Stability characterizations of feed-based bivalent vaccine containing inactivated Streptococcus agalactiae and Aeromonas hydrophila against streptococcosis and Aeromonas infections in red hybrid tilapia (Oreochromis sp.).

Feed-based bivalent vaccine (FBBV) containing killed whole organism (KWO) of Streptococcus agalactiae and Aeromonas hydrophila with 10% palm oil was previously proved to improve red hybrid tilapia's (Oreochromis sp.) immunity against streptococcosis and Aeromonas infections. This study characterized the FBBV's stability following the preparatory process and storage. The FBBV was prepared, and the KWO's stability was determined microscopically and molecularly. The efficacy of FBBV stored at room temperature (25 ± 2 °C) for 0, 30 and 60 days was investigated in red hybrid tilapia. The results indicated the addition of palm oil was not affecting the KWO's structure and helping in the FBBV's pelletization. In 1 g of FBBV contained 1.5 × 109 CFU/g of S. agalactiae and 4.9 × 109 CFU/g of A. hydrophila, respectively, even after 60 days of storage at room temperature. The KWO's structure in FBBV was not affected following in vitro acidic tolerance analysis, as noted from light and electron microscopies. The FBBV's carbohydrate, energy, moisture, total protein and total ash contents remained stable at 95% after 60 days of storage at room temperature, while the KWO's concentration was slightly reduced to 83.3% for S. agalactiae (1.25 × 109 CFU/g) and 80.6% for A. hydrophila (3.85 × 109 CFU/g), respectively. Fish vaccinated with FBBV that was stored for 0, 30 and 60 days did not show any significant differences (p ≥ 0.05) in the relative percent survival when challenged with pathogenic Streptococcus spp. and Aeromonas spp. These findings suggested that the FBBV is a stable vaccine, which underscores its potential application as aquatic vaccines in aquaculture.

Impact of Chaetomorpha aerea-enriched diet on growth, feed utilization, and haemato-immunological responses in Clarias batrachus challenged with Aeromonas hydrophila.

The bacteria Aeromonas hydrophila, which causes motile Aeromonas septicemia (MAS), is dangerous to aquaculture because it affects the fish's well-being and production. As the aquaculture industry seeks sustainable and effective methods to enhance fish immunity and growth, natural supplements such as marine algae have gained attention. This study explored the potential benefits of incorporating the green marine algae Chaetomorpha aerea into the fish diet, focusing on disease resistance, growth, feed utilization, and hematological and immunological responses. Five diets were prepared, varying concentrations of C. aerea (0 control, T1: 1 g/kg; T2: 2 g/kg: T3: 5 g/kg: and T4: 10 g/kg) and administered to fish over 30 days. Following the feeding trial, the fish were exposed to A. hydrophila, and their survival rates were observed for the next 14 days. The findings demonstrated that the final weight, weight gain, relative growth rate, specific growth rate, and daily growth rate were all positively impacted by a diet containing 5 g/kg of C. aerea. Additionally, fish in the 5 g/kg C. aerea group demonstrated improved feed conversion efficiency compared to the control group. While there were no significant changes in red and white blood cell counts on the initial day, serum lysozyme activity and overall resistance to infection were enhanced in fish receiving C. aerea at 2 and 5 g/kg. These results imply that C. aerea supplementation with fish supplements may be a useful immunostimulant, boosting improved health and growth in sustainable aquaculture practices.

Hypoxia-inducible factor-1α promotes macrophage functional activities in protecting hypoxia-tolerant large yellow croaker (Larimichthys crocea) against Aeromonas hydrophila infection.

The immune system requires a high energy expenditure to resist pathogen invasion. Macrophages undergo metabolic reprogramming to meet these energy requirements and immunologic activity and polarize to M1-type macrophages. Understanding the metabolic pathway switching in large yellow croaker (Larimichthys crocea) macrophages in response to lipopolysaccharide (LPS) stimulation and whether this switching affects immunity is helpful in explaining the stronger immunity of hypoxia-tolerant L. crocea. In this study, transcript levels of glycolytic pathway genes (Glut1 and Pdk1), mRNA levels or enzyme activities of glycolytic enzymes [hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase A (LDHA)], aerobic respiratory enzymes [pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (IDH), and succinate dehydrogenase (SDH)], metabolites [lactic acid (LA) and adenosine triphosphate (ATP)], levels of bactericidal products [reactive oxygen species (ROS) and nitric oxide (NO)], and transcripts and level changes of inflammatory factors [IL1ß, TNFα, and interferon (IFN) γ] were detected in LPS-stimulated L. crocea head kidney macrophages. We showed that glycolysis was significantly induced, the tricarboxylic acid (TCA) cycle was inhibited, and metabolic reprogramming occurred, showing the Warburg effect when immune cells were activated. To determine the potential regulatory mechanism behind these changes, LcHIF-1α was detected and found to be significantly induced and transferred to the nucleus after LPS stimulation. LcHif-1α interference led to a significant reduction in glycolytic pathway gene transcript expression, enzyme activity, metabolites, bactericidal substances, and inflammatory factor levels; a significant increase in the aerobic respiration enzymes; and decreased migration, invasion, and phagocytosis. Further ultrastructural observation by electron microscopy showed that fewer microspheres contained phagocytes and that more cells were damaged after LcHif-1α interference. LcHif-1α overexpression L. crocea head kidney macrophages showed the opposite trend, and promoter activities of Ldha and Il1ß were significantly enhanced after LcHif-1α overexpression in HEK293T cells. Our data showed that LcHIF-1α acted as a metabolic switch in L. crocea macrophages and was important in polarization. Hypoxia-tolerant L. crocea head kidney showed a stronger Warburg effect and inhibited the TCA cycle, higher metabolites, and bactericidal substance levels. These results collectively revealed that LcHif-1α may promote the functional activities of head kidney macrophages in protecting hypoxia-tolerant L. crocea from Aeromonas hydrophila infection.

Megalobrama amblycephala IL-22 attenuates Aeromonas hydrophila induced inflammation, apoptosis and tissue injury by regulating the ROS/NLRP3 inflammasome axis.

Mammalian interleukin-22 (IL-22) attenuates organismal injury by inhibiting reactive oxygen species (ROS) and impeding the NLRP3 inflammasome activation. However, the role of fish IL-22 in this process remains unclear. We characterized MaIL-22, an IL-22 homolog in blunt snout bream (Megalobrama amblycephala). Despite its low sequence identity, it shares conserved structures and close evolutionary relationships with other teleost IL-22s. Furthermore, Aeromonas hydrophila (A. hydrophila) infection leads to tissue injury in M. amblycephala immune organs and concomitantly altered Mail-22 mRNA expression, suggesting that MaIL-22 was involved in the antimicrobial immune response. To explore MaIL-22's biological functions, we produced recombinant MaIL-22 (rMaIL-22) protein and demonstrated it significantly enhanced the survival of M. amblycephala post-A. hydrophila infection. To unravel its protective mechanisms, we explored the ROS/NLRP3 inflammasome axis and its downstream signaling responses. The results showed that rMaIL-22 treatment significantly elevated antioxidant enzyme (T-SOD, CAT and GSH-PX) activities to inhibit MDA activity and scavenge ROS in visceral tissues. Meanwhile, rMaIL-22 impeded the activation of NLRP3 inflammasome by suppressing NLRP3 protein and mRNA expression. This indicated that rMaIL-22 contributed to inhibit A. hydrophila-induced activation of the ROS/NLRP3 inflammasome axis. Consistent with these findings, rMaIL-22 treatment attenuated the expression of proinflammatory cytokines (il-1ß, tnf-α and il-6) and proapoptotic genes (caspase-3 and caspase-8) while promoting antiapoptotic genes (bcl-2b and mcl-1a) expression, ultimately mitigating tissue injury in visceral tissues. In conclusion, our research underscores MaIL-22's key role in microbial immune regulation, offering insights for developing IL-22-targeted therapies and breeding programs.

Yorkie negatively regulates the Crustin expression during molting in Chinese mitten crab, Eriocheir sinensis.

Molting is a key biological process of crustaceans, which is mainly regulated by 20-hydroxyecdyone (20E). The molting cycle could be divided into three main stages including pre-molt, post-molt and inter-molt stages. The mechanism of immune regulation during molting process still requires further exploration. Yorkie (Yki) is a pivotal transcription factor in the Hippo signaling pathway, and it plays an essential role in regulating cell growth and immune response. In the present study, a Yki gene was identified from Eriocheir sinensis (designed as EsYki), and the regulatory role of EsYki in controlling the expression of antimicrobial peptide genes throughout the molting process was investigated. The mRNA expression level of EsYki was higher at the pre-molt stage compared to the post-molt stage and inter-molt stage. Following the injection of 20E, there was a notable and consistent rise in the EsYki mRNA expression in haemocytes. The increase was observed from 3 h to 48 h with the maximum level at 12 h. And the phosphorylation of Yki in the haemocytes was also significantly up-regulated at 3 h post 20E injection. Moreover, the levels of EsYki mRNA expression at three molting stages were significantly increased post Aeromonas hydrophila stimulation. The maximum level was detected at post-molt stage following A. hydrophila stimulation, while the lowest level was observed at inter-molt stage. The expression pattern of EsCrus was in contrast to EsCrus. After EsYki mRNA transcripts were inhibited by Yki inhibitor (CA3), the mRNA expression levels of EsCrus1 and EsCrus2 following A. hydrophila stimulation were significantly elevated. Furthermore, the phosphorylation level of NF-κB was also increased following the inhibition of Yki. Collectively, our findings indicated that EsYki could be induced by 20E and has a suppressive effect on the expression of EsCrus via inhibiting NF-κB during molting process. This research contributes to the understanding of the immunological regulation mechanism during molting process in crustaceans.

Fish IL-26 collaborates with IL-10R2 and IL-20R1 to enhance gut mucosal barrier during the antibacterial innate immunity.

IL-26 is a cytokine that is crucial for the maintenance and function of the gut mucosal barrier. IL-26 signaling pathway relies on a heterodimeric receptor complex, which is composed of two distinct subunits, IL-10R2 and IL-20R1. However, there are no reports on the antibacterial immunity of IL-26 and its receptors in fish. For this purpose, in this study we identified IL-26 and its receptors IL-10R2 and IL-20R1 in Carassius cuvieri × Carassius auratus red var. (named WR-IL-26, WR-IL10R2 and WR-IL20R1, respectively). Phylogenetic analysis confirmed the conservation of these genes, with shared structural motifs similar to those found in higher vertebrates. Upon exposure to Aeromonas hydrophila, a common fish pathogen, there was a significant upregulation of WR-IL-26, WR-IL10R2 and WR-IL20R1 in the gut, indicating a potential role in the immune response to infection. A co-immunoprecipitation assay revealed that WR-IL-26 formed complexes with WR-IL10R2 and WR-IL20R1. In vivo experiments demonstrated that administration of WR-IL-26 activated the JAK1-STAT3 signaling pathway and protected the gut mucosa barrier from A. hydrophila infection. Conversely, silencing WR-IL10R2 and WR-IL20R1 via RNA interference significantly attenuated the activation of WR-IL-26-mediated JAK1-STAT3 pathway. These results provided new insights into the role of IL-26 and its receptors in the gut mucosa barrier and could offer novel therapeutic strategies for managing bacterial infections in aquaculture.

Erythrocytes of the common carp are immune sentinels that sense pathogen molecular patterns, engulf particles and secrete pro-inflammatory cytokines against bacterial infection.

Introduction: Red blood cells (RBCs), also known as erythrocytes, are underestimated in their role in the immune system. In mammals, erythrocytes undergo maturation that involves the loss of nuclei, resulting in limited transcription and protein synthesis capabilities. However, the nucleated nature of non-mammalian RBCs is challenging this conventional understanding of RBCs. Notably, in bony fishes, research indicates that RBCs are not only susceptible to pathogen attacks but express immune receptors and effector molecules. However, given the abundance of RBCs and their interaction with every physiological system, we postulate that they act in surveillance as sentinels, rapid responders, and messengers. Methods: We performed a series of in vitro experiments with Cyprinus carpio RBCs exposed to Aeromonas hydrophila, as well as in vivo laboratory infections using different concentrations of bacteria. Results: qPCR revealed that RBCs express genes of several inflammatory cytokines. Using cyprinid-specific antibodies, we confirmed that RBCs secreted tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). In contrast to these indirect immune mechanisms, we observed that RBCs produce reactive oxygen species and, through transmission electron and confocal microscopy, that RBCs can engulf particles. Finally, RBCs expressed and upregulated several putative toll-like receptors, including tlr4 and tlr9, in response to A. hydrophila infection in vivo. Discussion: Overall, the RBC repertoire of pattern recognition receptors, their secretion of effector molecules, and their swift response make them immune sentinels capable of rapidly detecting and signaling the presence of foreign pathogens. By studying the interaction between a bacterium and erythrocytes, we provide novel insights into how the latter may contribute to overall innate and adaptive immune responses of teleost fishes.

Differential polyvalent passive immune protection of egg yolk antibodies (IgY) against live and inactivated Vibrio fluvialis in fish.

Egg yolk antibodies (IgY) can be prepared in large quantities and economically, and have potential value as polyvalent passive vaccines (against multiple bacteria) in aquaculture. This study prepared live and inactivated Vibrio fluvialis IgY and immunized Carassius auratus prior to infection with V. fluvialis and Aeromonas hydrophila. The results showed that the two IgY antibodies hold effective passive protective rates against V. fluvialis and A. hydrophila in C. auratus. Further, the serum of C. auratus recognized the two bacteria in vitro, with a decrease in the bacteria content of the kidney. The phagocytic activity of C. auratus plasma was enhanced, with a decrease in the expression of inflammatory and antioxidant factors. Pathological sections showed that the kidney, spleen, and intestinal tissue structures were intact, and apoptosis and DNA damage decreased in kidney cells. Moreover, the immunoprotection conferred by the live V. fluvialis IgY was higher than that of the inactivated IgY. Addition, live V. fluvialis immunity induced IgY antibodies against outer membrane proteins of V. fluvialis were more than inactivated V. fluvialis immunity. Furthermore, heterologous immune bacteria will not cause infection, so V. fluvialis can be used to immunize chickens to obtain a large amount of IgY antibody. These findings suggest that the passive immunization effect of live bacterial IgY antibody on fish is significantly better than that of inactivated bacterial antibody, and the live V. fluvialis IgY hold potential value as polyvalent passive vaccines in aquaculture.

Baseline and stress-induced changes in plasma bacterial killing ability against gram-negative bacteria are partially mediated by the complement system in Rhinella diptycha toads.

The plasma bacterial killing ability (BKA) is modulated by the stress response in vertebrates, including amphibians. The complement system is an effector mechanism comprised of a set of proteins present in the plasma that once activated can promote bacterial lysis. Herein, we investigated whether changes in plasma BKA as a result of the acute stress response and an immune challenge are mediated by the complement system in Rhinella diptycha toads. Additionally, we investigated whether the observed changes in plasma BKA are associated with changes in plasma corticosterone levels (CORT). We subjected adult male toads to a restraint or an immune challenge (with three concentrations of Aeromonas hydrophila heat inactivated), and then evaluated the plasma BKA against A. hydrophila, in vitro. We determined the complement system activity on plasma BKA, by treating the plasma (baseline, 1 h and 24 h post-restraint, and after the immune challenge) with ethylenediaminetetraacetic acid, heat, or protease. Our results showed increased CORT 1 h and 24 h after restraint and decreased plasma BKA 24 h post-restraint. The inhibitors of the complement system decreased the plasma BKA compared with untreated plasma at all times (baseline, 1 h, and 24 h after restraint), demonstrating that the plasma BKA activity is partially mediated by the complement system. The immune challenge increased CORT, with the highest values being observed in the highest bacterial concentration, compared with control. The plasma BKA was not affected by the immune challenge but was demonstrated to be partially mediated by the complement system. Our results demonstrated that restraint and the immune challenge activated the hypothalamus-pituitary-interrenal axis, by increasing plasma CORT levels in R. diptycha. Also, our results demonstrated the complement system is participative in the plasma BKA for baseline and post-stress situations in these toads.

Oral administration of recombinant outer membrane protein A-based nanovaccine affords protection against Aeromonas hydrophila in zebrafish.

Aeromonas hydrophila, an opportunistic warm water pathogen, has always been a threat to aquaculture, leading to substantial economic losses. Vaccination of the cultured fish would effectively prevent Aeromoniasis, and recent advancements in nanotechnology show promise for efficacious vaccines. Oral delivery would be the most practical and convenient method of vaccine delivery in a grow-out pond. This study studied the immunogenicity and protective efficacy of a nanoparticle-loaded outer membrane protein A from A. hydrophila in the zebrafish model. The protein was over-expressed, purified, and encapsulated using poly lactic-co-glycolic acid (PLGA) nanoparticles via the double emulsion method. The PLGA nanoparticles loaded with recombinant OmpA (rOmpA) exhibited a size of 295 ± 15.1 nm, an encapsulation efficiency of 72.52%, and a polydispersity index of 0.292 ± 0.07. Scanning electron microscopy confirmed the spherical and isolated nature of the PLGA-rOmpA nanoparticles. The protective efficacy in A. hydrophila-infected zebrafish after oral administration of the nanovaccine resulted in relative percentage survival of 77.7. Gene expression studies showed significant upregulation of immune genes in the vaccinated fish. The results demonstrate the usefulness of oral administration of nanovaccine-loaded rOmpA as a potential vaccine since it induced a robust immune response and conferred adequate protection against A. hydrophila in zebrafish, Danio rerio.

IL-20 is produced by CD3γδ T cells and induced in the mucosal tissues of grass carp during infection with Aeromonas hydrophila.

Interleukin (IL) 20 is a multifunctional cytokine and plays a vital role in regulating autoimmune diseases, inflammation, and immune responses. IL-20 homologs have been described in fish. However, due to the lack of antibodies, cellular sources and immunological functions of fish IL-20 in response to infections have not been fully characterized. In this study, a monoclonal antibody (mAb) was generated against the recombinant grass carp (Ctenopharyngodon idella) IL-20 protein and characterized by immunoblotting, immunofluorescent microscopy and flow cytometry. It was shown that the IL-20 mAb specifically recognized recombinant IL-20 proteins expressed in the E. coli cells and HEK293 cells. Using confocal microscopy, the IL-20+ cells were identified in the head kidney, gills and intestine of grass carp, and induced after infection with Aeromonas hydrophila. Moreover, the IL-20 protein was found to be secreted mainly by CD3γδ T cells which were located predominantly in the gill filaments and intestinal mucosa. Taken together, our results suggest that IL-20 producing T cells are required for the mucosal immunity against bacterial infection in fish.

Immune protection of grass carp by oral vaccination with recombinant Bacillus methylotrophicus expressing the heterologous tolC gene.

In the field of aquaculture, the enhancement of animal health and disease prevention is progressively being tackled using alternatives to antibiotics, including vaccines and probiotics. This study was designed to evaluate the potential of a recombinant Bacillus methylotrophicus, engineered to express the outer membrane channel protein TolC of Aeromonas hydrophila AH3 and the green fluorescent protein GFP, as an oral vaccine. Initially, the genes encoding tolC and GFP were cloned into a prokaryotic expression system, and anti-TolC mouse antiserum was generated. Subsequently, the tolC gene was subcloned into a modified pMDGFP plasmid, which was transformed into B. methylotrophicus WM-1 for protein expression. The recombinant B. methylotrophicus BmT was then administered to grass carp via co-feeding, and its efficacy as an oral vaccine was assessed. Our findings demonstrated successful expression of the 55 kDa TolC and 28 kDa GFP proteins, and the preparation of polyclonal antibodies with high specificity. The BmT exhibited stable expression of the GFP-TolC fusion protein and excellent genetic stability. Following oral immunization, significant elevations were observed in serum-specific IgM levels and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in grass carp. Concurrently, significant upregulation of immune-related genes, including IFN-I, IL-10, IL-1ß, TNF-α, and IgT, was noted in the intestines, head kidney, and spleen of the grass carp. Colonization tests further revealed that the BmT persisted in the gut of immunized fish even after a fasting period of 7 days. Notably, oral administration of BmT enhanced the survival rate of grass carp following A. hydrophila infection. These results suggest that the oral BmT vaccine developed in this study holds promise for future applications in aquaculture.

Immunoprotective efficacy evaluation of OmpTS subunit vaccine against Aeromonas hydrophila infection in Megalobrama amblycephala.

Bacterial septicemia in freshwater fish is mainly caused by Aeromonas hydrophila infection, which affects the development of aquaculture industry. In the context of sustainable aquaculture, subunit vaccines are of great values because they play positive roles in reducing the overuse of antibiotics and protecting aquatic animals against bacterial infection. In this study, the recombinant outer membrane protein OmpTS of A. hydrophila were used as subunit vaccine to immunize Megalobrama amblycephala, and its immunoprotective effect and host immune responses were evaluated. The survival rates of the vaccinated groups after bacterial infection were significantly higher than that of the control group, especially of the OmpTS high-dose vaccinated group. The better protective effects of vaccinated groups might be attributed to the increased levels of serum IgM-specific antibody titer, the reduced relative abundance of A. hydrophila in various tissues, the increased number of immune-positive cells with different epitopes, the up-regulated expression levels of immune-related genes, and the enhanced activities of antibacterial enzymes. In conclusion, OmpTS subunit vaccine could strongly induce host immune responses in M. amblycephala, thereby enhancing both cellular and humoral immunity, which exhibited excellent and effective immunoprotective efficacy.

DC-SIGN of Largemouth Bass (Micropterus salmoides) Mediates Immune Functions against Aeromonas hydrophila through Collaboration with the TLR Signaling Pathway.

C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of Micropterus salmoides was identified. MsDC-SIGN is classified as a type II transmembrane protein. The extracellular segment of MsDC-SIGN possesses a coiled-coil region and a carbohydrate recognition domain (CRD). The key amino acid motifs of the extracellular CRD of MsDC-SIGN in Ca2+-binding site 2 were EPN (Glu-Pro-Asn) and WYD (Trp-Tyr-Asp). MsDC-SIGN-CRD can bind to four pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), glucan, peptidoglycan (PGN), and mannan. Moreover, it can also bind to Gram-positive, Gram-negative bacteria, and fungi. Its CRD can agglutinate microbes and displays D-mannose and D-galactose binding specificity. MsDC-SIGN was distributed in seven tissues of the largemouth bass, among which the highest expression was observed in the liver, followed by the spleen and intestine. Additionally, MsDC-SIGN was present on the membrane of M. salmoides leukocytes, thereby augmenting the phagocytic activity against bacteria. In a subsequent investigation, the expression patterns of the MsDC-SIGN gene and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) exhibited an up-regulated expression response to the stimulation of Aeromonas hydrophila. Furthermore, through RNA interference of MsDC-SIGN, the expression level of the DC-SIGN signaling pathway-related gene (RAF1) and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) was decreased. Therefore, MsDC-SIGN plays a pivotal role in the immune defense against A. hydrophila by modulating the TLR signaling pathway.

Expression of immuno-transcriptome response in red hybrid tilapia (Oreochromis sp.) hindgut following vaccination with feed-based bivalent vaccine.

Streptococcosis and aeromoniasis are the main obstacles to sustainable tilapia production. Vaccination offered an effective method to control microbial infections. Previously, a feed-based bivalent vaccine (FBBV) containing killed whole organisms of Streptococcus agalactiae and Aeromonas hydrophila mixed with 10% palm oil was successfully developed, which provided good protection against streptococcosis and aeromoniasis in Oreochromis sp. However, the mechanisms of immunities in vaccinated fish still need clarification. Here, the hindgut transcriptome of vaccinated and control fish was determined, as the gut displays higher affinity towards antigen uptake and nutrient absorption. The efficacy of FBBV to improve fish immunity was evaluated according to the expression of immune-related genes in the vaccinated fish hindgut throughout the 8-week experimental period using RT-qPCR. The vaccinated fish hindgut at week 6 was further subjected to transcriptomic analysis due to the high expression of immune-related genes and contained killed whole organisms. Results demonstrated the expression of immune-related genes was in correlation with the presence of killed whole organisms in the vaccinated fish hindgut. Transcriptomic analysis has allowed the prediction of robust immune-related pathways, including innate and adaptive immunological responses in vaccinated fish hindgut than control fish. Pathways related to the regulation of lipid metabolism and modulation of the immune system were also significantly enriched (p ≤ .05). Overall, results offer a fundamental study on understanding the immunological response in Oreochromis sp. following vaccination with the FBBV pellet and support further application to prevent bacterial diseases in aquaculture.

Genome-wide identification of endosialin family of C-type lectins in common carp (Cyprinus carpio) and their response following Aeromonas hydrophila infection.

The endosialin family is the group XIV of C-type lectin, regulating several processes involved in innate immunity and inflammation. Endosialin family genes have been extensively studied in human and mammals, however, rarely reported in teleost. In the present study, a set of 8 endosialin family genes was identified across the entire common carp genome. Functional domain and motif prediction and phylogenetic analysis supported their annotation and orthologies. Through examining gene copy number across several vertebrates, endosialin family genes were found have undergone gene duplication. Most of the endosialin family genes were ubiquitously expressed during common carp early developmental stages, and presented tissue-specific expression patterns in various healthy tissues, with relatively high expression in intestine, liver, gill, spleen and kidney, indicating their likely essential roles in maintaining homeostasis and host immune response. After Aeromonas hydrophila infection, gene thbd-1, thbd-2 and cd93-2 were significantly up-regulated at one or more timepoints in spleen and kidney, while gene cd248a-1, cd248a-2, cd248b-1, cd248b-2, and cd93-1 were significantly down-regulated. Taken together, all these results suggested that endosialin family genes were involved in host immune response to A. hydrophila infection in common carp, and provided fundamental genomic resources for better understanding the critical roles of endosialin family on the primary innate immune processes in teleost.

The Elevated Expressions of Anti-lipopolysaccharide Factors After Priming Stimulation Confer Lastingly Humoral Protection in Crab Eriocheir sinensis.

Evidence of immune memory in invertebrates (immune priming) has accumulated in various organisms, and both cellular and humoral immune reactions are speculated to be involved in immune priming. However, there is a lack of understanding of the molecular mechanisms involved. In the present study, the protective effect of primed haemolymph was further validated by the increased survival rate of naïve crabs receiving a transfusion of primed haemolymph. By proteomic analysis, there were 474 proteins identified from the primed haemolymph, and most of them were functionally annotated in transport and metabolism classes. A total of 70 proteins were found to be differentially expressed in haemolymph at 12 hours and 7 days after priming stimulation with Aeromonas hydrophila, among which anti-lipopolysaccharide factor 1 (EsALF-1) and 3 (EsALF-3) were identified as the most significant (p < 0.05). After being challenged with A. hydrophila, EsALF-1 and EsALF-3 were highly expressed at both mRNA (in haemocytes) and protein (in haemolymph) levels compared with blank crabs, and the mRNA expressions of components in the EsTLR1-EsMyd88-EsPelle-EsALF pathway also increased significantly (p < 0.05). The EsALF-3 and EsMyd88 were even significantly higher expressed in response to the second A. hydrophila challenge, but their expressions all decreased (p < 0.05) when EsTLR1 was knocked down by RNAi. After the naïve crabs received an injection with the recombinant protein of EsALF-1 (rEsALF-1) or EsALF-3 (rEsALF-3), their survival rate increased significantly (p < 0.05) upon A. hydrophila stimulation. In contrast, the survival rate of the primed crabs reduced significantly (p < 0.05) after they received an injection with the antibody of EsALF-1 or EsALF-3. The enhanced expressions of EsALF-1 and EsALF-3 after A. hydrophilap riming stimulation could sustain for four weeks. All the results suggested that the EsTLR1-mediated productions of EsALF-1 and EsALF-3 in haemolymph played an indispensable role in the month-long humoral immune protection induced by A. hydrophila, which provides solid evidence of immune priming in crabs and a valuable reference for further understanding immune memory in invertebrates.

Transcriptome RNA-seq revealed lncRNAs activated by Edwardsiella anguillarum post the immunization of OmpA protecting European eel (Anguilla anguilla) from being infected.

Long noncoding RNAs (lncRNAs) play important roles in various biological activities as vital regulators. However, no study has focused on the lncRNA regulation of Outer membrane protein (OMP) immunization against aquatic bacterial infection. In this study, we examined the genome-wide expression of lncRNAs in the liver of European eel (Anguilla anguilla, Aa) administrated by a recombinant OmpA (rOmpA) from Edwardsiella anguillarum (Ea) to elucidate the functions of lncRNAs in the process of Ea infection and Aa anti-Ea infection using strand specific RNA-seq. Eels were challenged by Ea at 28 d post the immunization (dpi) of OmpA, and the result showed, compared to uninfected livers in the PBS group (Con group), the infected livers in the PBS group (Con_inf group) showed severe bleeding, hepatocyte atrophy and thrombi formed in the hepatic vessels; livers in the OmpA group (OmpA_inf) also formed slight thrombi in the hepatic vessels. The relative percent survival of eels in OmpA_inf vs Con_inf was 78.6%. Using high-throughput transcriptomics, we found 13405 lncRNAs in 3 compares of Con_inf vs Con, OmpA_inf vs Con and OmpA_inf vs Con_inf, of which 111, 129 and 158 DE-lncRNAs were ascertained. GO analysis of the DE-lncRNAs revealed the targeting DEGs were mainly involved in single-organism process, signaling, biological process and response to stimulus in BP, component of membrane in CC and binding in MF; KEGG pathways showed that the targeting DEGs in co-expression and co-location enriched in cell adhesion molecules. Finally, 54 DE-lncRNAs targeting 1675 DEGs were involved in an interaction network of 21692 co-expression and 483 co-location related links, of which 18 DE-lncRNAs appear to play crucial roles in anti-Ea infection. Thus, the interaction networks revealed crucial DE-lncRNAs underlying the process of Ea infection and Aa anti-Ea infection pre and post the immunization of OmpA.

An IL-1ß homologue induced inflammation and antibacterial immune defense in Siberian sturgeon (Acipenser baeri).

Interleukin-1ß is a key pro-inflammatory cytokine functioning in initiation of inflammatory responses against bacterial- and viral-infections. In the present study, a putative IL-1ß counterpart was identified from Siberian sturgeon (Acipenser baeri) and designated as AbIL-1ß. The Abil-1ß cDNA sequence consists of 1130 bp with an open reading frame (ORF) of 585 bp, which encodes a 194 amino acid (aa) protein. Multiple amino acid sequence alignment revealed that a possible mature peptide could start at Leu18, although no cut site for ICE (IL-1ß converting enzyme) enzyme was present in Siberian sturgeon IL-1ß. Even if AbIL-1ß shares a relative low identity (33.6%) with another sturgeon type II IL-1ß gene from Acipenser dabryanus, they still clustered together in phylogenetic tree. Endogenous Abil-1ß was highly expressed in brain, blood, head kidney and spleen of healthy Siberian sturgeon, and remarkably up regulated in head kidney, spleen, and liver upon Aeromonas hydrophila (A.h) challenge. Consistently, in vitro stimulation test using heat-killed A.h and LPS significantly increased Abil-1ß transcripts of primary spleen cells. To investigate the bactericidal capability of AbIL-1ß, recombinant AbIL-1ß (rAbIL-1ß) was generated by prokaryotes. Pre-injection of rAbIL-1ß reduced the bacterial load in sturgeon spleen after A.h infection. Further, rAbIL-1ß was served as feed additive and demonstrated to enhance hybrid sturgeon's defense against A.h infection by increased expressional levels of immune-related genes (IL-1ß, IL-6, IL-8, IgM and MHCIIß), elevated activities of serum lysosome, ACH50, and MPO, as well as higher percent survival. In summary, the current results suggested that AbIL-1ß functions in immune regulation and could improve sturgeon's resistance to bacterial infection.

Ferritin H can counteract inflammatory response in hybrid fish and its parental species after Aeromonas hydrophila infection.

Ferritin H can participate in the regulation of fish immunity. Tissue-specific analysis revealed that the highest expressions of Ferritin H in parental species were observed in spleen, while peaked level of Ferritin H mRNA in hybrid fish was observed in liver. In addition, A. hydrophila challenge could sharply enhance their Ferritin H mRNA expression in liver, kidney and spleen. To further investigate their roles in immune regulation, their Ferritin H fusion proteins were produced in vitro. Ferritin H fusion proteins could exhibit a direct binding activity to A. hydrophila and endotoxin in a dose-dependent manner, restrict dissemination of A. hydrophila to tissues and abrogate inflammatory cascades. Moreover, treatment with Ferritin H fusion proteins could reduce A. hydrophila-induced lipid peroxidation. These results indicated that Ferritin H in hybrid fish elicited a similar immune regulation of A. hydrophila-induced inflammatory signals in comparison with those of its parents.