes, and the innate (leukocyte respiratory burst, plasma lysozyme activity and hemolytic activity of the alternative complement pathway) and cellular (relative number of erythrocytes, lymphocytes and thrombocytes, and granulocytes such as monocytes and neutrophils) responses to these challenges. Overall, we only found a few minor effects of either tetracycline or florfenicol on immune gene expression or function at either temperature. Although several studies have reported that antibiotics may negatively affect fish immune responses, our results show that industry-relevant dietary tetracycline and florfenicol treatments do not substantially impact the salmon's innate immune responses. Currently, this is the most comprehensive study on the effects of antibiotics administrated according to industry protocols on immune function in Atlantic salmon.

Effects of pre-treatment with waterborne selenium on redox homeostasis and humoral innate immune parameters in African catfish, Clarias gariepinus (Burchell, 1822), experimentally challenged with Serratia marcescens.

Bacterial infections have been associated with immune dysfunction and oxidative stress in cultured fish species while essential elements could boost immunity and exhibit antioxidant properties in fish. This study was therefore aimed at determining the effects of pre-treatment with waterborne selenium on humoral immunity and redox status of Clarias gariepinus experimentally challenged with Serratia marcescens. Juveniles C. gariepinus were pre-treated with 50µg/L selenium for 14days after which they were challenged with 5 × 103CFU/mL of S. marcescens via oral gavage for 24 or 48h. The control fish were not pre-treated with selenium and not challenged with bacteria. Thereafter, fish were sacrificed, blood collected into EDTA bottles for the determination of plasma nitric oxide levels and respiratory burst, and the liver excised for the determination of reduced glutathione, lipid peroxidation, and activities of catalase, superoxide dismutase, and glutathione peroxidase. Fish that were pre-treated with selenium prior to bacterial challenge (Sel + Bact) had decreased levels of nitric oxide and lipid peroxidation but a significant increase in the levels of reduced glutathione (at 48-h post-infection period only) compared to the fish challenged with bacteria without prior selenium pre-treatment (Bact). The respiratory burst and catalase activity decreased significantly in the Sel + Bact group especially at 48-h post-infection period while the activity of glutathione peroxidase increased significantly in the Sel + Bact group (at 24-h post-infection period only) compared to the Bact group. The results from this study showed that infection with S. marcescens is capable of disrupting the immune system and redox homeostasis in C. gariepinus, while pre-treatment with selenium has the ability to improve the physiological status of fish that were challenged with bacteria probably through its antioxidant properties. HIGHLIGHT: The pre-treatment of Clarias gariepinus to waterborne selenium for 14days improved the redox homeostasis and innate immunity of fish that were experimentally challenged with the bacterium, Serratia marcescens.

MicroRNA-122 regulates NF-κB signaling pathway by targeting IκBα in miiuy croaker, Miichthys miiuy

The inhibitory protein IκBα plays a key role in the inflammatory process and immune response by regulating the activity of the transcription factor NF-κB. microRNA (miR) is a small non-coding RNA that can regulate many biochemical processes, such as cell growth, proliferation, and immune response. In this study, it was first predicted that IκBα is the target of miR-122 through bioinformatics, and it was confirmed by dual fluorescence experiments. Then we found that miR-122 can inhibit the expression of IκBα at the mRNA and protein levels, thereby promoting the p65-activated NF-κB pathway. It is speculated that miR-122 plays an important role in the innate immunity of teleost fish. This study will help to further understand miRNAs regulatory mechanism in teleost fish.

EIF3k inhibits NF-κB signaling by targeting MyD88 for ATG5-mediated autophagic degradation in teleost fish

Optimal activation of NF-κB signaling is crucial for the initiation of inflammatory responses and eliminating invading bacteria. Bacteria have likewise evolved the ability to evade immunity; however, mechanisms by which bacteria dysregulate host NF-κB signaling are unclear. In this study, we identify eukaryotic translation initiation factor eIF3k, a nonessential member of the eIF3 translation initiation complex, as a suppressor of the NF-κB pathway. Mechanistically, we show that eIF3k expression induced by Vibrio harveyi enhances E3 ligase Nrdp1-mediated K27-linked ubiquitination of MyD88, an upstream regulator of NF-κB pathway activation. Furthermore, we show that eIF3k acts as a bridge linking ubiquitin-tagged MyD88 and ATG5, an important mediator of autophagy. We demonstrate that the MyD88-eIF3k-ATG5 complex is transported to the autophagosome for degradation, and that innate immune signaling is subsequently terminated and does not attack invading V. harveyi. Therefore, our study identifies eIF3k as a specific inhibitor of the MyD88-dependent NF-κB pathway and suggests that eIF3k may act as a selective autophagic receptor that synergizes with ATG5 to promote the autophagic degradation of MyD88, which helps V. harveyi to evade innate immunity. We conclude that V. harveyi can manipulate a host's autophagy process to evade immunity in fish and also provide a new perspective on mammalian resistance to bacterial invasion.

Antigenicity and immunity of recombinant OmpAII from Aeromonas veronii and protection against virulent Aeromonas infections in Carassius auratus gibelio

Aeromonas spp. can infect a variety of aquatic and terrestrial animals including humans and cause motile aeromonad septicemia (MAS). OmpA is an important virulence factor of fish pathogens such as Aeromonas veronii and Aeromonas hydrophila. In the current study, we examined the antigenicity and immune efficiency of a recombinant protein OmpAII vaccine against a lethal challenge of A. veronii. The whole ompAII gene from A. veronii strain GZ09007 was successfully cloned and expressed in Escherichia coli. The affinity-purified rOmpAII emulsified with Montanide ISA 763 A adjuvant was used to immunize gibel carp(Carassius auratus gibelio). PBS-ISA763A emulsion and PBS were the controls. The relative survival of vaccinated versus PBS control fish against a lethal challenge of A. veronii strains GZ09007, YC170511, XZ180501 and A. hydrophila Hn099 on day 28 post immunization were 100, 100, 38 and 89%, respectively. The vaccinated fish produced continuously high specific antibody titers. The lysozyme(LZM) activity in the sera of rOmpAII-ISA763A immunized fish was significantly higher than the controls from 7d to 56d. However, the superoxide dismutase (SOD) activity of rOmpAII-ISA763A was significantly higher than the controls only on 14d, 42d and 56d. Therefore, The OmpAII from A. veronii GZ09007 displayed positive antigenicity and immunity against Aeromonas infection challenges and is a candidate as a commercial reagent.

Vitamin D impacts on the intestinal health, immune status and metabolism in turbot (Scophthalmus maximus L.).

Vitamin D (VD) plays a vital role in various physiological processes in addition to its classic functions on maintaining the balance of Ca and P metabolism. However, there still are gaps to understand in depth the issues on the precise requirement, metabolic processes and physiological functions of VD in fish. In this study, we investigated the effects of VD on the growth, intestinal health, host immunity and metabolism in turbot (Scophthalmus maximus L.), one important commercial carnivorous fish in aquaculture, through the supplementation of different doses of dietary VD3 (0, 200, 400, 800 and 1600 μg VD3/kg diet). According to our results, the optimal VD3 level in the feed for turbot growth was estimated to be around 400 IU/kg, whereas VD3 deficiency or overdose in diets induced the intestinal inflammation, lowered the diversity of gut microbiota and impaired the host resistance to bacterial infection in turbot. Moreover, the level of 1α,25(OH)2D3, the active metabolite of VD3, reached a peak value in the turbot serum in the 400 μg group, although the concentrations of Ca and phosphate in the turbot were stable in all groups. Finally, the deficiency of dietary VD3 disturbed the nutritional metabolism in turbot, especially the metabolism of lipids and glucose. In conclusion, this study evaluated the optimal dose of dietary VD3 for turbot and provided the evidence that VD has a significant impact on intestinal health, host immunity and nutritional metabolism in fish, which deepened our understanding on the physiological functions and metabolism of VD3 in fish.

Effect of dietary Spirulina ( Arthrospira platensis ) on the growth performance, immune‐related gene expression and resistance to Vibrio anguillarum in European seabass ( Dicentrarchus labrax )

This research was conducted to reveal the effects of dietary Spirulina (Arthrospira platensis) meal on the growth performance, immune responses and resistance against Vibrio anguillarum in European seabass (Dicentrarchus labrax). In the study, the fish (5.74 ± 0.02 g) were fed with control (C) and three different levels (1%, 2.5% and 5%) of Spirulina meal-containing diets (SP1, SP2.5 and SP5) for 60 days. The adaptive and innate immune responses of fish were determined every 30th day of the study. The results showed that respiratory burst activity (RBA) was significantly elevated on the SP2.5 and SP5 groups on the 30th day of the study. Lysozyme activity (LYS) was increased on the SP1 group on the 30th day and the SP5 group on the 60th day (p < 0.05). Myeloperoxidase activity (MPO) was raised on the 60th day of the SP2.5 and SP5 groups compared with the C (p < 0.05). IL-1β gene expression increased in the kidney and intestine of fish on the 30th day and in the spleen on the 60th day. IL-10 gene expression in the intestine of fish fed the Spirulina-containing diet was elevated at each sampling time relative to the C group (p < 0.05). Elevated IL-6 activity was determined in the kidney and the intestine on all Spirulina-fed groups compared with control at both sampling times. Similar to IL-6, IL-8 gene expressions were elevated in all Spirulina-fed groups (p < 0.05). TNF-α was up-regulated in the kidney and intestine of all Spirulina-fed groups compared with the C group on the 30th day of the study (p < 0.05). TGF-β gene expression was increased in the kidney and intestine of all Spirulina groups compared with the C group on all sampling times. COX-2 gene expression in the kidneys and intestines of fish fed with Spirulina diets was significantly elevated compared with the C diet at days 60th and 30th respectively. Hepcidin gene expression of all groups was elevated in all tissue samples on the 30th day of the study. The survival rate of fish infected with V. anguillarum was higher in all Spirulina-fed groups than in the C group. The feed conversion rate and protein efficiency ratio of fish fed with a 1% Spirulina diet was found to be better than those fed with other diets. These results show that up to the 5% inclusion of Spirulina meal in European seabass diets promotes some growth parameters and supports immunity in fish.

Identification and Characterization of Differentially Expressed IgM Transcripts of Channel Catfish Vaccinated with Antigens of Virulent Aeromonas hydrophila

Channel catfish (Ictalurus punctatus) is the top species produced in US aquaculture and motile Aeromonas septicemia, caused by virulent Aeromonas hydrophila (vAh), is one of the most severe diseases that afflict catfish farms. Previously, vaccination of fish with extracellular proteins (ECP) of vAh was shown to produce a robust antibody-mediated immune response against vAh infection. In this study, we analyzed IgM transcripts that were differentially expressed in the head kidney and liver of ECP-immunized and mock-immunized (control) fish with emphasis on a variable domain of heavy chain. Quantitative PCR analysis indicated that immunized fish produced significantly more IgM transcripts than control fish. Full-length IgM heavy chain cDNA was cloned, which encoded typical IgM peptide, including signal peptide, variable domain (VH), constant domain (CH), and carboxyl terminal peptide. Great sequence diversity was revealed in a VH segment, with the third complementarity diversity region (CDR3) being most variable. Using germline VH gene grouping method, variants (clones) of VH characterized in this study belonged to nine VH families. The most unique variants (approximately 49%) were found in the VH2 family. Vaccinated fish apparently had more unique variants than in the control fish. There were 62% and 79% of unique variants in the head kidney and liver of vaccinated fish, respectively, while 44% and 27% unique variants in the head kidney and liver of control fish, respectively. Among the unique variants in VH2 family, approximately 87% of them were found in vaccinated fish. Two-dimensional gel electrophoresis of semi-purified IgM protein confirmed that matured IgM protein was as variable as IgM transcripts identified in this study, with isoelectric points crossing from 6 to 10. Results of this study provided insight into the molecular and genetic basis of antibody diversity and enriched our knowledge of the complex interplay between antigens and antibodies in Ictalurid catfish.

Oral Administration of Nanopeptide CMCS-20H Conspicuously Boosts Immunity and Precautionary Effect Against Bacterial Infection in Fish.

Massive mortalities caused by bacterial infections in intensive aquaculture result in serious economic losses. In this study, a novel antimicrobial peptide gcIFN-20H was efficiently expressed in Pichia pastoris (GS115) and loaded on carboxylmethyl chitosan (CMCS) to prepare CMCS-20H nanoparticles. Through physical characterization assays (TEM, DLS, BCA, and Raman) and biological activity tests (antimicrobial activity and cytotoxicity), CMCS-20H nanopeptide was verified to be spherical nanoparticles with sustained release, antimicrobial activity, and negligible toxicity. CMCS-20H nanoparticles are more resistant to intestinal degradation than unloaded gcIFN-20H by indirect immunofluorescence assay. Oral administration was then carried out for 42 days. Complement C3 content, lysozyme, and total superoxide dismutase activities are highest in CMCS-20H group by serum biochemistry index assays. After challenge with Aeromonas hydrophila, the survival rate in CMCS-20H group is highest (46%), which is 64% higher than the control group (28%). Meanwhile, the tissue bacterial loads (intestine, spleen, head kidney, trunk kidney, hepatopancreas, muscle, and blood) in the CMCS-20H group are significantly lower than other groups. By PAS staining analysis, the number of intestinal villi goblet cells and the thickness of mucin in the CMCS-20H group obviously increased. CMCS-20H effectively enhances mRNA expressions of some important immune genes (IL-1β, IL-6, TNF-α, IL-2, IFN-γ2, and IgM). The minimal tissue lesions (Intestine, spleen, and trunk kidney) were seen in the CMCS-20H group by histopathological examination. 16S rRNA sequencing showed that oral CMCS-20H maintains the intestinal microbiome homeostasis in bacterial infection. The results indicate that the novel nanopeptide CMCS-20H as the immunopotentiator can remarkably boost fish immunity and precautionary effect by oral administration and address the theoretical mechanisms and insights into the promising application prospect in aquaculture.

Effects of hydroxy-selenomethionine on performance, innate immune system and antioxidant defense of tambaqui (Colossoma macropomum) exposed to a physical stressor

Selenium (Se) is a mineral with natural antioxidant properties that constitutes a number of enzymes with a fundamental role in the immunity and antioxidant systems and may confer a protective role against oxidative stress in fish following exposure to physical stressors. Adopting an integrated approach, this study investigated simultaneously the role of hydroxy-selenomethionine (OH-SeMet) supplementation in performance, hematological parameters, innate immune, antioxidant capacity and tissue Se retention of tambaqui (Colossoma macropomum) and the possible protective role of dietary selenium when fish are exposed to a physical stressor (transport). Juvenile specimens (15.71 ± 1.90 g) were fed one of five diets: a basal unsupplemented diet (0.0 mg kg−1 Se) or diets supplemented with OH-SeMet to provide 0.3, 0.6, 0.9 and 1.2 mg kg−1 Se of diet for 75 days prior to subjection of fish to transport stress. Dietary supplementation with Se in the form of OH-SeMet for 75 days did not affect the production performance of juvenile tambaqui, but increased innate immunity parameters (oxidative burst) from the Se inclusion level of 0.6 mg kg−1 and induced the activation of the antioxidant defense system (GPX, GSH and GST) especially at the Se inclusion level of 0.9 mg kg−1. In addition, the Se content in the fillet rose significantly, as the OH-SeMet contents in the diet were increased. The stress caused by transport resulted in alterations in hematological parameters, blood protein profile and immune and enzymatic responses in the species. However, Se supplementation at 0.9 mg kg−1 had a positive effect, increasing innate immunity and activating antioxidant defenses (CAT and GPx, especially) after this physical stressor was applied. These results demonstrate that, when submitted to transport stress, juvenile tambaqui use Se stored in the muscle and dietary supplementation with OH-SeMet at 0.9 mg kg−1 improves the innate immunity and antioxidant system parameters of fish after transport. These findings reinforce the need for supplementing hydroxy-selenomethionine in commercial diets for tambaqui to ensure tissue Se reserves as a contingency in cases of stress.