Enhancement of immunity and disease resistance in Litopenaeus vannamei through injection of tyramine formulated with polyethylene glycol.

This study investigates the prolonged effect of immune disease resistance in Litopenaeus vannamei through the administration of tyramine (TA) formulated with polyethylene glycol (PEG). Facing the challenges of intensive farming, environmental stress, and global climate changes, innovative approaches to improve shrimp health are essential. The research focuses on the role of biogenic amines in stress response and immune regulation, demonstrating that TA, especially when combined with PEG, significantly prolongs immunity and resistance against Vibrio alginolyticus. The experimental design included administering TA, PEG, and TA-PEG, followed by evaluations of immunity, lactate and glucose levels, and immune-related gene expressions. Results showed notable prolonged effects in total hemocyte count, phenoloxidase activity, and phagocytic activity in the TA-PEG group, indicating enhanced immune activation period. Additionally, the expression of prophenoloxidase system-related genes was significantly upregulated in the TA-PEG group. Furthermore, the TA-PEG group exhibited a significantly higher survival rate in a susceptibility test against V. alginolyticus. The results of this study confirm that the combined use of PEG can effectively extend the immunostimulatory duration of TA.

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.

Activation of the TCA Cycle to Provide Immune Protection in Zebrafish Immunized by High Magnesium-Prepared Vibrio alginolyticus Vaccine.

Vaccines are safe and efficient in controlling bacterial diseases in the aquaculture industry and are in line with green farming. The present study develops a previously unreported approach to prepare a live-attenuated V. alginolyticus vaccine by culturing bacteria in a high concentration of magnesium to attenuate bacterial virulence. Furthermore, metabolomes of zebrafish immunized with the live-attenuated vaccines were compared with those of survival and dying zebrafish infected by V. alginolyticus. The enhanced TCA cycle and increased fumarate were identified as the most key metabolic pathways and the crucial biomarker of vaccine-mediated and survival fish, respectively. Exogenous fumarate promoted expression of il1ß, il8, il21, nf-κb, and lysozyme in a dose-dependent manner. Among the five innate immune genes, the elevated il1ß, il8, and lysozyme are overlapped in the vaccine-immunized zebrafish and the survival from the infection. These findings highlight a way in development of vaccines and exploration of the underlying mechanisms.

Identification of Polyvalent Vaccine Candidates From Extracellular Secretory Proteins in Vibrio alginolyticus.

Bacterial infections cause huge losses in aquaculture and a wide range of health issues in humans. A vaccine is the most economical, efficient, and environment-friendly agent for protecting hosts against bacterial infections. This study aimed to identify broad, cross-protective antigens from the extracellular secretory proteome of the marine bacterium Vibrio alginolyticus. Of the 69 predicted extracellular secretory proteins in its genome, 16 were randomly selected for gene cloning to construct DNA vaccines, which were used to immunize zebrafish (Danio rerio). The innate immune response genes were also investigated. Among the 16 DNA vaccines, 3 (AT730_21605, AT730_22220, and AT730_22910) were protective against V. alginolyticus infection with 47-66.7% increased survival compared to the control, while other vaccines had lower or no protective effects. Furthermore, AT730_22220, AT730_22910, and AT730_21605 also exhibited cross-immune protective effects against Pseudomonas fluorescens and/or Aeromonas hydrophila infection. Mechanisms for cross-protective ability was explored based on conserved epitopes, innate immune responses, and antibody neutralizing ability. These results indicate that AT730_21605, AT730_22220, and AT730_22910 are potential polyvalent vaccine candidates against bacterial infections. Additionally, our results suggest that the extracellular secretory proteome is an antigen pool that can be used for the identification of cross-protective immunogens.

A novel C-type lectin from Crassostrea gigas involved in the innate defense against Vibrio alginolyticus.

C-type lectins (CTLs) are important immune molecules that participate in invertebrate defense response. In the present work, a novel structural CTL (CgLec-4E) was identified from Crassostrea gigas, which encodes 237 amino acids (aa) with an extra long chain of aa and in the C-type CRD domain with EPA, QPG and WHD mutated motifs respectively. rCgLec-4E could agglutinate and inhibit the growth of Vibrio alginolyticus, except Chlorella, which might be relevant to three mutated motifs. CgLec-4E was mainly expressed in digestive gland, and its expression level was significantly up-regulated post V. alginolyticus challenge, indicating that the high expression of CgLec-4E could provide necessary mucosal immune protections and might involve in food particle recognition for C. gigas. Moreover, the subcellular locations indicated that CgLec-4E might play different roles in the immune response. Taken together, our results enrich our understanding of the structures and function of CTLs in invertebrates.

pH-Controlled Release of Antigens Using Mesoporous Silica Nanoparticles Delivery System for Developing a Fish Oral Vaccine.

The development of effective vaccines and delivery systems in aquaculture is a long-term challenge for controlling emerging and reemerging infections. Cost-efficient and advanced nanoparticle vaccines are of tremendous applicability in prevention of infectious diseases of fish. In this study, dihydrolipoamide dehydrogenase (DLDH) antigens of Vibrio alginolyticus were loaded into mesoporous silica nanoparticles (MSN) to compose the vaccine delivery system. Hydroxypropyl methylcellulose phthalate (HP55) was coated to provide protection of immunogen. The morphology, loading capacity, acid-base triggered release were characterized and the toxicity of nanoparticle vaccine was determined in vitro. Further, the vaccine immune effects were evaluated in large yellow croaker via oral administration. In vitro studies confirmed that the antigen could be stable in enzymes-rich artificial gastric fluid and released under artificial intestinal fluid environment. In vitro cytotoxicity assessment demonstrated the vaccines within 120 µg/ml have good biocompatibility for large yellow croaker kidney cells. Our data confirmed that the nanoparticle vaccine in vivo could elicit innate and adaptive immune response, and provide good protection against Vibrio alginolyticus challenge. The MSN delivery system prepared may be a potential candidate carrier for fish vaccine via oral administration feeding. Further, we provide theoretical basis for developing convenient, high-performance, and cost-efficient vaccine against infectious diseases in aquaculture.

A New C-Type Lectin Homolog SpCTL6 Exerting Immunoprotective Effect and Regulatory Role in Mud Crab Scylla paramamosain.

C-type lectin (CTL), a well-known immune-related molecule, has received more and more attention due to its diverse functions, especially its important role in development and host defense of vertebrate and invertebrate. Since the research on crab CTLs is still lack, we screened a new CTL homolog, named SpCTL6 from mud crab Scylla paramamosain. The full-length cDNA sequence of SpCTL6 was 738 bp with a 486 bp of ORF, and the deduced amino acids were 161 aa. SpCTL6 was predicted to have a 17 aa signal peptide and its mature peptide was 144 aa (MW 16.7 kDa) with pI value of 5.22. It had typical CTL structural characteristics, such as a single C-type lectin-like domain, 4 conserved cysteines, similar tertiary structure to that of vertebrate CTLs and a mutated Ca2+ binding motif Gln-Pro-Thr (QPT), clustering into the same branch as the crustacean CTLs. SpCTL6 was highly expressed in the entire zoeal larval stages and widely distributed in adult crab tissues with the highest transcription level in testis. During the molting process of juvenile crabs, the expression level of SpCTL6 was remarkably increased after molting. SpCTL6 could be significantly upregulated in two larval stages (Z1 and megalopa) and adult crab testis under immune challenges. Recombinant SpCTL6 (rSpCTL6) was successfully obtained from eukaryotic expression system. rSpCTL6 exhibited binding activity with PAMPs (LPS, lipoteichoic acid, peptidoglycan, and glucan) and had a broad spectrum bacterial agglutination activity in a Ca2+-dependent manner. In addition, rSpCTL6 could enhance the encapsulation activity of hemocytes and has no cytotoxic effect on hemocytes. Although rSpCTL6 had no bactericidal activity on Vibrio alginolyticus, rSpCTL6 treatment could significantly reduce the bacterial endotoxin level in vitro and greatly improved the survival of S. paramamosain under V. alginolyticus infection in vivo. The immunoprotective effect of rSpCTL6 might be due to the regulatory role of rSpCTL6 in immune-related genes and immunological parameters. Our study provides new information for understanding the immune defense of mud crabs and would facilitate the development of effective strategies for mud crab aquaculture disease control.

Early immune response in large yellow croaker (Larimichthys crocea) after immunization with oral vaccine.

Mesoporous silica nanoparticles (MSNs) have been used in the field of biomedicine as antigen carriers and adjuvants for protective antigens. In the present study, an oral nanovaccine against Vibrio alginolyticus was prepared employing MSNs as carriers. The uptake of the dihydrolipoamide dehydrogenase (DLDH) antigens in the intestine of large yellow croaker was evaluated using an immunohistochemistry assay. Additionally, the effects of the nanovaccine on the early immune response in large yellow croaker were investigated via oral vaccination. The presence of the antigens was detected in the mucosa and lamina propria of the foregut, midgut, and hindgut of large yellow croaker at 3 h following oral immunization. The expression levels of cytokines (i.e., lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13) in the intestine, spleen, and head kidney tissues of large yellow croaker before and after the immune challenge were determined via RT-qPCR assay. The obtained results revealed that the expression levels of lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13 in the intestine and head kidney of the vaccinated large yellow croaker, as well as the expression of lysozyme, IL-1ß, and IL-10 in the spleen, exhibited time-dependent oscillation regulation patterns. Notably, the nanovaccine immunization could induce early (6 h) and high expression of IFN-γ in the spleen and kidney tissues after the bacterial infection. The current study supplements the available data on the early immune response to fish nanovaccines. It also provides a valuable theoretical basis for the future development of large yellow croaker oral vaccines.

Molecular cloning, inducible expression with SGIV and Vibrio alginolyticus challenge, and function analysis of Epinephelus coioides PDCD4.

Programmed cell death 4 (PDCD4) in mammals, a gene closely associated with apoptosis, is involved in many biological processes, such as cell aging, differentiation, regulation of cell cycle, and inflammatory response. In this study, grouper Epinephelus coioides PDCD4, EcPDCD4-1 and EcPDCD4-2, were obtained. The open reading frame (ORF) of EcPDCD4-1 is 1413 bp encoding 470 amino acids with a molecular mass of 52.39 kDa and a theoretical pI of 5.33. The ORF of EcPDCD4-2 is 1410 bp encoding 469 amino acids with a molecular mass of 52.29 kDa and a theoretical pI of 5.29. Both EcPDCD4-1 and EcPDCD4-2 proteins contain two conserved MA3 domains, and their mRNA were detected in all eight tissues of E. coioides by quantitative real-time PCR (qRT-PCR) with the highest expression in liver. The expressions of two EcPDCD4s were significantly up-regulated after Singapore grouper iridovirus (SGIV) or Vibrio alginolyticus infection. In addition, over-expression of EcPDCD4-1 or EcPDCD4-2 can inhibit the activity of the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), and regulate SGIV-induced apoptosis. The results demonstrated that EcPDCD4s might play important roles in E. coioides tissues during pathogen-caused inflammation.

Molecular characterization, expression and function analysis of Epinephelus coioides MKK4 response to SGIV and Vibrio alginolyticus infection.

Mitogen-activated protein kinase 4 (MKK4), a member of the MAP kinase family, play important roles in response to many environmental and cellular stresses in mammals. In this study, three MKK4 subtypes, EcMKK4-1, EcMKK4-2 and EcMKK4-3, were obtained from grouper Epinephelus coioides. The open reading frame (ORF) of EcMKK4s are obtained and the EcMKK4s proteins contain highly conserved domains: a S_TKc domain, a canonical diphosphorylation group and two conserved MKKK ATP binding motifs, Asp-Phe-Gly (DFG) and Ala-Pro-Glu (APE). EcMKK4s could be found both in the cytoplasmic and nuclear. The EcMKK4s mRNA were detected in all E. coioides tissues examined with the different expression levels, and the expression were up-regulated during SGIV (Singapore grouper iridescent virus) or Vibrio alginolyticus infection. EcMKK4 could significantly reduce the activation of AP-1 reporter gene. The results suggested that EcMKK4s might play important roles in pathogen-caused inflammation.