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1.
Fish Shellfish Immunol ; 94: 880-888, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31562894

RESUMO

The humpback grouper (Cromileptes altivelis) is a commercially valuable species of the family Epinephelidae; however, its marketization suffers from slow growth speed, low survival rate, and various pathogenic diseases. Lactococcus lactis and Schizochytrium limacinum are commonly used as immunostimulants due to their health benefits for the aquatic organisms. In the present study, we assessed the effects of dietary supplementation with L. lactis HNL12 combined with S. limacinum algal meal on the growth performances, innate immune response, and disease resistance of C. altivelis against Vibrio harveyi. The results showed that fish fed with a combination diet of L. lactis and S. limacinum exhibited significantly higher final weight, percent weight gain, and specific growth rate compared with groups fed with them alone. A bacterial challenge experiment indicated that the group fed with the L. lactis combined with S. limacinum diet achieved the highest relative percent of survival value (68.63%), suggesting that L. lactis and S. limacinum significantly improved the disease resistance against V. harveyi after a 4-week feeding trial. Moreover, the respiratory burst activity of macrophages of fish fed with a L. lactis combined with S. limacinum diet was significantly higher than that of fish fed the control diet after 1, 2, and 3 weeks of feeding. The serum superoxide dismutase of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to those fed the control diet after 1 and 2 weeks of feeding, while the serum alkaline phosphatase of fish fed with a L. lactis combined with S. limacinum diet after 2 and 4 weeks was significantly increased, compared to the control group. The serum lysozyme activities of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to the control group after 2 weeks of feeding. Furthermore, transcriptome sequencing of the C. altivelis head kidney was conducted to explore the immune-regulating effects of the L. lactis combined with S. limacinum diet on C. altivelis. A total of 86,919 unigenes, annotated by at least one of the reference databases (Nr, Swiss-Prot, GO, COG, and KEGG), were assembly yielded by de novo transcriptome. In addition, 157 putative differentially expressed genes (DEGs) were identified between the L. lactis combined with S. limacinum group and the control group. For pathway enrichment, the DEGs were categorized into nine KEGG pathways, which were mainly related to infective diseases, antigen processing and presentation, digestive system, and other immune system responses. The findings of this study suggest that the L. lactis combined with S. limacinum diet can induce positive effects on the growth, immunity, and disease resistance of C. altivelis against V. harveyi. This study expands our understanding of the synergistic combinations of probiotics and prebiotics in aquaculture.


Assuntos
Bass/imunologia , Doenças dos Peixes/imunologia , Imunidade Inata/efeitos dos fármacos , Lactococcus lactis/química , Prebióticos , Probióticos/farmacologia , Estramenópilas/química , Adjuvantes Imunológicos/farmacologia , Animais , Bass/crescimento & desenvolvimento , Resistência à Doença/efeitos dos fármacos , Resistência à Doença/imunologia , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
2.
Fish Shellfish Immunol ; 94: 634-642, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31533082

RESUMO

Long non-coding RNA refers to an RNA transcript of a non-coding protein with a sequence length greater than 200 bp. More and more reports indicated that lncRNA was involved in the regulation of gene expression as a signalling molecule, an inducing molecule, a leader molecule and a scaffold molecule. Previous studies have sequenced the draft genome and several transcriptome data sets for protein-coding genes of the large yellow croaker (Larimichthys crocea), but little is known about the expression and function of lncRNAs in this species. In order to obtain a catalogue of lncRNAs for this croaker, Vibrio parahaemolyticus infection challenge experiment was conducted and long non-coding RNA sequences were obtained. Using high-throughput sequencing of lncRNA, a total of 73,233 high-confidence transcripts were reconstructed in 32,726 loci, recovering most of the expressed reference transcripts, and 6473 novel expressed loci were identified. The tissue expression profile revealed that most lacunas were specifically enriched in distinct tissues. A set of 163 lncRNAs were identified as being specifically expressed in the spleen and may be involved in the immune response. It is the first time to identify specific lncRNAs in the L. crocea systematically in this croaker, aiming to benefit the future genomic study of this species.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , RNA Longo não Codificante/genética , Animais , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , RNA Longo não Codificante/imunologia , Distribuição Aleatória , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrioses/veterinária , Vibrio parahaemolyticus/fisiologia
3.
Fish Shellfish Immunol ; 93: 917-923, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31430560

RESUMO

This study investigated the effect of the dietary supplementation of bovine lactoferrin (LF) on growth performance, hematological and immunological parameters, antioxidant enzymes activity and disease resistance against Vibrio harveyi in yellowfin sea bream (Acanthopagrus latus) fingerling. The fish with initial body weight 10 ±â€¯0.3 g were randomly distributed at 10 fish per each 250 L fiberglass tank, and fed with four experimental diets (a control basal diet and three supplemented diets with 400, 800 and 1200 mg LF kg-1 diet) for 8 weeks. The obtained results showed that fish fed with LF supplemented diets had significantly higher final body weight as compared to control diet (P < 0.05). There were no significant differences between LF-treatments and the control group in white blood cell counts, red blood cell counts, hemoglobin and hematocrit. Total protein and complement activity (ACH50) in the serum of yellowfin sea bream were enhanced with increasing the dietary LF supplementation level (P < 0.05). The mucus lysozyme activity in fish fed on 800 and 1200 mg LF kg-1 was significantly higher than those fed on 400 mg LF kg-1 and control fish (P < 0.05). None of the antioxidant enzymes (catalase, glutathione reductase, glutathione S-transferase) was affected by LF supplementation (P > 0.05). Fish fed with dietary LF had a significantly higher survival rate than those fed with the control diet after challenge with Vibrio harveyi (P < 0.05). These results revealed that diet supplementation in A. latus especially with 1200 mg LF kg-1 improve fish growth performance and immune parameters, as well as survival rate against Vibrio harveyi.


Assuntos
Antioxidantes/metabolismo , Suplementos Nutricionais , Resistência à Doença/efeitos dos fármacos , Doenças dos Peixes/imunologia , Imunidade Inata/efeitos dos fármacos , Lactoferrina/farmacologia , Dourada/imunologia , Adjuvantes Imunológicos/farmacologia , Ração Animal/análise , Animais , Dieta/veterinária , Relação Dose-Resposta a Droga , Distribuição Aleatória , Dourada/sangue , Dourada/crescimento & desenvolvimento , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
4.
Fish Shellfish Immunol ; 93: 1018-1027, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31446082

RESUMO

In our previous study, a DNA plasmid encoding the VAA gene of Vibrio anguillarum was constructed and demonstrated to confer moderated protection against V. anguillarum challenge. Here, a bicistronic DNA vaccine (pVAA-IRES-IL2), co-expressing the VAA gene of V. anguillarum and Interleukin-2 (IL2) gene of flounder, was constructed to increase the protective efficacy of VAA DNA vaccine. The potential of pVAA-IRES-IL2 to express both VAA and IL2 in transfected HINAE cell lines was confirmed by immunofluorescence assay. Further, the variation of sIgM+, CD4-1+, CD4-2+ lymphocytes and production of VAA-specific antibodies in flounder, which was intramuscularly immunized with three DNA plasmids (pIRES, pVAA-IRES, pVAA-IRES-IL2), were investigated, respectively. The bacterial burden and relative percentage survival (RPS) of flounder exposed to V. anguillarum infection were both analyzed to evaluate the efficacy of bicistronic DNA plasmid. Our results revealed that the percentages of sIgM+, CD4-1+, CD4-2+ lymphocytes and antibodies specific to VAA were remarkably increased in pVAA-IRES or pVAA-IRES-IL2 immunized fish. Moreover, the co-expression of IL2 enhanced the immune response in response to VAA DNA vaccination, as shown by the higher percentages of sIgM+, CD4-1+, CD4-2+ lymphocytes and production of specific antibody. Importantly, the RPS in pVAA-IRES-IL2 and pVAA-IRES groups reached 64.1% and 51.3%, respectively, when compared with the 97.5% cumulative mortality in pIRES group. Furthermore, the number of V. anguillarum in liver, spleen and kidney of pVAA-IRES or pVAA-IRES-IL2 immunized flounder after V. anguillarum challenge was significantly reduced, as compared to that in pIRES group. These suggest that the bicistronic DNA vaccine can be an effective immunization strategy in inducing immune response against V. anguillarum infection and IL2 has the potential as the adjuvant for VAA DNA vaccine.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Doenças dos Peixes/imunologia , Linguados/imunologia , Interleucina-2/imunologia , Vibrio/imunologia , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Interleucina-2/genética , Vacinas de DNA/imunologia , Vibrioses/imunologia , Vibrioses/veterinária
5.
PLoS Pathog ; 15(8): e1007767, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31437245

RESUMO

The tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three tad loci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only the Δtad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the Δtad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. The Δtad123 mutant cells showed attenuated host cell adhesion, decreased biofilm formation, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. The Δtad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Complement depletion by treating with anti-C5 antibody rescued the viable count of Δtad123 in infected mouse bloodstream to the level comparable to wild type strain. Taken together, all three tad loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Ativação do Complemento/imunologia , Fímbrias Bacterianas/fisiologia , Vibrioses/microbiologia , Vibrio vulnificus/patogenicidade , Virulência , Animais , Aderência Bacteriana , Proteínas de Bactérias/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Camundongos , Camundongos Endogâmicos ICR , Óperon , Ratos , Ratos Sprague-Dawley , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/patologia , Vibrio vulnificus/genética , Vibrio vulnificus/crescimento & desenvolvimento
6.
Fish Shellfish Immunol ; 93: 1047-1055, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31425831

RESUMO

Nowadays, there is no suitable treatment for vibriosis in groupers. So an eco-efficient and environmentally friendly treatment is necessary for the grouper industry. Probiotic-feeding has been a promising strategy to control the bacterial pathogens in aquaculture. A new Bacillus velezensis strain named K2 was isolated from the intestinal tract of healthy grouper, and exhibited wide antimicrobial spectrum of against fish pathogens, including Vibrio harveyi, Vibrio alginolyticus, Aeromonas hydrophila, Aeromonas veronii, Aeromonas caviae, Enterococcus casseliflavus and Lactococcus garvieae. Moreover, results of the safety of B. velezensis K2 showed that intraperitoneal injection of K2 in healthy grouper did not cause any pathological abnormality or death, indicating this bacteria could be considered as a candidate probiotic in aquaculture. Groupers were fed with the diets containing 1 × 107 cfu/g of B. velezensis K2 for 4 weeks. Various immune parameters were examined at 1, 2, 3, and 4 weeks of post-feeding. Results showed that diets supplemented with K2 significantly increased serum acid phosphatase (ACP) activity (P < 0.05). Results of the mRNA expression of immune-related genes in the head kidney of hybrid grouper showed that the expression of lysozyme gene was significantly upregulated after 1 and 2 weeks of feeding (P < 0.05). A significant up-regulation of the expression of piscidin, IgM and MyD88 were detected at day 21, whereas the TLR3 and TLR5 showed lower expression compared to the controls during 21 days, and a significant decrease of TLR3 gene was found at day 28 (P < 0.05). After challenge with V. harveyi, the survival rate of fish administrated with the strain K2 for 28 days was signifiacantly higher than the controls without this strain (P < 0.05). These results collectively suggest that B. velezensis K2 is a potential probiotic species to improve health status and disease resistance and can be developed as a probiotic agent in grouper industry.


Assuntos
Bacillus/química , Bass/imunologia , Resistência à Doença/imunologia , Doenças dos Peixes/imunologia , Probióticos/farmacologia , Ração Animal/análise , Animais , Bass/crescimento & desenvolvimento , Dieta/veterinária , Distribuição Aleatória , Vibrio/efeitos dos fármacos , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
7.
Fish Shellfish Immunol ; 93: 863-870, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31422178

RESUMO

Interleukins (ILs) are a subgroup of cytokines, which are molecules involved in the intercellular regulation of the immune system. These cytokines have been extensively studied in mammalian models, but systematic analyses of fish are limited. In the current study, 3 IL genes from golden pompano (Trachinotus ovatus) were characterized. The IL-1ß protein contains IL-1 family signature motif, and four long helices (αA - αD) in IL-11 and IL-34, which were well conserved. All 3 ILs clustered phylogenetically with their respective IL relatives in mammalian and other teleost species. Under normal physiological conditions, the expression of IL-1ß, IL-11, and IL-34 were detected at varied levels in the 11 tissues examined. Most of the 3 ILs examined were highly expressed in liver, spleen, kidney, gill, or skin. Following pathogenic bacterial, viral, or parasitic challenge, IL-1ß, IL-11, and IL-34 exhibited distinctly different expression profiles in a time-, tissue-, and pathogen-dependent manner. In general, IL-1ß was expressed at higher levels following challenge with all pathogens examined than was observed for IL-11 and IL-34. Furthermore, Streptococcus agalactiae and Cryptocaryon irritans caused higher levels of IL-1ß and IL-11 expression than Vibrio harveyi and viral nervous necrosis virus (VNNV). The increased expression of IL-34 caused by VNNV and C. irritans were higher than that caused by V. harveyi and S. agalactiae. These results suggest that these 3 ILs in T. ovatus may play different effect pathogen type specific responses.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Animais , Cilióforos/fisiologia , Infecções por Cilióforos/imunologia , Infecções por Cilióforos/veterinária , Perfilação da Expressão Gênica/veterinária , Interleucina-11/genética , Interleucina-11/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucinas/genética , Interleucinas/imunologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae/fisiologia , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
8.
Fish Shellfish Immunol ; 93: 823-831, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31422181

RESUMO

Calreticulin (CRT) is a highly conserved and multi-functional protein with diverse localizations. CRT has lectin-like properties and possesses important immunological activities in mammalian. In teleost, very limited studies on CRT immunologic function have been documented. In the present study, a CRT homologue (SsCRT) was cloned, identified and characterized from black rockfish, Sebastes schlegeli, an important aquaculture species in East Asia. The full length of SsCRT cDNA is 2180 bp and encoded a polypeptide of 425 amino acids. SsCRT contains a signal peptide, three distinct structural and functional domains (N-, P- and C-domains), and an endoplasmic reticulum (ER) retrieval signal sequence (KDEL). The deduced amino acid sequence of SsCRT shares 89-92% overall sequence identities with the CRT proteins of several fish species. SsCRT was distributed ubiquitously in all the detected tissues and was highly expressed in the spleen, muscle and liver. After the infection of fish extracellular bacterial pathogen Vibrio anguillarum and intracellular bacterial pathogen Edwardsiella tarda, the mRNA transcripts of SsCRT in spleen, liver, and head kidney were significantly up-regulated. The expression patterns were time-dependent and tissue-dependent. Recombinant SsCRT (rSsCRT) exhibited apparent binding activities against different bacteria and PAMPs. In vivo studies showed that the expressions of multiple immune-related genes such as TNF13B, IL-1ß, IL-8, SAA, Hsp70, and ISG15 in head kidney were significantly enhanced when black rockfish were treated with rSsCRT. Furthermore, rSsCRT reduced pathogen dissemination and replication in fish kidney and spleen. These results indicated that SsCRT served as an immune receptor to recognize and eliminate the invading pathogens, which played a vital role in the immune response of Sebastes schlegeli. These findings provide new insights into understanding the roles of CRT proteins in immune response and pathogen infection in teleost.


Assuntos
Calreticulina/genética , Calreticulina/imunologia , Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Calreticulina/química , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Padrões Moleculares Associados a Patógenos/farmacologia , Perciformes/genética , Perciformes/imunologia , Filogenia , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
9.
Fish Shellfish Immunol ; 92: 782-791, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31288100

RESUMO

Toll-like receptor (TLR) genes are the earliest reported pathogen recognition receptors (PRRs) and have been extensively studied. These genes play pivotal roles in the innate immune defense against pathogen invasion. In this study, a total of 16 tlr genes were identified and characterized in spotted sea bass (Lateolabrax maculatus). The tlr genes of spotted sea bass were classified into five subfamilies (tlr1-subfamily, tlr3-subfamily, tlr5-subfamily, tlr7-subfamily, and tlr11-subfamily) according to the phylogenetic analysis, and their annotations were confirmed by a syntenic analysis. The protein domain analysis indicated that most tlr genes had the following three major TLR protein domains: a leucine-rich repeat (LRR) domain, a transmembrane region (TM) and a Toll/interleukin-1 receptor (TIR) domain. The tlr genes in spotted sea bass were distributed in 11 of 24 chromosomes. The mRNA expression levels of 16 tlr genes in response to Vibrio harveyi infection were quantified in the head kidney. Most genes were downregulated following V. harveyi infection, while only 5 tlr genes, including tlr1-1, tlr1-2, tlr2-2, tlr5, and tlr7, were significantly upregulated. Collectively, these results help elucidate the crucial roles of tlr genes in the immune response of spotted sea bass and may supply valuable genomic resources for future studies investigating fish disease management.


Assuntos
Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Genoma/imunologia , Imunidade Inata/genética , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
10.
Fish Shellfish Immunol ; 93: 153-160, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31319206

RESUMO

Cathepsins are the best-known group of proteases in lysosomes, playing a significant role in immune responses. Cathepsin K (CTSK) is abundantly and selectively expressed in osteoclasts, dendritic cells and monocyte-derived macrophages, where it is involved in ECM degradation and bone remodeling. A growing body of evidences have indicated the vital roles of cathepsin K in innate immune responses. Here, one CTSK gene was captured in turbot (SmCTSK) with a 993 bp open reading frame (ORF). The genomic structure analysis showed that SmCTSK had 7 exons similar to other vertebrate species. The syntenic analysis revealed that CTSK had the same neighboring genes across all the selected species, which suggested the synteny encompassing CTSK region was conserved during vertebrate evolution. Subsequently, SmCTSK was widely expressed in all the examined tissues, with the highest expression level in spleen and the lowest expression level in liver. In addition, SmCTSK was significantly down-regulated in intestine following Gram-negative bacteria Vibrio anguillarum immersion challenge, but up-regulated in three tissues (gill, skin and intestine) following Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmCTSK showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmCTSK played vital roles in fish innate immune responses against infection. However, the knowledge of SmCTSK is still limited in teleost species, further studies should be carried out to better characterize its comprehensive roles in teleost mucosal immunity.


Assuntos
Catepsina K/genética , Catepsina K/imunologia , Doenças dos Peixes/imunologia , Linguados/genética , Linguados/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Animais , Catepsina K/química , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus iniae/fisiologia , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
11.
Mol Immunol ; 112: 360-368, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31261021

RESUMO

Hemocyanin is primarily a respiratory copper-containing glycoprotein present in the hemolymph of mollusks and arthropods. Recently, hemocyanin has attracted huge research interest due to its multifunctionality and polymorphism. Most previous immune-related studies on shrimp hemocyanin have focused on the C-terminal. Moreover, we previously reported that the C-terminal domain of Litopenaeus vannamei hemocyanin possesses single nucleotide polymorphisms (SNPs), but little is known about the molecular diversity of the N-terminal domain. In the current study, diversity within the N-terminal domain of L. vannamei hemocyanin (LvHMC-N) was explored using bioinformatics and molecular biology techniques as well as immune challenge. Twenty-five LvHMC-N variants were identified using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing, with multiple sequence alignment showing that the 25 variants shared 87%-99 % sequence homology with LvHMC (AJ250830.1). In different shrimp individuals and different shrimp tissues (i.e., hemocytes, stomach, muscle and hepatopancreas), the LvHMC-N variants were expressed differently. Pathogen challenge could modulate the molecular diversity of LvHMC-N, as three LvHMC-Nr variants (LvHMC-Nr1, LvHMC-Nr2 and LvHMC-Nr3) were identified by sequencing following Vibrio parahaemolyticus challenge. Most importantly, recombinant proteins of these three variants (rLvHMC-Nr1, rLvHMC-Nr2 and rLvHMC- Nr3) had relatively high in vitro agglutinative activities against V. parahaemolyticus, Vibrio alginolyticus and Streptoccocus iniae. Our present data indicates that the N-terminus of L. vannamei hemocyanin also possess molecular diversity, which seems to be associated with immune resistance to pathogenic infections.


Assuntos
Hemocianinas/genética , Hemocianinas/imunologia , Imunidade/genética , Imunidade/imunologia , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Clonagem Molecular/métodos , Biologia Computacional/métodos , Hemócitos/imunologia , Hemócitos/microbiologia , Hemolinfa/imunologia , Hemolinfa/microbiologia , Hepatopâncreas/imunologia , Hepatopâncreas/microbiologia , Alinhamento de Sequência , Análise de Sequência de DNA , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrio parahaemolyticus/imunologia
12.
Fish Shellfish Immunol ; 93: 191-199, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31326589

RESUMO

Interleukin-6 (IL-6) is one of the most pleiotropic cytokines because of its wide range of effects on cells of the immune and non-immune systems in the body. However, the role of IL-6 in fish monocytes/macrophages (MO/MФ) is poorly understood. In this study, we cloned the cDNA sequence of the IL-6 gene from ayu (Plecoglossus altivelis) and demonstrated using a tissue distribution assay that ayu interleukin-6 (PaIL-6) mRNA is expressed in all tested tissues. Changes in expression were observed in immune tissues as well as in MO/MФ after a Vibrio anguillarum infection; subsequently, PaIL-6 was expressed and purified to prepare anti-PaIL-6 antibodies. Recombinant PaIL-6 protein (rPaIL-6) treatment enhanced pro-inflammatory cytokine expression. Ayu interleukin-6 receptor ß (PaIL-6Rß) knockdown resulted in decreased pro-inflammatory cytokine expression in MO/MФ treated with rPaIL-6, whereas no significant changes were observed after ayu interleukin-6 receptor α (PaIL-6Rα) knockdown in MO/MФ. PaIL-6 and PaIL-6Rß knockdown in MO/MФ inhibited the phosphorylation of signal transducer and activator of transcription 1. Moreover, PaIL-6Rß knockdown inhibited the phagocytic and bactericidal ability of ayu MO/MФ treated with rPaIL-6. These data indicate that PaIL-6 may be able to regulate the function of ayu MO/MФ.


Assuntos
Receptor gp130 de Citocina/genética , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Interleucina-6/genética , Osmeriformes/genética , Osmeriformes/imunologia , Sequência de Aminoácidos , Animais , Receptor gp130 de Citocina/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Técnicas de Silenciamento de Genes/veterinária , Interleucina-6/química , Interleucina-6/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Fagocitose/genética , Fagocitose/imunologia , Fosforilação , Filogenia , Fator de Transcrição STAT1/metabolismo , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
13.
Fish Shellfish Immunol ; 93: 28-38, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31302288

RESUMO

Eomesodermin (Eomes) is a member of T-box transcription factor family and plays an important role in the regulation of a wide variety of developmental processes and immune response in animals. Here we report cloning and characterization of the full-length cDNA of Atlantic cod Eomes (GmEomes), which possesses a TBOX_3 domain similar to its counterpart in mammals. The regulated expression was observed in head kidney and spleen in response to live Vibrio anguillarum infection in vivo, and spleen leukocytes in vitro after PMA and poly I:C stimulation. Furthermore, we determined a 694 bp sequence, upstream of the transcriptional start site (TSS), to contain a number of sequence motifs that matched known transcription factor-binding sites. Activities of the presumptive regulatory gene were assessed by transfecting different 5'-deletion constructs in CHSE-214 cells. The results showed that the basal promoters and positive transcriptional regulator activities of GmEomes were dependent by sequences located from -694 to -376 bp upstream of TSS. Furthermore, we found that some Eomes binding sites were present in the 5'-flanking regions of the cod IFNγ gene predicted by bioinformatics. However, Co-transfection of eomesodermin overexpression plasmids with INFγ reporter vector into CHSE-214 cells determined that Atlantic cod eomesodermin played a minor role in activation of the INFγ promoter.


Assuntos
Doenças dos Peixes/imunologia , Gadus morhua/genética , Gadus morhua/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência/veterinária , Proteínas com Domínio T/química , Acetato de Tetradecanoilforbol/farmacologia , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
14.
Fish Shellfish Immunol ; 93: 269-277, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31306762

RESUMO

As an isoform of Rho family GTPases, RhoB plays a pivotal role in cytoskeletal organization, cell proliferation, apoptosis and immune response. However, the regulatory mechanisms of RhoB expression in aquatic animals are still unknown. In the present study, we first construct Vibrio anguillarum infection model in S. maximus, including susceptible and resistant individuals. Then the temporal expression of RhoB was detected after V. anguillarum challenge using qRT-PCR and found that RhoB transcripts were significantly induced in the liver, gill and blood despite of differential expression levels and responsive time points. In addition, the mRNA levels of RhoB in resistant individuals were significantly higher than in susceptible ones. The length of 2083 bp sequences of RhoB promoter was cloned and characterized. Moreover, DNA methylation of the RhoB promoter was measured by bisulfite sequencing (BSP) and hypo-methylated was detected in the CpG islands. Three SNPs (-1590, -1575 and -1449) and two haplotypes in the promoter region of RhoB were identified to be associated with V. anguillarum resistance in turbot by association analysis in group 17-R and 17-S. Deletion analysis indicated that these SNPs could negatively mediate the activity of RhoB promoter. Site-directed mutagenesis and qRT-PCR of individuals with different genotypes demonstrated that -1575 T/A polymorphism affected promoter activity. Further study showed that this mutation altered the binding site of the transcription factor CREB. Co-transfection of SmCREB and RhoB promoter was performed in HEK293T cells which confirmed the -1575 allelic differences on transcriptional activity, with the susceptibility allele showing reduced activity. Taken together, our findings implicate that losing of binding of CREB to SmRhoB promoter due to -1575T/A polymorphisms enhances SmRhoB expression in resistant turbot, which provide insights into the effect of SmRhoB expression in response to V. anguillarum infection.


Assuntos
Doenças dos Peixes/imunologia , Linguados/genética , Polimorfismo de Nucleotídeo Único/imunologia , Vibrio/fisiologia , Proteína rhoB de Ligação ao GTP/imunologia , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/veterinária , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Linguados/imunologia , Regulação da Expressão Gênica/imunologia , Haplótipos/imunologia , Mutação , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , Vibrioses/imunologia , Vibrioses/veterinária , Proteína rhoB de Ligação ao GTP/genética
15.
Fish Shellfish Immunol ; 92: 680-689, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31271837

RESUMO

The Notch signaling pathway is known to regulate innate immunity by influencing macrophage function and interacting with the Toll-like receptor (TLR) signaling pathway. However, the comprehensive role of the Notch signaling pathway in the innate immune response remains unknown. To assess the function of Notch1a in immunity, we examined the innate immune responses to Vibrio parahaemolyticus strain Vp13 of wild-type (WT) and notch1a-/- zebrafish larvae generated using the clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system. The median lethal dose (LD50) of V. parahaemolyticus was significantly lower in notch1a-/- larvae than in WT larvae 3 days post fertilization (dpf). Transcriptome data analysis revealed 359 significantly differentially expressed genes (DEGs), including 246 significantly down-regulated genes and 113 significantly up-regulated genes, in WT infected groups compared with WT control groups. In contrast, 986 significantly DEGs were found in notch1a-/- infected groups compared with notch1a-/- control groups, of which 82 genes were significantly down-regulated and 904 genes were significantly up-regulated. These DEGs belonged to the tumor necrosis factor (TNF), complement, nuclear factor kappa B (NF-κB), cathepsin, interleukin (IL), chemokine, serpin peptidase inhibitor, matrix metallopeptidase, innate immune cells, pattern recognition receptor (PRR), and other cytokine families. Our results indicate that Notch1a plays roles in inhibiting many immunity-related genes and could comprehensively mediate the innate immune response by regulating TLRs, nucleotide-binding-oligomerization-domain-like receptors (NLRs), lectins, complement, ILs, chemokines, TNF, cathepsin, and serpin. Further studies are required to understand the specific mechanism of Notch1a in innate immunity.


Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Imunidade Inata/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Receptor Notch1/genética , Receptor Notch1/imunologia , Transdução de Sinais/imunologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Animais , Doenças dos Peixes/imunologia , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio parahaemolyticus/fisiologia
16.
Fish Shellfish Immunol ; 92: 450-459, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31207302

RESUMO

PIK3CA has been extensively investigated from its molecular mechanism perspective and association with its mutations in different types of cancers. However, little has been reported regarding the pathological significance of PIK3CA expression in teleost. Here, in our present study, three PIK3CA genes termed SmPIK3CAa, SmPIK3CAb and SmPIK3CA-like were firstly identified in the genome of turbot S. maximus. Although these three genes located in different chromosomes, all of them share the same five domains. Phylogenetic and synteny analysis indicated that SmPIK3CAa, SmPIK3CAb and SmPIK3CA-like were three paralogs that may originate from duplication of the same ancestral PIK3CA gene. Subcellular localization analysis confirmed the cytoplasm distribution of these three paralogs. All three SmPIK3CA were ubiquitously expressed in examined tissues in turbot, with the higher expression levels in immune-related tissues such as blood, spleen, kidney, gills and intestines. Upon Vibrio anguillarum challenge, SmPIK3CAa and SmPIK3CA-like transcripts were significantly induced in spleen, intestine and blood despite of differential expression levels and responsive time points. Additionally, individuals in resistant group showed significantly higher expression level of both two genes than in the susceptible group. Moreover, four SNPs (102, 2530, 3027 and 3060) and one haplotype (Hap2) located in exon region of SmPIK3CA-like were identified and confirmed to be associated with V. anguillarum resistance in turbot by association analysis in different populations. Taken together, these results suggested that functional differentiation occurred in three SmPIK3CA paralogs with Vibrio anguillarum resistance and SmPIK3CAa and SmPIK3CA-like probable play potential roles in innate immune response to pathogenic invasions in turbot.


Assuntos
Doenças dos Peixes/imunologia , Linguados/genética , Linguados/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Fosfatidilinositol 3-Quinase/química , Filogenia , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
17.
Fish Shellfish Immunol ; 92: 256-264, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31200076

RESUMO

NK-lysin (NKL) is a cationic host defense peptide that plays an important role in host immune responses against various pathogens. However, the immunomodulatory activity of NKL in fishes is rarely investigated. In this study, we characterized a cDNA sequence encoding an NK-lysin homolog (BpNKL) from the fish, mudskipper (Boleophthalmus pectinirostris). Sequence analysis revealed that BpNKL is most closely related to tiger puffer (Takifugu rubripes) NKL. BpNKL transcript was detected in all the tested tissues, with the highest level in the gill, followed by the spleen and kidney. Upon Edwardsiella tarda infection, the mRNA expression of BpNKL in the mudskipper was significantly upregulated in the spleen, kidney, and gill. A shortened peptide derived from BpNKL, BpNKLP40, was then chemically synthesized and its biological functions were investigated. BpNKLP40 exhibited a direct antibacterial activity against some Gram-negative bacteria, including E. tarda, Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio harveyi, and induced hydrolysis of E. tarda genomic DNA. Intraperitoneal injection of 1.0 µg/g BpNKLP40 significantly improved the survival of mudskipper following E. tarda infection and reduced the bacterial burden in tissues and blood. Moreover, 1.0 µg/ml BpNKLP40 treatment had an enhanced effect on the intracellular killing of E. tarda by monocytes/macrophages (MO/MФ) as well as on the mRNA expression of pro-inflammatory cytokines in MO/MФ. In conclusion, our study reveals that BpNKL plays a role against E. tarda infection in the mudskipper by not only directly killing bacteria but also through an immunomodulatory activity on MO/MФ.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteolipídeos/genética , Proteolipídeos/imunologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/farmacologia , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Fatores Imunológicos/farmacologia , Macrófagos/imunologia , Monócitos/imunologia , Filogenia , Proteolipídeos/química , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
18.
Fish Shellfish Immunol ; 92: 500-507, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31247318

RESUMO

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation.


Assuntos
Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , MAP Quinase Quinase 6/genética , MAP Quinase Quinase 6/imunologia , Sequência de Aminoácidos , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , MAP Quinase Quinase 6/química , Filogenia , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio alginolyticus/fisiologia
19.
Fish Shellfish Immunol ; 92: 377-383, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31202969

RESUMO

Vibrio mimicus (V. mimicus) is a significant pathogen in freshwater catfish, though knowledge of virulence determinants and effective vaccine is lacking. Multiplex genome editing by natural transformation (MuGENT) is an easy knockout method, which has successfully used in various bacteria except for V. mimicus. Here, we found V. mimicus strain SCCF01 can uptake exogenous DNA and insert it into genome by natural transformation assay. Subsequently, we exploited this property to make five mutants (△Hem, △TS1, △TS2, △TS1△TS2, and △II), and removed the antibiotic resistance marker by Flp-recombination. Finally, all of the mutants were identified by PCR and RT-PCR. The results showed that combination of natural transformation and FLP-recombination can be applied successfully to generate targeted gene disruptions without the antibiotic resistance marker in V. mimicus. In addition, the five mutants showed mutant could be inherited after several subcultures and a 668-fold decrease in the virulence to yellow catfish (Pelteobagrus fulvidraco). This study provides a convenient method for the genetic manipulation of V. mimicus. It will facilitate the identification and characterization of V. mimicus virulence factors and eventually contribute to a better understanding of V. mimicus pathogenicity and development of attenuated vaccine.


Assuntos
Vacinas Bacterianas/imunologia , Peixes-Gato , Doenças dos Peixes/imunologia , Edição de Genes/veterinária , Técnicas de Inativação de Genes/veterinária , Vibrio mimicus/imunologia , Animais , Técnicas de Inativação de Genes/métodos , Vacinas Atenuadas/imunologia , Vibrioses/imunologia , Vibrioses/veterinária
20.
Fish Shellfish Immunol ; 92: 111-118, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31176005

RESUMO

Apolipoproteins (Apos), which are the protein components of plasma lipoproteins, play important roles in lipid transport in vertebrates. It has been demonstrated that in teleosts, several Apos display antimicrobial activity and play crucial roles in innate immunity. Despite their importance, apo genes have not been systematically characterized in many aquaculture fish species. In our study, a complete set of 23 apo genes was identified and annotated from spotted sea bass (Lateolabrax maculatus). Phylogenetic and homology analyses provided evidence for their annotation and evolutionary relationships. To investigate their potential roles in the immune response, the expression patterns of 23 apo genes were determined in the liver and intestine by qRT-PCR after Vibrio harveyi infection. After infection, a total of 20 differentially expressed apo genes were observed, and their expression profiles varied among the genes and tissues. 5 apo genes (apoA1, apoA4a.1, apoC2, apoF and apoO) were dramatically induced or suppressed (log2 fold change >4, P < 0.05), suggesting their involvement in the immune response of spotted sea bass. Our study provides a valuable foundation for future studies aimed at uncovering the specific roles of each apo gene during bacterial infection in spotted sea bass and other teleost species.


Assuntos
Apolipoproteínas/genética , Apolipoproteínas/imunologia , Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Animais , Apolipoproteínas/química , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Família Multigênica/imunologia , Filogenia , Transcriptoma , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
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