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1.
Dis Aquat Organ ; 137(3): 167-173, 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31942862

RESUMO

Skin abrasions often occur in farmed fish following handling by labourers, injury by farm facilities, cannibalism and ectoparasites. Vibrio spp. are opportunistic pathogens that can invade host fish through damaged tissues and cause outbreaks of vibriosis. This study describes the effect of skin abrasions on the infectivity of V. harveyi using Asian seabass Lates calcarifer (Bloch, 1790) fingerlings as a case example and compares bacterial load and fish survival following immersion challenge with different doses. In total, 315 fish (6.67 ± 1.8 g) were divided into 3 treatments: skin abrasion followed by immersion infection, immersion infection only and an uninfected, uninjured control. Fish in the infection treatments were divided into 3 subgroups and exposed in triplicate to a 7 d immersion challenge with 106, 107 and 108 CFU ml-1 of live V. harveyi. No mortalities were observed in the control and immersion infection groups. However, fish in the skin abrasion treatment group that were infected with 108 CFU ml-1 of live V. harveyi showed signs of progressing disease throughout the experiment, which resulted in mortalities. Significantly higher bacterial loads (p < 0.05) were recorded in the intestine, liver and gills of the fish in this group. Fish in the skin abrasion treatment that were exposed to 107 and 108 CFU ml-1 of V. harveyi showed 100% mortality by Days 5 and 4, respectively. These findings confirm that skin injuries increase the susceptibility of seabass fingerlings to V. harveyi infection.


Assuntos
Bass , Doenças dos Peixes , Perciformes , Vibrioses , Vibrio , Animais , Vibrioses/veterinária
2.
Zool Res ; 41(1): 39-50, 2020 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-31709785

RESUMO

D-dopachrome tautomerase (DDT), a member of the macrophage migration inhibitory factor (MIF) protein superfamily, is a newly described cytokine with chemokine-like characteristics. However, research on fish DDT remains limited. In this study, we identified a DDT homolog (LjDDT) from the Japanese sea bass, Lateolabrax japonicus. Sequence analysis showed that LjDDT had typical sequence features of known DDT and MIF homologs and was most closely related to DDT of rock bream ( Oplegnathus fasciatus). LjDDT transcripts were detected in all tested tissues of healthy Japanese sea bass, with the highest expression found in the liver. Upon infection with Vibrio harveyi, LjDDT transcripts were significantly down-regulated in the three tested tissues, including the liver, spleen, and head kidney. Recombinant LjDDT (rLjDDT) and the corresponding antibody (anti-rLjDDT) were subsequently prepared. The administration of 100 µg/g anti-rLjDDT had a statistically significant protective effect on the survival of V. harveyi-infected fish. Moreover, rLjDDT was able to induce the migration of monocytes/macrophages (MO/MФ) and lymphocytes both in vitro and in vivo, but without significant influence on the migration of neutrophils. rLjDDT exhibited chemotactic activity for lipopolysaccharide (LPS) -stimulated M1-type MO/ MΦ in vitro, but not for cAMP-stimulated M2-type MO/MΦ. Furthermore, the knockdown of LjCD74, but not LjCXCR4, significantly down-regulated the rLjDDT-enhanced migration of MO/MΦ and relieved the rLjMIF-inhibited migration of MO/MΦ. These results indicate that LjCD74 may be the major chemotactic receptor of LjDDT and LjMIF in Japanese sea bass MO/MΦ. Combined rLjDDT+ rLjMIF treatment had no significant effect on the migration of MsiRNA, LjCD74si-, or LjCXCR4sitreated MO/MΦ compared to the control group, suggesting that the roles of LjDDT and LjMIF may be antagonistic. In conclusion, our study demonstrates for the first time that DDT may play a role in the immune responses of fish against bacterial infection through chemotactic recruitment of MO/MΦ via mediation of CD74 as an antagonist of MIF.


Assuntos
Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Sequência de Aminoácidos , Animais , Movimento Celular , Relação Dose-Resposta a Droga , Peixes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/fisiologia , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/farmacologia , RNA Mensageiro , Vibrio , Vibrioses/enzimologia , Vibrioses/microbiologia , Vibrioses/veterinária
3.
Emerg Microbes Infect ; 8(1): 1604-1618, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31711375

RESUMO

The incidence of Vibrio alginolyticus infections has increased in recent years due to the influence of climate change and rising sea temperature. Vibrio virulence regulatory RNA 1 (Vvrr1) is a newly found noncoding RNA (ncRNA) predicted to be closely related to the adhesion ability of V. alginolyticus based on the previous RNA-seq. In this study, the target genes of Vvrr1 were fully screened and verified by constructing Vvrr1-overexpressing strains and using the proteome sequencing technology. Pyruvate kinase I (pykF) gene was predicted to be a chief target gene of Vvrr1 involved in virulence regulation. The adhesion ability, biofilm formation and virulence were significantly reduced in the Vvrr1-overexpressing and the pykF-silenced strain compared with the wild strains. Similar to the overexpression of Vvrr1, the silencing of pykF also reduced the expression level of virulence genes, such as ndk, eno, sdhB, glpF, and cysH. Meanwhile, by constructing the "pykF-GFP" fusion expression plasmid and using the GFP reporter gene analysis in Escherichia coli, the fluorescence intensity of the strain containing Vvrr1 whole ncRNA sequence vector was found to be significantly weakened. These indicated that Vvrr1 participated in the virulence regulation mechanism of V. alginolyticus by interacting with the virulence gene pykF.


Assuntos
Doenças dos Peixes/microbiologia , RNA Bacteriano/genética , RNA não Traduzido/genética , Vibrioses/veterinária , Vibrio alginolyticus/genética , Vibrio alginolyticus/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Peixes , Regulação Bacteriana da Expressão Gênica , Proteômica , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , RNA Bacteriano/metabolismo , RNA não Traduzido/metabolismo , Vibrioses/microbiologia , Vibrio alginolyticus/metabolismo , Virulência
4.
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
5.
Fish Shellfish Immunol ; 94: 746-751, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31546040

RESUMO

The present study evaluated the biofilm (BF) of Vibrio anguillarum for oral vaccination of Asian seabass, Lates calcarifer. An 80-day experiment was carried out in circular fiber-reinforced plastic (FRP) tanks using free cell (FC) and BF of Vibrio anguillarum with triplicate in each. Heat-inactivated FC and BF cells at 107, 1010 and 1013 CFU/g fish/d were fed to fish for 20 days, agglutination antibody titer estimated at each 10 days interval up to 60-day post vaccination. As compared to FC and control there was a significant increase in agglutinating antibody titer in the biofilm vaccinated fishes. Among the 3 doses, BF at 1010 cfu/g fish/d was considered the ideal dose for vaccination. Relative percentage survival (RPS) was higher in biofilm vaccinated fish (85.4%) compared to that with free cells (27.0%). The study demonstrated the better performance of V. anguillarum biofilm oral vaccine compared that with free cell vaccine in L. calcarifer. The study further supports better performance of biofilm vaccine model with one more bacterial pathogen in a high carnivore fish.


Assuntos
Vacinas Bacterianas/farmacologia , Bass , Biofilmes , Doenças dos Peixes/prevenção & controle , Vacinação/veterinária , Vibrioses/veterinária , Vibrio/fisiologia , Administração Oral , Animais , Vacinas Bacterianas/administração & dosagem , Temperatura Alta , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/farmacologia , Vibrio/imunologia , Vibrioses/prevenção & controle
6.
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
7.
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
8.
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
9.
J Fish Dis ; 42(11): 1523-1529, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31448425

RESUMO

As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)-based enzyme-linked apta-sorbent assay (VA2-ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2-ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2-ELASA could specifically identify V. alginolyticus, but not other non-target bacterial strains. VA2-ELASA could detect V. alginolyticus at the concentration of 5 × 104 /ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2-ELASA in this study. It took less than one hour to accomplish the detection process by VA2-ELASA. The characteristics of specificity, sensitivity and easy operation make VA2-ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Bacteriológicas/veterinária , Doenças dos Peixes/diagnóstico , Peixes , Vibrioses/veterinária , Vibrio alginolyticus/isolamento & purificação , Animais , Doenças dos Peixes/microbiologia , Vibrioses/diagnóstico , Vibrioses/microbiologia
10.
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
11.
J Fish Dis ; 42(10): 1447-1455, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418903

RESUMO

Vibrio ordalii is an extracellular, Gram-negative bacterium that produces vibriosis in salmonids. While pathogenesis is not fully understood, this bacterium has numerous likely genes for adhesion, colonization, invasion factors and, as recently suggested, intracellular behaviour. Therefore, this study aimed to clarify possible intracellular behaviour for V. ordalii Vo-LM-18 and ATCC 33509T in the fish-cell lines SHK-1 and CHSE-214. Confocal microscopy revealed Vo-LM-18 and ATCC 33509T inside cytoplasm in both fish-cell lines at 4 hr post-inoculation (hpi). At 8 and 16 hpi, the proportion of fish cells invaded by both strains increased. Moreover, intracellular V. ordalii were observed after 8 hpi inside mouse embryonic fibroblasts (MEF), demonstrating that entry was not due to a cellular phagocytosis process. Flow cytometry confirmed immunocytochemistry results, with both V. ordalii evidencing statistically significant differences in the number of infected cells between 8 and 16 hpi. Interestingly, V. ordalii infection did not significantly damage fish cells, as determined by LDH liberation. Viable counts at 8 hpi detected, on average for both lines, 176 ± 47 CFU/ml of culturable intracellular Vo-LM-18 and ATCC 33509T cells. These in vitro findings support the facultative intracellular behaviour of V. ordalii and may be of importance for understanding pathogenicity and survival in aquatic environments.


Assuntos
Doenças dos Peixes/microbiologia , Salmão , Vibrioses/veterinária , Vibrio/fisiologia , Animais , Linhagem Celular , Citometria de Fluxo/veterinária , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Microscopia Confocal/veterinária , Vibrioses/microbiologia
12.
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
13.
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
14.
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
15.
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
16.
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
17.
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
18.
J Basic Microbiol ; 59(9): 936-949, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31347191

RESUMO

Vibrio harveyi is a marine luminous pathogen, which causes biofilm-mediated infections, pressures the search for an innovative alternate approach to strive against vibriosis in aquaculture. This study anticipated to explore the effect of glycolipid biosurfactant as an antipathogenic against V. harveyi to control vibriosis. In this study, 27 bacterial strains were isolated from marine soil sediments. Out of these, 11 strains exhibited surfactant activity and the strain MK3 showed high emulsification index. The potent strain was identified as Vibrio natriegens and named as V. natriegens MK3. The extracted biosurfactant was purified using high-performance liquid chromatography and it was efficient to decrease the surface tension of the growth medium up to 21 mN/m. The functional group and composition of the biosurfactant were determined by Fourier-transform infrared spectroscopy and nuclear magnetic resonance spectroscopy spectral studies and the nature of the biosurfactant was identified as glycolipid. The surfactant was capable of reducing the biofilm formation, bioluminescence, extracellular polysaccharide synthesis, and quorum sensing in marine shrimp pathogen V. harveyi. The antagonistic effect of biosurfactant was evaluated against V. harveyi-infected brine shrimp Artemia salina. This study reveals that biosurfactant can be considered for the management of biofilm-related aquatic infections.


Assuntos
Biofilmes/efeitos dos fármacos , Tensoativos/farmacologia , Vibrio/química , Vibrio/efeitos dos fármacos , Virulência/efeitos dos fármacos , Animais , Aquicultura , Artemia/microbiologia , Biofilmes/crescimento & desenvolvimento , Poluição por Petróleo , Percepção de Quorum/efeitos dos fármacos , Tensoativos/isolamento & purificação , Vibrio/crescimento & desenvolvimento , Vibrio/patogenicidade , Vibrioses/prevenção & controle , Vibrioses/veterinária
19.
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
20.
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
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