RESUMEN

Largemouth bass ranavirus (LMBV) causes disease outbreaks and high mortality at all stages of largemouth bass farming. Therefore, live vaccine development is critical for largemouth bass prevention against LMBV by immersion immunization. Herein, an attenuated LMBV strain with good immunogenicity, designated as LMBV-2007136, was screened from the natural LMBV strains bank through challenge assay and immersion immunization experiment. After determing the safe concentration range of LMBV-2007136, the minimum immunizing dose of immersion immunization was verified. When largemouth bass were vaccinated by immersion at the lowest concentration of 102.0 TCID50/mL, all of fish were survival post virulent LMBV challenge, and the relative percent survival (RPS) was 100 %. And the immune gene expression levels of IL-10, IL-12, IFN-γ, and IgM in the spleen and kidney post-vaccination were significantly up-regulated compared to the control group, but TNF-α expression showed no significant changes. The safety and efficacy of LMBV-2007136 at passages P8, P13, and P18 were futher assessed, and no death of largemouth bass was observed within 21 days post-immunization and RPS of three vaccination groups was 100 %, suggesting that the safety and efficacy of the attenuated strain at different passages was stable. Furthermore, in the virulence reversion test, the attenuated strain was propagated through 5 times in largemouth bass by intraperitoneal injection and no abnormality and mortality were observed, further proving the attenuated vaccine candidate LMBV-2007136 was safe. These results proved that LMBV-2007136 could be a promising candidate for a live vaccine to protect largemouth bass from LMBV disease.

Asunto(s)

Lubina , Infecciones por Virus ADN , Enfermedades de los Peces , Ranavirus , Vacunas Atenuadas , Vacunas Virales , Animales , Lubina/inmunología , Ranavirus/inmunología , Enfermedades de los Peces/prevención & control , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/prevención & control , Infecciones por Virus ADN/inmunología , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/administración & dosificación , Inmunización/veterinaria , Inmersión , Vacunación/veterinaria

RESUMEN

Largemouth bass ranavirus (LMBV) seriously affects the development of largemouth bass (Micropterus salmoides) industry and causes huge economic losses. Oral vaccine can be a promising method for viral disease precaution. In this study, MCP2α was identified as a valuable epitope region superior to MCP and MCP2 of LMBV by neutralizing antibody experiments. Then, recombinant Lactobacillus casei expressing the fusion protein MCP2αC (MCP2α as antigen, C represents flagellin C from Aeromonas hydrophila as adjuvant) on surface was constructed and verified. Further, PLA microsphere vaccine loading recombinant MCP2αC L. casei was prepared. The PLA microspheres vaccine were observed by scanning electron microscopy and showed a smooth, regular spherical surface with a particle size distribution between 100 and 200 µm. Furthermore, we evaluated the tolerance of PLA-MCP2αC vaccine in simulated gastric fluid and simulated intestinal fluid, and the results showed that PLA-MCP2αC can effectively resist the gastrointestinal environment. Moreover, the protective effect of PLA-MCP2αC against LMBV was evaluated after oral immunization and LMBV challenge. The results showed that PLA-MCP2αC effectively up-regulated the activity of serum biochemical enzymes (T-SOD, T-AOC, LZM, complement C3) and induced the mRNA expression of representative immune genes (IL-1ß, TNF-α, IFN-γ, MHC-IIα, Mx, IgM) in spleen and head kidney tissues. The survival rate of largemouth bass vaccinated with PLA-MCP2αC increased from 24 % to 68 %. Meanwhile, PLA-MCP2αC inhibited the LMBV burden in spleen, head kidney and liver tissues and attenuated tissue damage in spleen. These results suggested that PLA-MCP2αC can be used as a candidate oral vaccine against LMBV infection in aquaculture.

Asunto(s)

Lubina , Infecciones por Virus ADN , Enfermedades de los Peces , Lacticaseibacillus casei , Microesferas , Animales , Lubina/inmunología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/prevención & control , Lacticaseibacillus casei/inmunología , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/prevención & control , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Poliésteres/administración & dosificación , Iridoviridae

RESUMEN

Probiotics play an essential role in the largemouth bass (Micropterus salmoides) aquaculture sector. They aid the fish in sickness prevention, intestinal structure improvement, food absorption, and immune system strengthening. In this experiment, Bacillus subtilis (BS, 107 CFU/g) and Lactobacillus reuteri (LR, 107 CFU/g) were added to the feed and then fed to M. salmoides for 35 days. The effects of two probiotics on the growth, immunity, and metabolism of M. salmoides organisms were studied. The results revealed that the BS group significantly increased the growth rate and specific growth rate of M. salmoides, while both the BS and LR groups significantly increase the length of villi M. salmoides intestines. The BS group significantly increased the levels of AKP, T-AOC, and CAT in the blood of M. salmoides, as well as AKP levels in the intestine. Furthermore, the BS group significantly increased the expression of intestinal genes Nrf2, SOD1, GPX, and CAT, while significantly decreasing the expression of the keap1 gene. M. salmoides gut microbial analysis showed that the abundance of Planctomycetota was significantly different in both control and experimental groups. Analyzed at the genus level, the abundance of Citrobacter, Paracoccus, Luedemannella， Sphingomonas, Streptomyces and Xanthomonas in the both control and experimental groups were significantly different. The BS group's differentially expressed genes were predominantly enriched in oxidative phosphorylation pathways in the intestine, indicating that they had a good influence on intestinal metabolism and inflammation suppression. In contrast, differentially expressed genes in the LR group were primarily enriched in the insulin signaling and linoleic acid metabolism pathways, indicating improved intestine metabolic performance. In conclusion, B. subtilis and L. reuteri improve the growth and health of M. salmoides, indicating tremendous potential for enhancing intestinal metabolism and providing significant application value.

Asunto(s)

Alimentación Animal , Bacillus subtilis , Lubina , Suplementos Dietéticos , Microbioma Gastrointestinal , Limosilactobacillus reuteri , Probióticos , Animales , Probióticos/administración & dosificación , Lubina/inmunología , Lubina/crecimiento & desarrollo , Lubina/microbiología , Limosilactobacillus reuteri/inmunología , Limosilactobacillus reuteri/fisiología , Microbioma Gastrointestinal/inmunología , Intestinos/inmunología , Intestinos/microbiología , Acuicultura , Proteínas de Peces/metabolismo , Proteínas de Peces/genética

RESUMEN

Our previous study has demonstrated that supplementation of yeast ß-glucan improves intestinal health in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀), accompanied by the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we investigated the effects of perturbing p38 MAPK activity using an inhibitor on the intestinal health of ß-glucan-injected pearl gentian grouper to elucidate the potential molecular mechanism underlying the protective effects of ß-glucan on the fish gut. The pearl gentian grouper was categorized into four groups: PBS injected (CD group), ß-glucan injected at a dose of 80 mg/kg (ßG group), p38 MAPK inhibitor SB203580 injected at a dose of 1 mg/kg (SB203580 group), and a combination of ß-glucan (80 mg/kg) and SB203580 (1 mg/kg) injected together (ßG + SB203580 group). The results revealed that the introduction of SB203580 significantly suppressed the ß-glucan-induced increase in p38α and p38ß mRNA expression, as well as the phosphorylation of p38 MAPK. Both the ßG group and SB203580 group exhibited reduced plica height and muscularis thickness. The ßG + SB203580 group displayed a significant reduction in mucin cell level; interleukin 1ß (il1ß) mRNA expression; induced nitric oxide synthase, tumor necrosis factor α, and IL1ß concentration; catalase and total antioxidant capacity activities. Additionally, there was a significant increase in the levels of intestinal malondialdehyde in the ßG + SB203580 group compared to the ßG group. The inhibition of the p38 MAPK signaling halted the trend of apoptosis-related caspase molecular expression induced by ß-glucan. In conclusion, ß-glucan injection resulted in elevated levels of mucous cells, nonspecific immunity, antioxidant capacity, and anti-apoptosis in grouper by modulating the p38 MAPK pathway. This study offers insights into the potential molecular mechanism underlying the protective effects of ß-glucan on intestinal health in pearl gentian grouper.

Asunto(s)

Intestinos , beta-Glucanos , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , beta-Glucanos/farmacología , beta-Glucanos/administración & dosificación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Intestinos/efectos de los fármacos , Imidazoles/farmacología , Imidazoles/administración & dosificación , Piridinas/farmacología , Lubina/inmunología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Suplementos Dietéticos/análisis , Alimentación Animal/análisis , Inmunidad Innata/efectos de los fármacos

RESUMEN

Singapore grouper iridovirus (SGIV) always causes high transmission efficiency and mortality in the larval and juvenile stages of grouper in aquaculture industry. Although inactivated virus and recombinant DNA vaccines administered via intraperitoneal injection have shown efficacy in protection against SGIV, their potential applications in field testing were limited due to the vaccine delivery methods. Here, we developed an immersion vaccine containing inactivated virus and Montanide IMS 1312 adjuvant (IMS 1312) and evaluated its protective efficacy against SGIV infection. Compared to the PBS group, fish vaccinated with immersion inactivated vaccine with or without IMS 1312 were significantly protected against SGIV, with a relative percent survival (RPS) of 57.69 % and 38.47 %, respectively. Furthermore, the transcripts of viral core genes were reduced, and the histopathological severity caused by SGIV were relatively mild in multiple tissues of the IMS + V group. The immersion vaccine activated the AKP and ACP activities and increased the mRNA levels of IFN and inflammation-associated genes. The transcriptome analysis showed that a total of 731 and 492 genes were significantly regulated in the spleen and kidney from the IMS + V group compared to the PBS group, respectively. Among them, 129 DEGs were co-regulated, and enriched in the KEGG pathways related to immune and cell proliferation, including MAPK signaling, JAK-STAT signaling and PI3K-Akt signaling pathways. Similarly, the DEGs specially regulated in the kidney and spleen upon vaccine immunization were significantly enriched in the KEGG pathways related to interferon and inflammation response. Together, our results elucidated that the immersion vaccine of inactivated SGIV with IMS 1312 induced a protective immune response of grouper against SGIV.

Asunto(s)

Infecciones por Virus ADN , Enfermedades de los Peces , Ranavirus , Vacunas de Productos Inactivados , Vacunas Virales , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/prevención & control , Enfermedades de los Peces/virología , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/administración & dosificación , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/prevención & control , Ranavirus/fisiología , Ranavirus/inmunología , Lubina/inmunología , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/administración & dosificación , Inmunidad Innata , Inmersión

RESUMEN

Interferon-related developmental regulator 1 (IFRD1) is a viral responsive gene associated with interferon-gamma. Herein, we identified the IFRD1 gene (EaIFRD1) from red-spotted grouper (Epinephelus akaara), evaluated its transcriptional responses, and investigated its functional features using various biological assays. EaIFRD1 encodes a protein comprising 428 amino acids with a molecular mass of 48.22 kDa. It features a substantial domain belonging to the interferon-related developmental regulator superfamily. Spatial mRNA expression of EaIFRD1 demonstrated the highest expression levels in the brain and the lowest in the skin. Furthermore, EaIFRD1 mRNA expression in grouper tissues exhibited significant modulation in response to immune stimulants, including poly (I:C), LPS, and nervous necrosis virus (NNV) infection. We analyzed downstream gene regulation by examining type Ⅰ interferon pathway genes following EaIFRD1 overexpression. The results demonstrated a significant upregulation in cells overexpressing EaIFRD1 compared to the control after infection with viral hemorrhagic septicemia virus (VHSV). A subcellular localization assay confirmed the nuclear location of the EaIFRD1 protein, consistent with its role as a transcriptional coactivator. Cells overexpressing EaIFRD1 exhibited increased migratory activity, enhancing wound-healing capabilities compared to the control. Additionally, under H2O2 exposure, EaIFRD1 overexpression protected cells against oxidative stress. Overexpression of EaIFRD1 also reduced poly (I:C)-mediated NO production in RAW267.4 macrophage cells. In FHM cells, EaIFRD1 overexpression significantly reduced VHSV virion replication. Collectively, these findings suggest that EaIFRD1 plays a crucial role in the antiviral immune response and immunological regulation in E. akaara.

Asunto(s)

Secuencia de Aminoácidos , Lubina , Enfermedades de los Peces , Proteínas de Peces , Regulación de la Expresión Génica , Inmunidad Innata , Filogenia , Poli I-C , Animales , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Lubina/inmunología , Lubina/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Poli I-C/farmacología , Nodaviridae/fisiología , Alineación de Secuencia/veterinaria , Perfilación de la Expresión Génica/veterinaria , Novirhabdovirus/fisiología , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/veterinaria , Lipopolisacáridos/farmacología

RESUMEN

Aeromonas salmonicida is a common pathogenic bacterial species found in both freshwater and marine fish, leading to significant economic losses in the aquaculture industry. YidC is an accessory to SecYEG and is essential for the SecYEG transporter to insert into the bacterial membrane. However, the roles of the yidC gene on the host immune response remain unclear. Here, we compared the pathogenicity of yidC gene-deleted (ΔyidC) strain and wild-type (SRW-OG1) strain of mesophilic A. salmonicida to Orange-spotted grouper (Epinephelus coioides), and explored the impacts of yidC gene on the immune response of E. coioides to mesophilic A. salmonicida infection by using Red/ET recombineering. In this study, the E. coioides in the Secondary infected group had a 53.9 % higher survival rate than those in the Primary infected group. In addition, the adhesion ability of ΔyidC strain decreased by about 83.36 % compared with that of the wild-type (SRW-OG1) strain. Further comparison of the biological phenotype of SRW-OG1 and ΔyidC revealed that this yidC gene could regulate the expression of genes related to iron metabolism and have no effect on bacterial growth under the limited iron concentration. In the low concentration of Fe3+ and Fe2+ environment, SRW-OG1 can obtain iron ions by regulating yidC. Based on the above results, yidC gene contributed to the pathogenicity of mesophilic A. salmonicida to E. coioides, deletion of yidC gene promoted the inflammation and immune response of E. coioides to mesophilic A. salmonicida infection.

Asunto(s)

Aeromonas salmonicida , Proteínas Bacterianas , Enfermedades de los Peces , Infecciones por Bacterias Gramnegativas , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Virulencia , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Infecciones por Bacterias Gramnegativas/microbiología , Aeromonas salmonicida/fisiología , Aeromonas salmonicida/patogenicidad , Proteínas Bacterianas/genética , Lubina/inmunología , Lubina/genética , Inmunidad Innata/genética

RESUMEN

Scavenger receptors (SRs) are integral to the innate immune system and function as pattern-recognition receptors that facilitate pathogen clearance and mediate anti-inflammatory responses. However, the role of SRs in the immune response of Lateolabrax maculatus against Aeromonas veronii is unclear. Here, we cloned scavenger receptor B1 from L. maculatus (LmSRB1) and performed bioinformatics analysis to study its potential functions. The open reading frame spans 1530 base pairs and encodes a 509-amino acid protein with a molecular mass of 57.44 kDa. Comparative analysis revealed high sequence conservation among fish species. Expression profiling revealed strong LmSRB1 transcription in various tissues, especially in head kidney and spleen. Following A. veronii exposure, LmSRB1 expression initially increased, peaking after 4-8 h, with a notable secondary peak at 72 h. Fluorescence in situ hybridization indicated that LmSRB1 mainly localized to the cytoplasm, and subcellular-localization studies confirmed LmSRB1 protein expression in the cytoplasm and cell membrane. Enzyme-linked immunosorbent assay data showed dose-dependent binding of LmSRB1 to A. veronii. Modulating LmSRB1 expression significantly altered the levels of IL-8, IL-1ß, TRAF6, and NIK. These results highlight the crucial role of LmSRB1 in L. maculatus's innate immune response to A. veronii and offer insights into improving the management of bacterial infections in aquaculture.

Asunto(s)

Secuencia de Aminoácidos , Lubina , Enfermedades de los Peces , Proteínas de Peces , Perfilación de la Expresión Génica , Infecciones por Bacterias Gramnegativas , Inmunidad Innata , Filogenia , Alineación de Secuencia , Animales , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Enfermedades de los Peces/inmunología , Lubina/inmunología , Lubina/genética , Inmunidad Innata/genética , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Perfilación de la Expresión Génica/veterinaria , Alineación de Secuencia/veterinaria , Regulación de la Expresión Génica/inmunología , Aeromonas veronii/fisiología

RESUMEN

T-cell/transmembrane immunoglobulin and mucin domain-containing (TIM) protein family has attracted particular attention because of their broad immune functions and the response to viral infections. TIM-1, a member of the TIM family, has been demonstrated to play an important role in viral infections. However, its roles during fish nodavirus infection still remained largely unknown. In this study, a homolog of TIM-1 from orange-spotted grouper (Epinephelus coioides) (EcTIM-1) was identified, and characterized. EcTIM-1 encoded a 217-amino acids protein, containing one Immunoglobulin domain. Homology analysis showed that EcTIM-1 shared 98.62 % and 42.99 % identity to giant grouper (E. lanceolatus) and human (Homo sapiens). Quantitative Real-time PCR analyses indicated that EcTIM-1 was expressed in all examined tissues, with higher expression in liver, spleen, skin, and heart, and was significantly up-regulated in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. EcTIM-1 was distributed in the cytoplasm, and partly co-localized with Golgi apparatus and lysosomes in vitro. The ectopic expression of EcTIM-1 promoted RGNNV replication by increasing the level of viral genes transcription and protein synthesis. Besides, overexpression of EcTIM-1 decreased the luciferase activity of type I interferon (IFN1), interferon stimulated response elements (ISRE) and nuclear factor kappa-B (NF-κB) promoters, as well as the transcription of pro-inflammatory factors and interferon related genes. EcTIM-1 significantly suppressed the luciferase activity of IFN1, ISRE and NF-κB promoters evoked by Epinephelus coioides melanoma differentiation-associated gene 5 (EcMDA5), mitochondrial antiviral signaling protein (EcMAVS), stimulator of IFN genes (EcSTING) or TANK-binding kinase 1 (EcTBK1). Collectively, EcTIM-1 negatively regulated interferon and inflammatory response to promote RGNNV infection. These results provide a basis for a better understanding of the innate immune response of TIM-1 in fish.

Asunto(s)

Lubina , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Nodaviridae , Filogenia , Infecciones por Virus ARN , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/veterinaria , Nodaviridae/fisiología , Lubina/inmunología , Lubina/genética , Inmunidad Innata/genética , Regulación de la Expresión Génica/inmunología , Inflamación/inmunología , Inflamación/veterinaria , Inflamación/genética , Secuencia de Aminoácidos , Perfilación de la Expresión Génica/veterinaria , Alineación de Secuencia/veterinaria

RESUMEN

The Asian seabass (Lates calcarifer) faces significant disease threats, which are exacerbated by intensive farming practices and environmental changes. Therefore, understanding its immune system is crucial. The current study presents a comprehensive analysis of immune-related genes in Asian seabass peripheral blood leukocytes (PBLs) using Iso-seq technology, identifying 16 key pathways associated with 7857 immune-related genes, comprising 634 unique immune-related genes. The research marks the first comprehensive report on the entire immunoglobulin repertoire in Asian seabass, revealing specific characteristics of immunoglobulin heavy chain constant region transcripts, including IgM (Cµ, ighm), IgT (Cτ, ight), and IgD (Cδ, ighd). The study confirms the presence of membrane-bound form, ighmmb, ightmb, ighdmb of IgM, IgT and IgD and secreted form, ighmsc and ightsc of IgM and IgT, respectively, with similar structural patterns and conserved features in amino acids across immunoglobulin molecules, including cysteine residues crucial for structural integrity observed in other teleost species. In response to bacterial infections by Flavobacterium covae (formerly F. columnare genomovar II) and Streptococcus iniae, both secreted and membrane-bound forms of IgM (ighmmb and ighmsc) and IgT (ightmb and ightsc) show significant expression, indicating their roles in systemic and mucosal immunity. The expression of membrane-bound form IgD gene, ighdmb, predominantly exhibits targeted upregulation in PBLs, suggesting a regulatory role in B cell-mediated immunity. The findings underscore the dynamic and tissue-specific expression of immunoglobulin repertoires, ighmmb, ighmsc, ightmb, ightsc and ighdmb in Asian seabass, indicating a sophisticated immune response to bacterial pathogens. These findings have practical implications for fish aquaculture, and disease control strategies, serving as a valuable resource for advancing research in Asian seabass immunology.

Asunto(s)

Enfermedades de los Peces , Proteínas de Peces , Infecciones por Flavobacteriaceae , Flavobacterium , Inmunoglobulina D , Inmunoglobulina M , Inmunoglobulinas , Infecciones Estreptocócicas , Streptococcus iniae , Animales , Enfermedades de los Peces/inmunología , Flavobacterium/fisiología , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/veterinaria , Inmunoglobulina M/inmunología , Inmunoglobulina M/genética , Infecciones por Flavobacteriaceae/inmunología , Infecciones por Flavobacteriaceae/veterinaria , Infecciones por Flavobacteriaceae/genética , Inmunoglobulina D/genética , Inmunoglobulina D/inmunología , Inmunoglobulina D/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Streptococcus iniae/fisiología , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Inmunidad Innata/genética , Lubina/inmunología , Lubina/genética

RESUMEN

NLRP3 has an important role in the immune response and viral infection as an essential inflammasome component. However, it is unclear whether the grouper immune system is regulated by NLRP3 inflammasome. In this study, we cloned the NLRP3 gene from Epinephelus coioides. Ec-NLRP3 encodes 893 amino acids and contains two major structural domains, the NACHT domain (69-234aa) and the LRR domain (477-893aa). Tissue distribution analysis showed that Ec-NLRP3 was expressed in all tissues tested, with the spleen exhibiting the highest expression. Additionally, after being infected with SGIV, the expression of the Ec-NLRP3 gene was significantly increased. The results of subcellular localization revealed that Ec-NLRP3 was distributed throughout GS cells. In addition, Ec-NLRP3 co-localized with Ec-ASC and was observed as a cytosolic speck. Ec-NLRP3 overexpression significantly inhibited SGIV infection, which was further inhibited by co-overexpression of Ec-NLRP3 and Ec-ASC. Further studies revealed that overexpression of Ec-NLRP3 significantly upregulated caspase-1 activity, and co-overexpression of Ec-NLRP3 and Ec-ASC further upregulated caspase-1 activity. In addition, inhibition of Caspase-1 activity with VX-765 significantly increased the infection of SGIV. Furthermore, the NLRP3 inflammasome activator Nigericin was able to inhibit the infection of SGIV significantly. The above findings suggest that Ec-NLRP3 inhibits SGIV infection by upregulating caspase-1 activity.

Asunto(s)

Lubina , Caspasa 1 , Enfermedades de los Peces , Proteínas de Peces , Regulación de la Expresión Génica , Proteína con Dominio Pirina 3 de la Familia NLR , Filogenia , Alineación de Secuencia , Regulación hacia Arriba , Animales , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Lubina/inmunología , Lubina/genética , Alineación de Secuencia/veterinaria , Regulación de la Expresión Génica/inmunología , Caspasa 1/genética , Caspasa 1/inmunología , Caspasa 1/metabolismo , Secuencia de Aminoácidos , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/veterinaria , Inmunidad Innata/genética , Perfilación de la Expresión Génica/veterinaria , Iridoviridae

RESUMEN

Viral nervous necrosis (VNN) presents a significant challenge to aquaculture due to its potential for causing mass fish mortality and resulting in substantial economic losses. Therefore, the urgent need to find antiviral drugs is paramount. This study found that oleanolic acid (OA) exhibited anti-nervous necrosis virus (NNV) activity both in vivo and in vitro. The RT-qPCR results demonstrated that OA at 10.95 µM had an inhibition rate of 99.97 %. The prevention experiments also showed that OA pretreatment effectively inhibited the replication of NNV. Furthermore, the results of indirect immunofluorescence and flow cytometry suggest that OA's anti-NNV effect may be due to its ability to inhibit NNV-induced apoptosis. The in vivo study revealed a 30 % survival rate in the OA treatment group, compared to only 10 % in the control group. Additionally, RT-qPCR results demonstrated that OA treatment upregulated immune gene expression in grouper and effectively suppressed NNV replication in the host. This study demonstrates the potential of OA as an antiviral therapeutic agent for NNV. It exerts its antiviral effect by upregulating immune gene expression. These findings provide valuable insights into the development of novel antiviral treatment strategies.

Asunto(s)

Antivirales , Enfermedades de los Peces , Nodaviridae , Ácido Oleanólico , Infecciones por Virus ARN , Animales , Nodaviridae/fisiología , Nodaviridae/efectos de los fármacos , Ácido Oleanólico/farmacología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Enfermedades de los Peces/tratamiento farmacológico , Antivirales/farmacología , Infecciones por Virus ARN/veterinaria , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/virología , Lubina/inmunología , Replicación Viral/efectos de los fármacos

RESUMEN

Probiotic Bacillus pumilus SE5, heat-inactivated (HSE5) or active (ASE5), were supplemented to high soybean meal (HSM) (36 %) diet at whole term (0-56 days) and middle term (29-56 days) to investigate the preventing and repairing effects of B. pumilus SE5 in ameliorating the adverse effects of HSM in Epinephelus coioides. The results suggested that the HSM significantly decreased the weight gain rate (WGR), specific growth rate (SGR), and increased the feed conversion rate (FCR) at day 56 (P < 0.05), while HSE5 and ASE5 promoted the growth performance. The HSE5 and ASE5 showed preventive and reparative functions on the antioxidant capacity and serum immunity, with significantly increased the total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-PX) activities, and reduced malondialdehyde (MDA) level, and increased acid phosphatase (ACP), alkaline phosphatase (AKP), immunoglobulin M (IgM) and complement 3 (C3). The HSM impaired the intestinal health (destroyed the intestinal structure, significantly increased the contents of serum D-lactic acid and diamine oxidase, and reduced the expressions of claudin-3 and occludin), while HSE5 and ASE5 improved them at whole term and middle term. The HSM impaired the intestinal microbiota and reduced its diversity, and the HSE5 or ASE5 improved the intestinal microbiota (especially at whole term). HSE5 and ASE5 improved the intestinal mRNA expressions of anti-inflammatory genes (il-10 and tgf-ß1) and reduced the expressions of pro-inflammatory genes (il-1ß, il-8, il-12), and promoted the expressions of humoral immune factor-related genes (cd4, igm, mhcII-α) and antimicrobial peptide genes (ß-defensin, epinecidin-1 and hepcidin-1), and decreased the expressions of NF-κB/MAPK signaling pathway-related genes (ikk-α, nf-κb, erk-1), and improved the expressions of MAPK signaling pathway-related gene p38-α (P < 0.05). In conclusion, the heat-inactivated and active B. pumilus SE5 effectively prevented and repaired the suppressive effects of soybean meal in E. coioides, which underscored the potential of B. pumilus SE5 as a nutritional intervention agent in HSM diet in aquaculture.

Asunto(s)

Alimentación Animal , Bacillus pumilus , Lubina , Dieta , Glycine max , Probióticos , Animales , Lubina/inmunología , Alimentación Animal/análisis , Dieta/veterinaria , Probióticos/administración & dosificación , Probióticos/farmacología , Bacillus pumilus/inmunología , Bacillus pumilus/química , Glycine max/química , Calor/efectos adversos , Inmunidad Innata , Distribución Aleatoria , Microbioma Gastrointestinal/efectos de los fármacos

RESUMEN

Largemouth bass virus (LMBV) infections has resulted in high mortality and economic losses to the global largemouth bass industry and has seriously restricted the healthy development of the bass aquaculture industry. There are currently no antiviral therapies available for the control of this disease. In this study, we developed three types of vaccine against LMBV; whole virus inactivated vaccine (I), a subunit vaccine composed of the major viral capsid protein MCP (S) as well as an MCP DNA vaccine(D), These were employed using differing immunization and booster strategies spaced 2 weeks apart as follows: II, SS, DD and DS. We found that all vaccine groups induced humoral and cellular immune responses and protected largemouth bass from a lethal LMBV challenge to varying degrees and DD produced the best overall effect. Specifically, the levels of specific IgM in serum in all immunized groups were elevated and significantly higher than those in the control group. Moreover, the expression of humoral immunity (CD4 and IgM) and cellular immunity (MHCI-α) as well as cytokines (IL-1ß) was increased, and the activity of immunity-related enzymes ACP, AKP, LZM, and T-SOD in the serum was significantly enhanced. In addition, the relative percent survival of fish following an LMBV lethal challenge 4 weeks after the initial immunizations were high for each group: DD(89.5 %),DS(63.2 %),SS(50 %) and II (44.7 %). These results indicated that the MCP DNA vaccine is the most suitable and promising vaccine candidate for the effective control of LMBV disease.

Asunto(s)

Lubina , Infecciones por Virus ADN , Enfermedades de los Peces , Vacunas de ADN , Vacunas Virales , Animales , Vacunas de ADN/inmunología , Vacunas de ADN/administración & dosificación , Enfermedades de los Peces/prevención & control , Enfermedades de los Peces/inmunología , Lubina/inmunología , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/prevención & control , Infecciones por Virus ADN/inmunología , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/administración & dosificación , Inmunidad Humoral , Ranavirus/inmunología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/administración & dosificación , Inmunidad Celular

RESUMEN

Spotted seabass (Lateolabrax maculatus) is the second largest maricultural fish species in China and is the main trigger of food-related allergic reactions. Nevertheless, studies on the allergens of L. maculatus are limited. This study aimed to characterize pan-allergen parvalbumin from L. maculatus. Two proteins of about 11 kDa were purified and confirmed as parvalbumins by mass spectrometry. The IgG- and IgE-binding activities were evaluated through an immunoblotting assay. The molecular characteristics of ß-parvalbumin were investigated by combining proteomics, genomics, and immunoinformatics approaches. The results indicated that ß-parvalbumin consists of 109 amino acids with a molecular weight of 11.5 kDa and is the major allergen displaying strong IgE-binding capacity. In silico analysis and a dot blotting assay confirmed seven linear B cell epitopes distributed mainly on α-helixes and the calcium-binding loops. In addition, the cross-reactivity among 26 commonly consumed fish species was analyzed. The in-house generated anti-L. maculatus parvalbumin polyclonal antibody recognized 100% of the 26 fish species, demonstrating cross-reactivity and better binding capacity than the anticod parvalbumin antibody. Together, this study provides an efficient protocol to characterize allergens with multiomics methods and supports parvalbumin from L. maculatus as a candidate for fish allergen determination and allergy diagnosis.

Asunto(s)

Alérgenos , Reacciones Cruzadas , Proteínas de Peces , Hipersensibilidad a los Alimentos , Inmunoglobulina E , Parvalbúminas , Parvalbúminas/inmunología , Parvalbúminas/química , Parvalbúminas/genética , Animales , Alérgenos/inmunología , Alérgenos/genética , Alérgenos/química , Proteínas de Peces/inmunología , Proteínas de Peces/química , Proteínas de Peces/genética , Inmunoglobulina E/inmunología , Hipersensibilidad a los Alimentos/inmunología , Lubina/inmunología , Lubina/genética , Epítopos/inmunología , Epítopos/química , Humanos , Proteómica , Inmunoglobulina G/inmunología , Secuencia de Aminoácidos , Multiómica

RESUMEN

Chinese seabass (Lateolabrax maculatus) stands out as one of the most sought-after and economically significant species in aquaculture within China. Diseases of L. maculatus occur frequently due to the degradation of the germplasm, the aggravation of environmental pollution of water, and the reproduction of pathogenic microorganisms, inflicting considerable economic losses on the Chinese seabass industry. The Myxovirus resistance (Mx) gene plays pivotal roles in the antiviral immune response ranging from mammals to fish. However, the function of the Mx gene in L. maculatus is still unknown. Firstly, the origin and evolutionary history of Mx proteins was elucidated in this study. Subsequently, an Mx gene from L. maculatus (designed as LmMxA gene) was identified, and its functions in combating antiviral and antibacterial threats were investigated. Remarkably, our findings suggested that while Mx group genes were present in chordates, DYN group genes were present in everything from single-celled animals to humans. Furthermore, our investigation revealed that the LmMxA mRNA level increased in the kidney, spleen and liver subsequent to Vibrio anguillarum and poly(I:C) challenged. Immunofluorescence analysis indicated that LmMxA is predominantly localization in the nucleus and the cytoplasm. Notably, the expression of MAVS, IFN1 and Mx1 increased when LmMxA was overexpression within the EPC cells. Moreover, through assessment via cytopathic effect (CPE), virus titer, and antibacterial activity, it becomes evident that LmMxA exerts a dual role in bolstering both antiviral and antibacterial immune responses. These compelling findings laid the foundation for further exploring the mechanism of LmMxA in response to innate immunity of L. maculatus.

Asunto(s)

Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Proteínas de Resistencia a Mixovirus , Filogenia , Animales , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , Proteínas de Resistencia a Mixovirus/inmunología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Inmunidad Innata/genética , Regulación de la Expresión Génica/inmunología , Vibriosis/inmunología , Vibriosis/veterinaria , Vibrio/fisiología , Secuencia de Aminoácidos , Alineación de Secuencia/veterinaria , Poli I-C/farmacología , Lubina/inmunología , Lubina/genética , Perfilación de la Expresión Génica/veterinaria , Evolución Molecular

RESUMEN

Aquaculture is a prosperous economic sector threatened by viral infections. Among the viruses threatening fish culture, Betanodavirus (NNV) is extremely important in the Mediterranean Sea affecting to highly traded species as European sea bass. In this context, application of antimicrobial peptides (AMPs) has arisen as a potential biotechnological tool. The aim of this work was to evaluate the therapeutic application of two European sea bass-derived AMPs, NK-lysin (Nkl) and dicentracin (Dic), against NNV infections. Synthetic Dic peptide was able to significantly reduce NNV-induced mortalities while Nkl failed to do so. Although neither Dic nor Nkl peptides were able to alter the transcriptional levels of NNV and the number of infected cells, Nkl seemed to increase the viral load per cell. Interestingly, both Nkl and Dic peptides showed immunomodulatory roles. For instance, our data revealed an interplay among different AMPs, at both gene and protein levels. Otherwise, Nkl and Dic peptides provoked an anti-inflammatory balance upon NNV infection, as well as the recruitment of macrophages and B cells to the target site of the infection, the brain. In conclusion, Dic can be proposed as a therapeutic candidate to combat NNV.

Asunto(s)

Lubina , Enfermedades de los Peces , Nodaviridae , Infecciones por Virus ARN , Nodaviridae/fisiología , Animales , Lubina/inmunología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/tratamiento farmacológico , Enfermedades de los Peces/virología , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/veterinaria , Infecciones por Virus ARN/tratamiento farmacológico , Proteolípidos/farmacología , Proteolípidos/inmunología , Proteínas de Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/farmacología , Proteínas de Peces/química , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/química , Agentes Inmunomoduladores/farmacología , Agentes Inmunomoduladores/química

RESUMEN

Singapore grouper iridovirus (SGIV) belongs to the family Iridoviridae and the genus Ranavirus, which is a large cytoplasmic DNA virus. Infection of grouper with SGIV can cause hemorrhage and swelling of the spleen of the fish. Previous work on genome annotation demonstrated that SGIV contained numerous uncharacterized or hypothetical open reading frames (ORFs), whose functions remained largely unknown. In the present study, the protein encoded by SGIV ORF128 (VP128) was identified. VP128 is predominantly localized within the endoplasmic reticulum (ER). Overexpression of VP128 significantly promoted SGIV replication. VP128 inhibited the interferon (IFN)-3 promoter activity and mRNA level of IFN-related genes induced by poly(I:C), Epinephelus coioides cyclic GMP/AMP synthase (EccGAS)/stimulator of IFN genes (EcSTING), and TANK-binding kinase 1 (EcTBK1). Moreover, VP128 interacted with EcSTING and EcTBK1. The interaction between VP128 and EcSTING was independent of any specific structural domain of EcSTING. Together, our results demonstrated that SGIV VP128 negatively regulated the IFN response by inhibiting EcSTING-EcTBK1 signaling for viral evasion.

Asunto(s)

Infecciones por Virus ADN , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Ranavirus , Transducción de Señal , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Ranavirus/fisiología , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/veterinaria , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Transducción de Señal/inmunología , Inmunidad Innata/genética , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Evasión Inmune , Lubina/inmunología , Regulación de la Expresión Génica/inmunología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos

RESUMEN

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-ß (IFN-ß) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-ß (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

Asunto(s)

Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Lubina , Enfermedades de los Peces , Proteínas de Peces , FN-kappa B , Filogenia , Animales , Lubina/inmunología , Lubina/genética , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , FN-kappa B/genética , FN-kappa B/metabolismo , FN-kappa B/inmunología , Enfermedades de los Peces/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Transducción de Señal/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Alineación de Secuencia/veterinaria , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/química , Perfilación de la Expresión Génica/veterinaria , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo , Secuencia de Bases

RESUMEN

E3 ubiquitin ligases, key components of the ubiquitin proteasome system, orchestrate protein degradation through ubiquitylation and profoundly impact cellular biology. Small HERC E3 ligases (HERC3-6) have diverse functions in mammals, including roles in spermatogenesis, protein degradation, and immunity. Until now, only mammals' HERC3, HERC5, and HERC6 are known to participate in immune responses, with major involvement in the antiviral response. Interestingly, an exclusive HERC7 has been characterized in fish showing great molecular conservation and antiviral roles. Thus, this study identifies and characterizes the herc7 gene in the European sea bass teleost. The European sea bass herc7 gene and the putative protein show good conservation of the promoter binding sites for interferons and the RCC1 and HECT domains characteristic of HERC proteins, respectively. The phylogenetic analysis shows a unique cluster with the fish-exclusive HERC7 orthologues. During ontogeny, the herc7 gene is expressed from 3 days post-fertilization onwards, being constitutively and widely distributed in adult tissues. In vitro, stimulated leucocytes up-regulate the herc7 gene in response to mitogens and viruses, pointing to a role in the immune response. Furthermore, sea bass herc7 expression is related to the interferon response intensity and viral load in different tissues upon in vivo infection with red-grouper betanodavirus (RGNNV), suggesting the potential involvement of fish HERC7 in ISGylation-based antiviral activity, similarly to mammalian HERC5. This study broadens the understanding of small HERC proteins in fish species and highlights HERC7 as a potential contributor to the immune response in European sea bass, with implications for antiviral defense mechanisms. Future research is needed to unravel the precise actions and functions of HERC7 in teleost fish immunity, providing insights into direct antiviral activity and viral evasion.

Asunto(s)

Lubina , Enfermedades de los Peces , Proteínas de Peces , Filogenia , Ubiquitina-Proteína Ligasas , Animales , Lubina/inmunología , Lubina/genética , Lubina/virología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/metabolismo , Enfermedades de los Peces/virología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/genética , Nodaviridae , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/virología , Infecciones por Virus ARN/genética , Infecciones por Virus ARN/veterinaria