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1.
Fish Shellfish Immunol ; 91: 78-86, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31039439

RESUMO

Type I interferons, as a class of multipotent cytokines, play a key role in host antiviral immune responses. In this study, a type I IFN coding gene of gibel carp, Carassius auratus gibelio, CagIFNa was cloned and sequenced. The full-length cDNA sequence of CagIFNa consists of 724 nucleotides that encode a predicted protein of 183 amino acids. CagIFNa has two highly conserved cysteine residues in the deduced protein, which is mostly conserved in the fish group I type I IFNs. CagIFNa was identified as a member of the IFNa subgroup of group I type I IFNs by phylogenetic analysis. CagIFNa transcripts were detected in all investigated tissues with higher levels in the liver, intestine, spleen and head kidney of gibel carp. Following injection with Cyprinid herpesvirus 2 (CyHV-2), CagIFNa gene expression was significantly inhibited in the spleen but delayed and then increased in head kidneys. Similarly, while CagIFNa expression was rapidly induced in gibel carp brain (GiCB) cells by poly I:C stimulation and its high induction level was delayed following CyHV-2 infection. CagIFNa overexpression in GiCB cells drastically reduced virus CPE and titer. Furthermore, several genes associated with type I IFN signaling pathway including IRF3, IRF7, IRF9, STAT1, Mx1 and PKR were induced in GiCB cells overexpressing CagIFNa upon CyHV-2 infection. These results show that CagIFNa plays a role in antiviral immune system in gibel carp.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Carpa Dourada/genética , Carpa Dourada/imunologia , Imunidade Inata/genética , Interferon Tipo I/genética , Interferon Tipo I/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 , Herpesviridae , Infecções por Herpesviridae/imunologia , Interferon Tipo I/química , Filogenia , Poli I-C/farmacologia , Distribuição Aleatória , Alinhamento de Sequência/veterinária
2.
Fish Shellfish Immunol ; 89: 141-148, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30926477

RESUMO

Cadmium (Cd) is the most common heavy metal and is easily detected in aquatic environments worldwide. The genus Bacillus was one of dominant probiotics, which was commonly used in aquaculture. The present study was undertaken to explore the effects of Bacillus cereus (B. cereus) supplementation on hematological parameters and the immune response of Carassius auratus gibelio (C. gibelio) following Cd exposure. Fish were exposed to waterborne Cd (0, 1 and 2 mg/L) and/or treated with dietary B. cereus at 108 cfu/g for four weeks. The hematological disturbances observed after exposure of waterborne Cd included significant decreases in red blood cell (RBC) count, hemoglobin (Hb) concentration and hematocrit (HCT). While significant elevation (P < 0.05) of RBC count, HCT and Hb levels in the 1 and 2 mg/L Cd-B. cereus administration group at 4 weeks, compared with the Cd-only group. Among serum enzymatic activities, aspartate aminotransferase (AST) and alanine transaminase (ALT) activities by Cd exposure were significantly higher than controls, but this increase was effectively inhibited in Cd-B. cereus administration groups. In the Cd-B. cereus administration group, significant down-regulation of Hsp70, Hsp90, IL-1ß, IL-6, IL-10 and TNF-α in conjunction with the up-regulation of IgM and LZM in the spleen indicated that B. cereus alleviated the Cd-induced damage to the immune system to some degree. The results of this study suggested that B. cereus has the potential to countermeasure Cd-induced hematological disturbances and immunosuppression in C. gibelio.


Assuntos
Bacillus cereus/química , Cádmio/efeitos adversos , Carpa Dourada/imunologia , Tolerância Imunológica , Probióticos/farmacologia , Poluentes Químicos da Água/efeitos adversos , Ração Animal/análise , Animais , Dieta/veterinária , Testes Hematológicos/veterinária , Distribuição Aleatória
3.
Dev Comp Immunol ; 94: 11-15, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30659854

RESUMO

Hepcidin is an antimicrobial peptide and an iron regulatory protein that prevents the release of excess iron in the blood. There is evidence suggesting that teleost hepcidin is a major player in antimicrobial defense against various bacteria species, but little is known regarding the effects of teleost hepcidin in protozoan parasitic infections. We examined the role of hepcidin during the course of infection of goldfish with Trypanosoma carassii. Quantitative real-time PCR was used to determine the expression of hepcidin in goldfish immune organs during the course of T. carassii infection. During the acute phase of the T. carassii infection, the mRNA levels of hepcidin were up-regulated in liver and kidney. In contrast, an up-regulation of hepcidin mRNA expression in spleen was observed during the chronic phase of the infection. Furthermore, a synthetic goldfish hepcidin peptide induced trypanosome lysis in vitro, and parasite surface disruption was confirmed by scanning electron microscopy (SEM) analysis. These results suggest that, in addition to well-characterized direct antibacterial activities, teleost hepcidin also exhibits trypanocidal activity.


Assuntos
Anti-Infecciosos/metabolismo , Doenças dos Peixes/imunologia , Carpa Dourada/imunologia , Hepcidinas/metabolismo , Macrófagos/imunologia , Trypanosoma/imunologia , Tripanossomíase/imunologia , Animais , Citocinas/metabolismo , Imunidade Inata , Transcriptoma , Regulação para Cima
4.
Fish Shellfish Immunol ; 86: 913-921, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30550991

RESUMO

The present study was conducted to evaluate dietary inosine 5'-monophosphate (5'-IMP) on growth, immune genes expression and disease resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS Ⅲ) (initial body weight: 7.48 g). Six diets were formulated containing exogenous 5'-IMP at three gradient levels (0, 0.1% and 0.2%) in the high dietary fishmeal group (15% fishmeal: D1, D2, D3) and in the high dietary soybean meal group (33% soybean meal: D4, D5, D6). Each diet was randomly allotted to triplicate tanks in a recirculating system. After the feeding trial, fish were exposed to Aeromonas hydrophila challenge. Hematological and immunological responses were analyzed before and after challenge. The results indicated that feeding rate in all 5'-IMP supplemented treatments (D2, D3, D5 and D6) and daily growth coefficient in D5 and D6 were reduced compared with those of respective control treatments (D1 and D4) without 5'-IMP addition (P < 0.05). The cumulative survival rates were numerically improved by dietary 5'-IMP supplementation (P > 0.05). Compared with the respective control treatment, in the high fishmeal group, plasma SOD and MPO were significantly elevated in D3 at the end of feeding trial (P < 0.05), plasma SOD and lysozyme were significantly increased in D3 after bacterial challenge (P < 0.05); in high soybean meal group, plasma lysozyme activity was significantly elevated in D5 post bacterial challenge (P < 0.05). Most of the expression of immune related genes (intelectin, major histocompatibility complex class II ß (MHC II ß), Complement 3 (C3), Complement component C7-1 (ccC7), lysozyme C, Interleukin 1ß (IL-1ß), Tumor necrosis factor α1 (TNF-α1), Transforming growth factor-beta (TGF-ß) and Interleukin 8 (IL-8)) in spleen, kidney and liver of the fish were significantly affected by supplementation of 5'-IMP at the end of feeding trial and post bacterial challenge. Additionally, adding 5'-IMP in high soybean meal diets exerted further effects of promoting immunity than counterparts in high fishmeal diets. Considering enhanced disease resistance, the immunopotentiation of 5'-IMP was manifested when the addition level was 0.1% in high soybean meal diets and 0.2% in high fishmeal diets.


Assuntos
Dieta/veterinária , Regulação da Expressão Gênica/imunologia , Carpa Dourada/genética , Carpa Dourada/imunologia , Inosina Monofosfato/metabolismo , Aeromonas hydrophila/fisiologia , Ração Animal/análise , Animais , Dieta/classificação , Suplementos Nutricionais/análise , Resistência à Doença/efeitos dos fármacos , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Carpa Dourada/crescimento & desenvolvimento , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Inosina Monofosfato/administração & dosagem
5.
Dev Comp Immunol ; 91: 93-100, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385316

RESUMO

In this study, we investigated the immune responses against Mycobacterium gordonae in ginbuna crucian carp. Cumulative mortality of ginbuna injected with 2.0 × 107 CFU of M. gordonae was 50% at 170 days post-infection. CD4-1, CD8α, T-bet and IFNγ2 gene expression levels were significantly upregulated in ginbuna injected with 1.9 × 108 CFU of M. gordonae at 21 and 28 days post-infection. The CD4-2 level did not change during the experiment. Granulomatous responses consisted of central macrophage accumulation and surrounding lymphocytes, and Ziehl-Neelsen-positive bacteria were observed in the trunk kidney of the challenged fish. Immunohistochemistry using anti-ginbuna IFNγs and anti-ginbuna CD4-1 polyclonal antibody revealed that the marginal lymphocytes were positive for CD4-1, and the IFNγ-producing cells surrounded the mycobacterial cell-laden phagocytes. These results suggest that CD4-1+ cells and IFNγ2 play important roles in the granulomatous inflammation against Mycobacterial infections in teleosts.


Assuntos
Doenças dos Peixes/imunologia , Carpa Dourada/imunologia , Granuloma/imunologia , Inflamação/imunologia , Linfócitos/imunologia , Macrófagos/imunologia , Infecções por Micobactéria não Tuberculosa/imunologia , Micobactérias não Tuberculosas/fisiologia , Animais , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Proteínas de Peixes/metabolismo , Imuno-Histoquímica , Interferon gama/metabolismo , Proteínas com Domínio T/metabolismo
6.
Fish Shellfish Immunol ; 79: 265-273, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29775741

RESUMO

The present study examined the effect of dietary spirulina, Arthrospira platensis on growth performance, blood physiological indices, immune-related gene expressions and resistance of juvenile gibel carp against Aeromonas hydrophila infection. Four isonitrogenous (360 g kg-1) and isolipidic (90 g kg-1) diets were formulated with containing different levels of spirulina powder of 0 g (SP0, the control diet), 3.38 g (SP3.38), 6.76 g (SP6.76) and 13.52 g (SP13.52) per 100 g diet to replace 0%, 25%, 50% and 100% of fishmeal protein, respectively. And each diet was randomly assigned to triplicate tanks (150-L capacity per each) and each tank was stocked with 22 fish (15.37 ±â€¯0.06 g). Fish were fed one of the tested diets up to satiation twice a day for 46 days. A challenge test was carried out after the feeding trial by injecting Aeromonas hydrophila intraperitoneally for 7 days. The results showed that fish growth, feeding rate in groups SP3.38 and SP6.76 were significantly higher than those of groups SP0 and SP13.52 (P < 0.05). Feed efficiency and protein retention rate had no significant difference among all tested groups. Plasma superoxide dismutase and phagocyte activity of blood leukocytes significantly increased in the spirulina-fed fish groups at 12-h post the bacterial challenge (P < 0.05). Both pre and post challenge test, plasma lysozyme activities in spirulina-fed groups were significantly higher than that in the control group (P < 0.05). Plasma malondialdehyde got the lowest value in the SP13.52 group before and after the challenge test. The transcriptional levels of TLR2 (Toll like receptor 2), myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), interleukin-1ß (IL-1ß) and tumor necrosis factor-α1 (TNF-α1) in spleen and kidney significantly increased post the bacterial challenge compared to the pre challenge. And the relative expressions of the immune-related genes of spirulina-fed fish groups were higher than those of the control group before and after the challenge test. The 7-day cumulative survival rate after the bacterial challenge was highest in the SP3.38 group (P < 0.05). The present results indicated that low dietary inclusion of spirulina significantly enhanced the immune response of gibel carp partly through TLR2 pathway and 3.38% of dietary spirulina was recommended for the juveniles based on the growth and immune response.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica/imunologia , Carpa Dourada/genética , Carpa Dourada/imunologia , Spirulina/química , Aeromonas hydrophila/fisiologia , Ração Animal/análise , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Resistência à Doença/imunologia , Proteínas de Peixes/metabolismo , Carpa Dourada/crescimento & desenvolvimento , Infecções por Bactérias Gram-Negativas/imunologia
7.
Fish Shellfish Immunol ; 78: 322-330, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29702236

RESUMO

This study evaluated the probiotic potential of B. velezensis JW through experimental and genomic analysis approaches. Strain JW showed antimicrobial activity against a broad range of fish pathogenic bacteria including Aeromonas hydrophila, Aeromonas salmonicida, Lactococcus garvieae, Streptococcus agalactiae, and Vibrio Parahemolyticus. Fish (Carassius auratus) were fed with the diets containing 0 (control), 107, and 109 cfu/g of B. velezensis JW for 4 weeks. Various immune parameters were examined at 1, 2, 3, and 4 weeks of post-feeding. Results showed that JW supplemented diets significantly increased acid phosphatase (ACP), alkaline phosphatase (AKP), and glutathione peroxidase (GSH-PX) activity. The mRNA expression of immune-related genes in the head kidney of C. auratus was measured. Among them, the interferon gamma gene (IFN- γ) and tumor necrosis factor-α (TNF-α) showed higher expression after 3 and 4 weeks of feeding (P < 0.05). The expression of interleukin-1 (IL-1) only being significantly upregulated by 109 cfu/g of JW after 1 week of feeding (P < 0.05). The upregulation of interleukin-4 (IL-4) increased over time from 1st to 4th week. The expression of interleukin-10 (IL-10) and interleukin-12 (IL-12) showed an opposite expression pattern with IL-10 significantly upregulated and IL-12 significantly downregulated by JW containing diets at 2, 3, and 4 weeks of post-feeding (P < 0.05). Moreover, fish fed with JW supplemented diets showed significantly improved survival rate after A. hydrophila infection. The analysis of the genome of JW revealed several features aiding host health and being relevant to the GIT adaptation. Four bacteriocins, three Polyketide Synthetase (PKS), and five Nonribosomal Peptide-Synthetase (NRPS) gene clusters were identified in the genome. In summary, the above results clearly proved that B. velezensis JW has the potential to be developed as a probiotic agent in aquaculture.


Assuntos
Bacillus/química , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Carpa Dourada/imunologia , Imunidade Inata/genética , Probióticos/farmacologia , Ração Animal/análise , Animais , Infecções Bacterianas/imunologia , Fenômenos Fisiológicos Bacterianos , Dieta/veterinária , Proteínas de Peixes/genética , Carpa Dourada/genética
8.
Fish Shellfish Immunol ; 76: 101-109, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29481848

RESUMO

The manganese contamination has become a global problem, recently, because it is perceived as a real threat to the human health and the environment. It is well-known that overexposure to Mn2+ may have negative physiological effects on fish and other organisms inhabiting heavy metal polluted waters. To the best of our knowledge, studies relating with manganese effects on fish antioxidant enzyme response in the blood, immunocompetence and erythron profile alteration, are scarce. In this study, the acute sub-lethal effects of manganese on blood antioxidant response, immune status and erythron profile were determined by exposing the freshwater model organism, Carassius auratus, to two doses of this metal (3.88 ±â€¯0.193 mg/L and 7.52 ±â€¯0.234 mg/L Mn2+) for 96 h. Significant increases in blood antioxidant enzyme activity like superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST), were observed in fish exposed to manganese. Furthermore, plasmatic glucose and cortisol levels increased, while total protein decreased significantly. White blood cell differential count revealed a significant increase in monocyte and neutrophil number and a significant decrease of lymphocyte's number in fish exposed to manganese compared with those of control group. That can be considered as a clear evidence of altered immune system. Measured of erythron profile revealed a significant increasing of cellular and nuclear alteration of red blood cells, with karryorhectic, dividing and micronucleated erythrocytes in exposed fish, indicating the cytotoxic and genotoxic effects Mn2+ ions. Our data shown also that manganese could trigger antioxidant response, modulate immune response and induce erythron profile modification leading to eryptosis, compromising the blood oxygen carrying capacity, and overall health status in fish. This may suggest those parameters consider as useful biomarkers for monitoring effects of sub-lethal metal exposure on fish.


Assuntos
Antioxidantes/metabolismo , Eritrócitos/metabolismo , Carpa Dourada/imunologia , Imunidade Inata , Manganês/efeitos adversos , Poluentes Químicos da Água/efeitos adversos , Albânia , Animais , Relação Dose-Resposta a Droga , Enzimas/sangue , Células Precursoras Eritroides/metabolismo , Distribuição Aleatória , Rios
9.
Fish Shellfish Immunol ; 76: 35-40, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29486350

RESUMO

Crucian carp (Carassius auratus gibelio) is a popular food fish in Asia, and cyprinid herpesvirus 2 (CyHV-2) is the only known viral pathogen for crucian carp. Type I interferon genes are induced up on host cell recognition of viral nucleic acids and well recognized for their crucial roles in providing local or systemic protection against the viruses in various organisms. In a transcriptome analysis to uncover differentially expressed genes in crucian carp in response to CyHV-2 challenge, a partial interferon transcript was identified to be significantly up-regulated in the kidney of infected fish, which was named as crucian carp IFNc (ccIFNc). The complete ORF of ccIFNc was further determined by RACE technique, which spanned over 546 bp and encoded a polypeptide containing 182 amino acids. Phylogenetic analysis revealed that ccIFNc clustered with known type I IFN genes from other aquatic organisms. Quantitative RT-PCR analysis demonstrated that ccIFNc was constitutively expressed in all investigated tissues with a comparably higher expression level in spleen, gill, kidney, and muscle. Following challenge with CyHV-2, the transcriptional levels of ccIFNc were dramatically up-regulated in all of the tested tissues, especially in the spleen and gill with increased folds of 436 and 158, respectively. The intramuscular (i.m.) injection of a eukaryotic expression plasmid encoding ccIFNc (pEGFP-cIFNc) resulted in increased ccIFNc expression and reduced the mortality after the CyHV-2 challenge significantly. In summary, our data suggested that the ccIFNc belongs to the type I interferon family with a potential role in countering CyHV-2 infection in crucian carp.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Carpa Dourada/genética , Carpa Dourada/imunologia , Imunidade Inata/genética , Interferon Tipo I/genética , Interferon Tipo I/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 , Herpesviridae/fisiologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/veterinária , Interferon Tipo I/química , Filogenia , Alinhamento de Sequência/veterinária
10.
Dev Comp Immunol ; 82: 1-6, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29305165

RESUMO

miRNAs (microRNAs), a small endogenous non-coding RNAs, play crucial roles in post-transcriptional regulator of genes expression in various biological processes. Cyprinid Herpesvirus-2 (CyHV-2) is a highly pathogenic member of the alloherpesviridae that causes acute mass mortalities in populations of Carassius auratus gibelio and Carassius auratus auratus. However, the molecular mechanisms underlying the pathogenicity of CyHV-2 have not been fully determined. Here, miRNA expression profiles were identified via high-throughput sequencing in the kidney of Carassius auratus gibelio infected or uninfected with CyHV-2. The results showed that a total number of 840 known miRNAs and 48 putative novel miRNAs were identified. Then we compared the expression patterns of miRNAs in the two groups, 23 miRNAs were significantly differentially expressed between the uninfected and infected groups. Further, the expressions of 23 miRNAs were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR), the results showed that the expression patterns were basically the same with the sequencing. Prediction of targets of differentially expressed miRNAs revealed that the miRNAs participated in the regulation of multiple immune-related signaling pathways, including Chemokine signaling pathway, Apoptosis, Jak-STAT signaling pathway and MAPK signaling pathway. Taken together, these data provide insight into the regulatory mechanisms of miRNA and highlight the function of miRNA in the regulation of the immune response during the interaction between host and virus pathogens.


Assuntos
Carpa Dourada/genética , Infecções por Herpesviridae/genética , Herpesviridae/imunologia , Imunidade/genética , MicroRNAs/genética , Animais , Apoptose/genética , Quimiocinas/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Carpa Dourada/imunologia , Carpa Dourada/virologia , Infecções por Herpesviridae/imunologia , Imunomodulação , Janus Quinases/genética , Janus Quinases/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/genética , Transcriptoma
11.
Dev Comp Immunol ; 79: 1-10, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28988993

RESUMO

The NLRC3-like (NLRC3L) molecule from the goldfish transcriptome database was identified and characterized. Quantitative gene expression analysis revealed the highest mRNA levels of NLRC3L were in the spleen and intestine, with lower mRNA levels observed in muscle and liver. Goldfish NLRC3L was differentially expressed in goldfish immune cell populations with highest mRNA levels measured in PBLs and macrophages. We generated a recombinant form of the molecule (rgfNLRC3L) and an anti-CT-NLRC3L IgG. Treatment of goldfish primary kidney macrophages in vitro with ATP, LPS and heat-killed Aeromonas salmonicida up-regulated the NLRC3L mRNA and protein. Confocal microscopy and co-immunoprecipitation assays indicated that goldfish rgfNLRC3L interacted with apoptosis-associated spec-like protein (ASC) in eukaryotic cells, indicating that NLRC3L may participate in the regulation of the inflammasome responses. The dual-luciferase reporter assay showed that NLRC3L over-expression did not cause the activation of NF-κB, but that it cooperated with RIP2 to down-regulate NF-κB activation. Our results indicate that the NLRC3L may function as a regulator of NLR pathways in teleosts.


Assuntos
Aeromonas salmonicida/imunologia , Proteínas de Peixes/genética , Carpa Dourada/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Animais , Células Cultivadas , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Rim/patologia , NF-kappa B/metabolismo
12.
Fish Shellfish Immunol ; 73: 75-83, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29196031

RESUMO

The present study was conducted to evaluate the effects of various Bacillus coagulans feeding patterns on growth, antioxidant parameter and Nrf2 pathway in juvenile gibel carp. The similar size of gibel carp (initial weight: 14.33 ± 0.15 g) were subjected to three levels of B. coagulans supplementation (0, 500, and 1000 mg/kg) and two feeding modes (supplementing B. coagulans continuously or for two days of B. coagulans after 5 days of a basal diet) according to a 3 × 2 factorial design. The fish that were continuously fed 500 mg/kg B. coagulans (P2) and those fed the first basal diet for 5 days followed by 500 mg/kg or 1000 mg/kg B.coagulans for 2 days (P4 or P5) showed higher weight gain rate and specific growth rate than the other groups. Blood respiratory burst (RB), myeloperoxidase (MPO), and anti-superoxide anion free radical (AFASER) activities in the P4 group were higher than those of the control. White blood cell count (WBC), RB activity, MPO activity, and glutathione (GSH) content in the P5 group were also higher than those of the control. A similar higher trend was observed in the gene expressions of NADPH oxidase 2 (NOX2), NFE2-related factor (Nrf2), Kelch-like-ECH-associated protein(Keap1) in the P4 and NOX2, NRF2, CNC homolog 1 (Bach1), peroxiredoxin 2 (Prx2) in the P5 group compared with the control. Additionally, we observed a significantly lower level of plasma aspartate aminotransferase (AST), lower activity of alanine aminotransferase (ALT), a higher level of MPO, higher GPX activity, and increased NRF2 and Prx2 expression were all observed in the P2 treatment group compared with the control. Furthermore, the malondialdehyde (MDA) content in the P2, P3, and P4 groups was lower than that of the control. These results indicate that a diet supplemented with appropriate levels of B.coagulans could improve the growth, immune response, and antioxidant capability of gibel carp. We concluded that the pattern of two days of 500 or 1000 mg/kg B. coagulans after 5 days of a basal diet was recommended for gibel carp.


Assuntos
Antioxidantes/metabolismo , Bacillus coagulans/química , Carpa Dourada/fisiologia , Imunidade Inata , Probióticos/farmacologia , Transdução de Sinais , Ração Animal/análise , Animais , Dieta/veterinária , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Carpa Dourada/crescimento & desenvolvimento , Carpa Dourada/imunologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Probióticos/administração & dosagem
13.
Fish Shellfish Immunol ; 72: 629-638, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29183810

RESUMO

Toll-like receptors (TLRs) are important components of innate immunity. TLRs recognize pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways in response. In present study, we report the identification of two TLRs from gibel carp (Carassius auratus gibelio), TLR2 and TLR3 (designated CagTLR2 and CagTLR3, respectively). We report on the genomic structures and mRNA expression patterns of CagTLR2 and CagTLR3. Five exons and four introns were identified from the genomic DNA sequence of CagTLR3 (4749 bp in total length); this genomic organization is similar to that of TLR3 in zebrafish and human. However, only one intron was identified from the CagTLR2 genomic locus (3166 bp in total length); this unique genomic organization of CagTLR2 is different from that of TLR2 in fish and humans. The cDNAs of CagTLR2 and CagTLR3 encoded 791 and 904 amino acid residues, respectively. CagTLR2 and CagTLR3 contained two distinct structural/functional motifs of the TLR family: a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The positions of critical amino acid residues involed in PAMP recognition and signaling pathway transduction in mammalian TLRs were conserved in CagTLR2 and CagTLR3. Phylogenetic analysis revealed a closer clustering of CagTLR2 and CagTLR3 with TLRs from freshwater fish than with marine fish species. In healthy gibel carp, transcripts of these genes were detected in all examined tissues, and high expression levels of CagTLR2 and CagTLR3 were observed in liver and brain, respectively. Following injection with CyHV-2, expression levels of CagTLR2 and CagTLR3 were significantly upregulated in the spleens of gibel carp after three days, and CagTLR3 transcript levels were rapidly increased in head kidney after 12 h. These results suggest that CagTLR2 and CagTLR3 are functionally involved in the induction of antiviral immune response.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Carpa Dourada/genética , Carpa Dourada/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência/veterinária , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 3 Toll-Like/química , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia
14.
Fish Shellfish Immunol ; 70: 372-380, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28916356

RESUMO

The polymeric immunoglobulin receptor (pIgR) plays a pivotal role in mucosal immune response by transporting polymeric immunoglobulins onto the surface of mucosal epithelia to protect animals from invading pathogens. In this study, the full-length cDNA of pIgR was firstly cloned in Qihe crucian carp (Carassius auratus), hereafter designated as CapIgR, by using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. The molecular characterization and expression of CapIgR were investigated. The full-length cDNA sequence of CapIgR was composed of 1409 bp, which included a 112 bp 5'-untranslated region (UTR), a 984 bp ORF, and a 313 bp 3'-UTR, with a putative polyadenylation signal sequence AATAAA located upstream of the poly(A) tail. The deduced amino acid sequence indicated that CapIgR was a single-spanning transmembrane protein with 327 amino acids and possessed a signal peptide, an extracellular region containing two immunoglobulin-like domains, a transmembrane region, and an intracellular region. The mRNA expression levels of CapIgR were detected in different tissues of healthy C. auratus by quantitative real-time PCR, and the highest expression level was found in the liver. After Aeromonas hydrophila challenge, CapIgR expression was upregulated in different tissues at certain time points, and temporal expression changes of CapIgR fluctuated in a time-dependent manner. CapIgR exhibited rapid immune response to A. hydrophila challenge and played an important role in the immune defense of fish. These findings provided insights into the structure, function, and immune defense mechanism of CapIgR in C. auratus. This study can serve as a basis for developing disease control strategies in aquaculture.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Carpa Dourada/genética , Carpa Dourada/imunologia , Imunidade Inata/genética , Receptores de Imunoglobulina Polimérica/genética , Receptores de Imunoglobulina Polimérica/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Filogenia , Receptores de Imunoglobulina Polimérica/química , Alinhamento de Sequência/veterinária
15.
BMC Genomics ; 18(1): 561, 2017 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-28738780

RESUMO

BACKGROUND: Gibel carp is an important aquaculture species in China, and a herpesvirus, called as Carassius auratus herpesvirus (CaHV), has hampered the aquaculture development. Diverse gynogenetic clones of gibel carp have been identified or created, and some of them have been used as aquaculture varieties, but their resistances to herpesvirus and the underlying mechanism remain unknown. RESULTS: To reveal their susceptibility differences, we firstly performed herpesvirus challenge experiments in three gynogenetic clones of gibel carp, including the leading variety clone A+, candidate variety clone F and wild clone H. Three clones showed distinct resistances to CaHV. Moreover, 8772, 8679 and 10,982 differentially expressed unigenes (DEUs) were identified from comparative transcriptomes between diseased individuals and control individuals of clone A+, F and H, respectively. Comprehensive analysis of the shared DEUs in all three clones displayed common defense pathways to the herpesvirus infection, activating IFN system and suppressing complements. KEGG pathway analysis of specifically changed DEUs in respective clones revealed distinct immune responses to the herpesvirus infection. The DEU numbers identified from clone H in KEGG immune-related pathways, such as "chemokine signaling pathway", "Toll-like receptor signaling pathway" and others, were remarkably much more than those from clone A+ and F. Several IFN-related genes, including Mx1, viperin, PKR and others, showed higher increases in the resistant clone H than that in the others. IFNphi3, IFI44-like and Gig2 displayed the highest expression in clone F and IRF1 uniquely increased in susceptible clone A+. In contrast to strong immune defense in resistant clone H, susceptible clone A+ showed remarkable up-regulation of genes related to apoptosis or death, indicating that clone A+ failed to resist virus offensive and evidently induced apoptosis or death. CONCLUSIONS: Our study is the first attempt to screen distinct resistances and immune responses of three gynogenetic gibel carp clones to herpesvirus infection by comprehensive transcriptomes. These differential DEUs, immune-related pathways and IFN system genes identified from susceptible and resistant clones will be beneficial to marker-assisted selection (MAS) breeding or molecular module-based resistance breeding in gibel carp.


Assuntos
Perfilação da Expressão Gênica , Carpa Dourada/imunologia , Carpa Dourada/virologia , Herpesviridae/fisiologia , Animais , Cruzamento , Resistência à Doença/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Carpa Dourada/genética , Hibridização Genética
16.
Fish Shellfish Immunol ; 67: 382-392, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28602683

RESUMO

C-type lectins (CTLs), as the members of pattern-recognition receptors (PRRs), play the significant roles in innate immunity through binding with pathogen-associated molecular patterns (PAMPs) on the surface of microbe. In the present study, a novel CTL, Nattectin-like protein (named as CaNTC), was investigated in Qihe crucian carp Carassius auratus. The full-length cDNA of CaNTC was composed of 776 bp, with a 152 bp 5'-untranslated region (UTR), a 492 bp ORF encoding a 163-aa protein, and a 132 bp 3'-UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of CaNTC contained a signal peptide, a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys57-Cys150, Cys126-Cys142), and an EPN/WND motif required for carbohydrate-binding specificity. With regard to the mRNA transcript of CaNTC, it was predominately expressed in liver. The temporal expressions of CaNTC were obviously up-regulated in liver, spleen and head-kidney after challenged by Aeromonas hydrophila and poly I: C, respectively, and the change pattern was in the time-depended manner. The recombinant CaNTC (rCaNTC) purified from Escherichia coli BL21 (DE3), exhibited strong binding ability with LPS and PGN, as well as all tested bacteria in a Ca2+-independent manner. With regard to the agglutinating activity of rCaNTC, rCaNTC was able to agglutinate rabbit erythrocytes and three kinds of bacteria (Gram-negative bacteria, Escherichia coli and A. hydrophila, and Gram-positive bacteria Staphylococcus aureus) in a Ca2+-dependent manner. These findings collectively demonstrated that CaNTC, as a PRR, could be involved in the innate immunity and play an important role in immune defense of C. auratus.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Carpa Dourada/genética , Carpa Dourada/imunologia , Imunidade Inata/genética , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Aeromonas hydrophila/fisiologia , Aglutinação , Sequência de Aminoácidos , Animais , Sequência de Bases , Metabolismo dos Carboidratos , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Lectinas Tipo C/química , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência/veterinária
17.
Dev Comp Immunol ; 74: 1-9, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28389237

RESUMO

Intracellular bacterial and viral diseases are widespread in the aquaculture industry and cause serious economic losses. Development of effective vaccines and adjuvants that can induce cell-mediated immunity is urgently needed for prevention of these diseases. Here we report the immunostimulatory effects of probiotic bacteria ''E. faecalis'' in ginbuna crucian carp Carassius auratus langsdorfii. Intraperitoneal injection of heat-killed E. faecalis induced an increase in CD4-1+ lymphocytes, CD8α+ lymphocytes and macrophages in vivo. Expression of Th1 cytokine genes was enhanced by exposure to the bacteria in vitro. We identified the leukocyte subsets that expressed specific Th1 cytokine genes: granulocytes and macrophages produced IL12 and IFNγrel2, respectively, while lymphocytes produced IFNγs including IFNγ1 and IFNγ2. Finally, expression of Th1 cytokines was also enhanced by intraperitoneal injection of heat-killed E. faecalis in vivo, while expression of Th2 cytokine was unchanged. Together, these findings suggest that heat-killed E. faecalis can induce cell-mediated immunity in fish.


Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Enterococcus faecalis/imunologia , Carpa Dourada/imunologia , Linfócitos/imunologia , Macrófagos/imunologia , Animais , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Proliferação de Células , Proteínas de Peixes/metabolismo , Temperatura Alta , Imunidade Celular , Interferon gama/metabolismo , Células Th1/imunologia
18.
Dev Comp Immunol ; 67: 300-309, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27623236

RESUMO

Transferrin is an evolutionary conserved protein that in addition to having a critical role in iron transport also has been shown to have a crucial role in host defence, by depriving iron from invading pathogens. Recently cleaved transferrin products was shown to activate macrophages in vitro. We now use an in vivo model of self-resolving peritonitis in goldfish, coupled with gene expression and protein analysis to evaluate the contributions of cleaved transferrin to acute inflammation. We show, for the first time, that cleaved transferrin products are produced in vivo early during an acute inflammatory response. These cleaved transferrin fragments were produced during pathogen-induced, but not sterile, inflammation. Both macrophages and neutrophils were able to contribute to transferrin cleavage. However, only macrophages contributed to this innate process through inducible expression of transferrin. The appearance of transferrin cleavage products in vivo correlated with the influx of leukocytes but did not necessarily correlate the induction of robust respiratory burst and nitric oxide responses. Overall, this study adds to a growing body of work highlighting the role of transferrin as an immune regulator during acute inflammation. Given the significant conservation of this and related molecules, these findings have potentially broad implications for host defences and inflammation control across evolution.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Carpa Dourada/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Peritonite/imunologia , Transferrina/metabolismo , Doença Aguda , Animais , Evolução Biológica , Movimento Celular , Células Cultivadas , Regulação da Expressão Gênica , Ferro/metabolismo , Proteólise
19.
Dev Comp Immunol ; 67: 377-384, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27581741

RESUMO

We report on the expression analysis and functional characterization of IL-4/13A and IL-4/13B in goldfish. Quantitative analysis indicated the highest expression in the heart, spleen, brain, and kidney, with comparable expression patterns for both IL-4/13A and IL-4/13B. The mRNA levels of IL-4/13A and IL-4/13B in the immune cells examined were highest in macrophage and monocytes. Assessment of spleen mRNA following infection with Trypanosoma carassii, a prominent protozoan pathogen of fish, revealed decrease in IL-4/13B and arginase expression 14 days post infection, followed by an increase in IL-4/13B and arginase-2 at 28 days post infection. Recombinant forms of IL-4/13A and IL-4/13B induced an increase in arginase activity in macrophages in a dose-dependent manner. Recombinant IL-4/13A and IL-4/13B also induced significant increase in mRNA levels of arginase -2 in macrophages at 6, 12, 18 and 24 h after treatment. Furthermore, treatment with both IL-4/13 recombinants interfered with the IFNγ-induced nitric oxide response of macrophages. Our results suggest a conserved role of IL-4/IL-13 in induction of alternative activation phenotype in teleost macrophages.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Carpa Dourada/imunologia , Macrófagos/imunologia , Proteínas Recombinantes/metabolismo , Trypanosoma/imunologia , Tripanossomíase/imunologia , Animais , Arginase/genética , Arginase/metabolismo , Evolução Biológica , Células Cultivadas , Regulação para Baixo , Imunidade Inata , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Ativação de Macrófagos , Óxido Nítrico/metabolismo , Cultura Primária de Células
20.
Fish Shellfish Immunol ; 59: 149-154, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27769690

RESUMO

The objective of the present study was to investigate the effect of dietary Lactobacillus acidophilus on skin mucus protein pattern, immune and appetite related genes expression as well as growth performance in gold fish (Carassius auratus gibelio). Three hundred healthy gold fish (2.5 ± 0.05) juveniles were randomly distributed in 12 glass aquariums (400-L; 25 fish per aquaria) and fed experimental diets contain different levels of L. acidophilus (0, 1.5 × 108, 3 × 108 and 6 × 108) for 8 weeks. SDS-PAGE analysis of skin mucus protein profile at the end of the feeding trial revealed differences in protein profile of probiotic fed fish and control group; even three new bands were observed in L. acidophilus treated groups. Furthermore, fish fed 6 × 108 CFU g-1 supplemented diet showed up-regulation of both TNF-1α and TNF-2α gene expression (P < 0.05). Evaluation of appetite related gene expression showed down-regulation of ghrelin in probiotic fed fish compared those of control treatment (P < 0.05). However, administration of different levels of L. acidophilus had no significant effects on growth performance (P > 0.05). These results demonstrated that while no beneficial effects on growth performance, dietary L. acidophilus affects immune and appetite related genes expression as well as skin mucus protein profile.


Assuntos
Proteínas de Peixes/genética , Regulação da Expressão Gênica , Carpa Dourada/genética , Carpa Dourada/imunologia , Imunidade Inata , Lactobacillus acidophilus , Probióticos , Ração Animal/análise , Animais , Apetite/genética , Dieta/veterinária , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida/veterinária , Proteínas de Peixes/metabolismo , Grelina/genética , Grelina/metabolismo , Lactobacillus acidophilus/imunologia , Muco/imunologia , Distribuição Aleatória , Pele/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
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