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1.
BMC Genomics ; 22(1): 653, 2021 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-34511071

RESUMO

BACKGROUND: As non-coding RNA molecules of more than 200 bp in length, long non-coding RNAs (lncRNAs) play a variety of roles in biological processes, including regulating the immune responses to bacterial infections. In recent years, there have been many in-depth studies on mammalian lncRNAs, but the relevant studies in fish are very limited. Meanwhile, since lncRNAs are not conserved among species, it is difficult to apply the existing results directly to unstudied species. RESULTS: To obtain the information of lncRNAs in Megalobrama amblycephala, one of the most economically important freshwater fish in China, also to better understand the biological significance of lncRNAs in the immunity system, the fish liver at 0, 4, 12, 24, and 72 h post Aeromonas hydrophila infection (hpi) were obtained for lncRNA-sequencing (lncRNA-seq). A total of 14,849 lncRNAs were identified, and 2196 lncRNAs showed significant differences at different time points post A. hydrophila infection. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the target genes of the differentially expressed lncRNAs were enriched in several pathways related to immune such as apoptosis, inflammation, and immune response. Time-specific modules were then identified, using weighted correlation network analysis (WGCNA), and 28 modules significantly correlated with different time point after infection were found. Furthermore, four immune-related genes and six lncRNAs in the time-specific modules were subsequently verified by RT-qPCR. CONCLUSIONS: The above findings reveal the discovery of widespread differentially expressed lncRNAs in the M. amblycephala liver post A. hydrophila infection, suggesting that lncRNAs might participate in the regulation of host response to bacterial infection, enriching the information of lncRNAs in teleost and providing a resources basis for further studies on the immune function of lncRNAs.


Assuntos
Cyprinidae , RNA Longo não Codificante , Aeromonas hydrophila , Animais , Cyprinidae/genética , Fígado , RNA Longo não Codificante/genética , RNA Mensageiro/genética
2.
Ecotoxicol Environ Saf ; 225: 112786, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34555717

RESUMO

The occurrence of immuno-compromised status in animals with zearalenone (ZEA) exposure may be a critical contributor to associated mucosal (gastrointestinal tract) diseases. However, it is difficult to assess the associated risks with limited reference data. This study comprehensively discussed the effects of ZEA on intestinal immune components, cytokines and molecular mechanism of juvenile grass carp infected with Aeromonas hydrophila. Specifically, the fish were fed six graded levels of dietary ZEA (0-2507 µg kg-1 diet) for 70 d. The results pointed out that the average residual amount of ZEA in the intestines increased with dose level after ZEA feeding. We further performed an infection assay using A. hydrophila. After 14 d, ZEA groups increased enteritis morbidity rate compared with controls. The acid phosphatase (ACP), lysozyme (LZ) activities and immunoglobulin M (IgM) content were significantly decreased in three intestinal segments. Furthermore, ZEA could reduce the transcription of ß-defensin-1, Hepcidin, liver expressed antimicrobial peptide 2A/2B (LEAP-2A/2B) and Mucin-2. We next confirmed the loss of these immune components accompanied by the invasion of the intestinal barrier by bacteria, as indicated by activation of the nuclear factor κB (NF-κB) and the expression of downstream cytokines. Notably, the phosphorylated target of rapamycin (TOR) plays an important role in regulating these genes, thus indicating a possible target caused by ZEA. In summary, the extensive inhibition of immune components by ZEA promotes the spread of pathogens, which may increase the possibility of intestinal mucosa exposure and the risk of transforming disease.


Assuntos
Carpas , Zearalenona , Aeromonas hydrophila , Animais , NF-kappa B/genética , Sirolimo , Zearalenona/toxicidade
3.
Braz J Biol ; 83: e249913, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34550293

RESUMO

Aeromonas hydrophila is a cause of infectious disease outbreaks in carp species cultured in South Asian countries including Pakistan. This bacterium has gained resistance to a wide range of antibiotics and robust preventive measures are necessary to control its spread. No prior use of fish vaccines has been reported in Pakistan. The present study aims to develop and evaluate inactivated vaccines against local strain of A. hydrophila in Pakistan with alum-precipitate as adjuvant. The immunogenic potential of vaccine was evaluated in two Indian major carps (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) and a Chinese carp (Grass carp: Ctenopharyngodon idella). Fish were vaccinated intraperitoneally followed by a challenge through immersion. Fish with an average age of 4-5 months were randomly distributed in three vaccinated groups with three vaccine concentrations of 108, 109 and 1010 colony forming unit (CFU)/ml and a control group. Fixed dose of 0.1ml was applied to each fish on 1st day and a booster dose at 15 days post-vaccination (DPV). Blood samples were collected on 14, 28, 35, 48 and 60 DPV to determine antibody titers in blood serum using compliment fixation test (CFT). Fish were challenged at 60 DPV with infectious A. hydrophila with 108 CFU/ml through immersion. Significantly higher levels of antibody titers were observed from 28 DPV in all vaccinated groups as compared to those in the control group. In challenge experiment the average RPS (relative percent survivability) was 71% for groups vaccinated with 109 and 1010 CFU/ml and 86% for 108 CFU/ml. Vaccine with 108 CFU/ml induced highest immune response followed by 109 and 1010 CFU/ml. The immune response of L. rohita and C. idella was better than that of C. mrigala. In general, normal histopathology was observed in different organs of vaccinated fish whereas minor deteriorative changes were found in fish vaccinated with higher concentrations of the vaccine.


Assuntos
Carpas , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Aeromonas hydrophila , Compostos de Alúmen , Animais , Vacinas Bacterianas , Doenças dos Peixes/prevenção & controle , Infecções por Bactérias Gram-Negativas/prevenção & controle , Infecções por Bactérias Gram-Negativas/veterinária , Imersão
4.
Food Res Int ; 147: 110484, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34399480

RESUMO

Aeromonas hydrophila is an emerging foodborne pathogen capable of causing human gastroenteritis, and the main reservoir is the aquatic environment. In this study, the prevalence and virulence of A. hydrophila in seafoods and ready-to-eat (RTE) sushi distributed in various conditions (refrigerated, dried, or frozen) or seasons was investigated. Strains were isolated from seafood (refrigerated or frozen oysters, sashimi, and processed fish; n = 333) and RTE sushi (n = 88) samples collected in South Korea and then genetically analyzed for gastroenteritis-related virulence genes (aer, ast, and alt). Raw oysters showed the highest prevalence of A. hydrophila (57.1%; 47/91) among all seafoods. Among the sashimi samples, flatfish sashimi (54.8%; 34/62) and salmon sushi (51.4%; 18/35) were the most prevalent. A. hydrophila was not detected in the oysters or anchovies distributed as either frozen or dried products. Seasonal investigations of sashimi and sushi showed that the summer prevalence of A. hydrophila with putative virulence genes was significantly lower in sashimi but highest in sushi. These results indicated that sushi could have been contaminated from several sources during the manufacturing or distribution processes. Significant correlations among the prevalence of putative virulence genes were confirmed, although no combination of genes presented a Phi correlation coefficient above 0.5 (0.26-0.43). To our knowledge, this is the first study to investigate the prevalence of A. hydrophila in various types of retail seafoods and RTE sushi in the East Asia region and then relate the prevalence to the distribution conditions of the samples. This study provides background information on the level of potential risk posed by A. hydrophila in retail seafoods and RTE sushi.


Assuntos
Aeromonas hydrophila , Microbiologia de Alimentos , Aeromonas hydrophila/genética , Animais , Humanos , Alimentos Marinhos , Estações do Ano , Virulência
5.
BMC Res Notes ; 14(1): 313, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399833

RESUMO

OBJECTIVES: Aeromonads cause severe diseases in farmed aquatic organisms. Herein, we examined 28 isolates causing disease in farmed aquatic organisms from India (n = 24) and Taiwan (n = 4) to gain insight of their genotypic and phenotypic properties. RESULTS: API 20NE biochemical phenotyping showed ≥ 90% similarity classifying all isolates as Aeromonas hydrophila. 16S rRNA genotyping showed ≥ 98% homology among all isolates with A. sobria (NR119044.1ATCC), A. veronii (MK990549.1), A. caviae (NR029252.1) and A. hydrophila (MG984625.1ATCC) and other reference strains. In contrast, gyrB showed a higher intraspecies diversity (≥ 96%) than 16S rRNA delineating the 28 isolates into three groups. Group-I consisted of seven Indian isolates clustered with A. sobria (MK484163.1ATCC), group-II comprised of five Indian and two Taiwanese isolates clustered with A. veronii AF417626.1ATCC while group-III had 11 Indian and three Taiwanese isolates grouped with A. hydrophila (AY987520.1 and DQ519366.1) reference strains. None of our isolates clustered with A. caviae (AJ868400.1ATCC) reference strain. These findings suggest that A. sobria, A. veronii and A. hydrophila could be the etiological agents of diseases observed in farmed fish and soft-shelled turtles (Pelodiscus sinensis) examined in this study. Overall, our findings accentuate the importance of combining phenotyping with genotyping for correct taxonomic classification of Aeromonas spp. in Aquaculture.


Assuntos
Aeromonas , Aeromonas/genética , Aeromonas hydrophila/genética , Animais , Índia , RNA Ribossômico 16S/genética , Taiwan
6.
Fish Shellfish Immunol ; 117: 291-298, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34419600

RESUMO

This study investigated the immunomodulatory effects of Sargassum polycystum extract administration in rainbow trout (Oncorhynchus mykiss). S. polycystum methanolic extract was administered orally using feeding needles to individual rainbow trout at the dose of 0 (control), 1 (S1), 3 (S3) and 5 (S5) mg/100 µl/per fish twice a day for 7 days. On 1st, 5th, 3rd and 7th day, blood and tissues were collected from the fish and changes in humoral immune responses and immune-related gene expressions were determined. The result of oxidative radical production showed no difference during early stage of the experiment and was lately decreased (P < 0.05). Lysozyme activity increased on 3rd and 7th day of the study in S5 fish group and on 5th day in S3 group compared to control (P < 0.05). Myeloperoxidase activity had an increased level on the 1st and 3rd day in S1, S5 and S5 fish groups, respectively. IL-1ß gene was significantly up-regulated in kidney and intestine in all experimental groups (except on the 1st day, in the intestine of S5 fish group) compared to control (P < 0.05). IL-8 gene expression was elevated on 1st and 3rd day in kidney of all experimental fish groups. IL-6 transcript enhanced in a dose-dependent manner on 3rd and 7th day. IL-10 and IL-12 genes were also up-regulated. Survival in all treated fish groups challenged with Aeromonas hydrophila was significantly increased compared to that of control. The highest survival rate was recorded in S5 fish group (83.65%) followed by S3 fish group (82.62%). Our results suggest that S. polycystum aqueous methanolic extract is an effective immunostimulant and provide protection against A. hydrophila infection in rainbow trout at a dose of 3-10 mg/20 g body weight/day.


Assuntos
Adjuvantes Imunológicos/farmacologia , Aeromonas hydrophila , Misturas Complexas/farmacologia , Doenças dos Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Oncorhynchus mykiss/imunologia , Sargassum , Administração Oral , Animais , Citocinas/genética , Citocinas/imunologia , Doenças dos Peixes/mortalidade , Infecções por Bactérias Gram-Negativas/mortalidade , Infecções por Bactérias Gram-Negativas/veterinária , Muramidase/imunologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/microbiologia , Peroxidase/imunologia
7.
Biomolecules ; 11(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34356657

RESUMO

The aims of this research were to perform molecular characterization and biofunctional analyses of giant river prawn Hsp40 and Hsp90 genes (Mr-hsp40 and Mr-hsp90) under various stress conditions. Comparisons of the nucleotide and amino acid sequences of Mr-hsp40 and Mr-hsp90 with those of other species showed the highest similarity scores with crustaceans. Under normal conditions, expression analysis using quantitative real-time RT-PCR (qRT-PCR) indicated that Mr-hsp40 was highly expressed in the gills and testis, and Mr-hsp90 expression was observed in all tissues, with the highest expression in the ovary. The expression patterns of Mr-hsp40 and Mr-hsp90 transcripts under Aeromonas hydrophila challenge and heat-cold shock conditions were examined in gills, the hepatopancreas and hemocytes, at 0, 3, 6, 12, 24, 48 and 96 h by qRT-PCR. Under bacterial challenge, Mr-hsp40 displayed variable expression patterns in all tissues examined during the tested periods. In contrast, upregulated expression of Mr-hsp90 was quickly observed from 3 to 12 h in the gills and hepatopancreas, whereas obviously significant upregulation of Mr-hsp90 was observed in hemocytes at 12-96 h. Under temperature shock conditions, upregulation of Mr-hsp40 expression was detected in all tested tissues, while Mr-hsp90 expression was quickly upregulated at 3-48 h in all tissues in response to 35 °C conditions, and conditions of 35 and 25 °C stimulated its expression in gills and the hepatopancreas at 12 and 48 h, respectively. Silencing analyses of these two genes were successfully conducted under normal, high-temperature (35 °C) and A. hydrophila infection conditions. Overall, knockdown of Mr-hsp40 and Mr-hsp90 effectively induced more rapid and higher mortality than in the PBS control and GFP induction groups in temperature and infectious treatments. Evidence from this study clearly demonstrated the significant functional roles of Mr-hsp40 and Mr-hsp90, which are crucially involved in cellular stress responses to both temperature and pathogenic bacterial stimuli.


Assuntos
Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP90/genética , Palaemonidae/fisiologia , Aeromonas hydrophila/patogenicidade , Animais , Resposta ao Choque Frio/fisiologia , Resistência à Doença/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Resposta ao Choque Térmico/fisiologia , Palaemonidae/microbiologia , Filogenia , Temperatura
8.
Front Immunol ; 12: 633324, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34262558

RESUMO

In the process of microbial invasion, the inflammation reaction is induced to eliminate the pathogen. However, un-controlled or un-resolved inflammation can lead to tissue damage and death of the host. MicroRNAs (miRNAs) are the signaling regulators that prevent the uncontrolled progress of an inflammatory response. Our previous work strongly indicated that miR-142a-3p is related to the immune regulation in grass carp. In the present study, we found that the expression of miR-142a-3p was down-regulated after infection by Aeromonas hydrophila. tnfaip2 and glut3 were confirmed as be the target genes of miR-142a-3p, which were confirmed by expression correlation analysis, gene overexpression, and dual luciferase reporter assay. The miR-142a-3p can reduce cell viability and stimulate cell apoptosis by targeting tnfaip2 and glut3. In addition, miR-142a-3p also regulates macrophage polarization induced by A. hydrophila. Our results suggest that miR-142a-3p has multiple functions in host antibacterial immune response. Our research provides further understanding of the molecular mechanisms between miRNAs and their target genes, and provides a new insights for the development of pro-resolution strategies for the treatment of complex inflammatory diseases in fish.


Assuntos
Apoptose/genética , Carpas/genética , Citocinas/genética , Transportador de Glucose Tipo 3/genética , Infecções por Bactérias Gram-Negativas/veterinária , Macrófagos/fisiologia , MicroRNAs/genética , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/patogenicidade , Animais , Carpas/imunologia , Carpas/microbiologia , Células Cultivadas , Citocinas/classificação , Infecções por Bactérias Gram-Negativas/imunologia , Inflamação/imunologia , Rim/citologia , Rim/microbiologia , Macrófagos do Fígado/microbiologia , Ativação de Macrófagos , MicroRNAs/classificação , Transdução de Sinais
9.
Fish Shellfish Immunol ; 116: 98-106, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34252543

RESUMO

Medicinal herbs are used for growth promotion, disease control and other health benefits in aquaculture industry. Here, we examined the effect of dietary laurel-leaf cistus (Cistus laurifolius) ethanolic extract on growth performance, digestive enzyme activity, haematological profile and nonspecific immune responses in common carp (Cyprinus carpio). In addition, resistance against Aeromonas hydrophila infection was examined. Common carp was fed diets containing 0 (Control), 0.1 (CL0.1), 0.5 (CL0.5) and 1 (CL1) g kg-1 laurel-leaf cistus extract for 45 days. After 30 days, superoxide anion production (SAP) increased in CL0.1 and CL0.5 fish groups and at the end of the study all experimental fish groups had higher SAP compared to that of the control (P ˂ 0.05). Lysozyme activity (LA) was elevated in CL0.5 and CL1 treated groups on 30th day (P < 0.05), and this increase was only observed in C0.1 fish group at the end of study compared to control (P ˂ 0.05). Myeloperoxidase activity was significantly increased in CL0.5 and CL1 fish groups at the end of study. IL-1ßgene expression was significantly increased in treated fish in a dose-depended manner. Similar results were observed for transcription of IL-6 and IL-8 (P < 0.05). Anti-inflammatory cytokines, IL-10 and TGF-ß were highly up-regulated in the intestine and head kidney of CL treated fish groups compared to control (P < 0.05). At the end of experiment, significantly higher final body weight, weight gain, and specific growth rate were obtained in CL0.1 treated fish group compared to control. However, growth was negatively affected in CL1 fish group (P < 0.05). CL1 fish group had also a significantly higher FCR. Amylase activity was significantly increased in all experimental fish groups compared to control (P ˂ 0.05). Trypsin activity was decreased in CL0.1 and CL1 fish groups (P ˂ 0.05). WBC and RBC were significantly increased (P ˂ 0.05) in CL0.5 and CL1 fish groups, whereas haemoglobin, haematocrit, mean cell, mean cell haemoglobin contents were no significantly changed among control and treatment groups. Result of challenge test with A. hydrophila exhibited that survival rate in all treatment groups was significantly higher than that of control. These findings demonstrated that laurel-leaf cistus at 0.1 g kg-1 can be a suitable candidate for growth promotion, immune system induction and infection control in fish.


Assuntos
Carpas , Cistus , Doenças dos Peixes/prevenção & controle , Infecções por Bactérias Gram-Negativas/prevenção & controle , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Extratos Vegetais/farmacologia , Aeromonas hydrophila , Amilases/metabolismo , Animais , Contagem de Células Sanguíneas , Carpas/sangue , Carpas/genética , Carpas/imunologia , Carpas/metabolismo , Citocinas/genética , Etanol/química , Infecções por Bactérias Gram-Negativas/veterinária , Rim Cefálico/citologia , Rim Cefálico/imunologia , Lipase/metabolismo , Muramidase/imunologia , Folhas de Planta/química , Solventes/química , Superóxidos/imunologia , Tripsina/metabolismo
10.
Mol Immunol ; 137: 114-123, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34242920

RESUMO

Toll-like receptors (TLRs) represent first line of host defence against microbes. Amongst different TLRs, TLR22 is exclusively expressed in non-mammalian vertebrates, including fish. The precise role of TLR22 in fish-immunity remains abstruse. Herein, we used headkidney macrophages (HKM) from Clarias gariepinus and deciphered its role in fish-immunity. Highest tlr22 expression was observed in the immunocompetent organ - headkidney; nonetheless expression in other tissues suggests its possible involvement in non-immune sites also. Aeromonas hydrophila infection up-regulates tlr22 expression in HKM. Our RNAi based study suggested TLR22 restricts intracellular survival of A. hydrophila. Inhibitor and RNAi studies further implicated TLR22 induces pro-inflammatory cytokines TNF-α and IL-1ß. We observed heightened caspase-1 activity and our results suggest the role of TLR22 in activating TNF-α/caspase-1/IL-1ß cascade leading to caspase-3 mediated apoptosis of A. hydrophila-infected HKM. We conclude, TLR22 plays critical role in immune-surveillance and triggers pro-inflammatory cytokines leading to caspase mediated HKM apoptosis and pathogen clearance.


Assuntos
Aeromonas hydrophila/imunologia , Apoptose/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Receptores Toll-Like/imunologia , Animais , Caspases/imunologia , Peixes-Gato/imunologia , Peixes-Gato/microbiologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Rim Cefálico/imunologia , Rim Cefálico/microbiologia , Inflamação/microbiologia , Interleucina-1beta/imunologia , Macrófagos/microbiologia , Fator de Necrose Tumoral alfa/imunologia
11.
Mol Immunol ; 137: 145-154, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34247100

RESUMO

Previous studies have found that the expression level of Megalobrama amblycephala intelectin (MaINTL) increased significantly post Aeromonas hydrophila infection, and recombinant MaINTL (rMaINTL) protein could activate macrophages and enhance the phagocytosis and killing activity of macrophages. In order to reveal the immune regulatory mechanisms of MaINTL, primary M. amblycephala macrophages were treated with endotoxin-removed rMaINTL and GST-tag proteins, then total RNA were extracted and used for comparative Digital Gene Expression Profiling (DGE). 1247 differentially expressed genes were identified by comparing rMaINTL and GST-tag treated macrophage groups, including 482 up-regulated unigenes and 765 down-regulated unigenes. In addition, eleven randomly selected differentially expressed genes were verified by qRT-PCR, and most of them shared the similar expression patterns as that of DGE results. GO enrichment revealed that the differentially expressed genes were mainly concentrated in the membrane part and cytoskeleton of cellular component, the binding and signal transducer activity of molecular function, the cellular process, regulation of biological process, signaling and localization of biological process, most of which might related with the phagocytosis and killing activity of macrophages. KEGG analysis revealed the activation and involvement of differentially expressed genes in immune related pathways, such as Tumor necrosis factor (TNF) signaling pathway, Interleukin 17 (IL-17) signaling pathway, Toll-like receptor signaling pathway, and NOD like receptor signaling pathway, etc. In these pathways, TNF-ɑ, Activator protein-1 (AP-1), Myeloid differentiation primary response protein MyD88 (MyD88), NF-kappa-B inhibitor alpha (ikBɑ) and other key signaling factors were significantly up-regulated. These results will be helpful to clarify the immune regulatory mechanisms of fish intelectin on macrophages, thus providing a theoretical basis for the prevention and control of fish bacterial diseases.


Assuntos
Aeromonas hydrophila/imunologia , Cyprinidae/imunologia , Cyprinidae/microbiologia , Infecções por Bactérias Gram-Negativas/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Animais , Regulação para Baixo/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/métodos , Infecções por Bactérias Gram-Negativas/microbiologia , Fatores Imunológicos/imunologia , Macrófagos/microbiologia , Transdução de Sinais/imunologia , Transcriptoma/imunologia , Fator de Necrose Tumoral alfa/imunologia , Regulação para Cima/imunologia
12.
Fish Shellfish Immunol ; 116: 1-11, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34174452

RESUMO

NK-lysin, an effector of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), not only exhibits cytotoxic effect in fish cells, but also participates in the immune defense against pathogenic infection. In this study, ORF sequences of RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin were 369 bp. Tissue-specific analysis revealed that the highest expressions of RCC-NK-lysin and WCC-NK-lysin were observed in gill, while the peaked level of WR-NK-lysin mRNA was observed in spleen. A. hydrophila infection sharply increased RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin mRNA expression in liver, trunk kidney and spleen. In addition, elevated levels of NK-lysin mRNA were observed in cultured fin cell lines of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) after Lipopolysaccharide (LPS) challenge. RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin exerted regulatory roles in inducing ROS generation, modulating mitochondrial membrane potential, decreasing fish cell viability and antagonizing survival signalings, respectively. RCC/WCC/WR-NK-lysin-overexpressing fish could up-regulate expressions of inflammatory cytokines and decrease bacterial loads in spleen. These results indicated that NK-lysin in hybrid fish contained close sequence similarity to those of its parents, possessing the capacities of cytotoxicity and immune defense against bacterial infection.


Assuntos
Aeromonas hydrophila , Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Proteolipídeos/imunologia , Nadadeiras de Animais/citologia , Animais , Carpas/genética , Sobrevivência Celular , Células Cultivadas , Quimera , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Expressão Gênica , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Rim/metabolismo , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Potencial da Membrana Mitocondrial , Proteolipídeos/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Baço/metabolismo , Baço/microbiologia
13.
Vet Res ; 52(1): 88, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34130754

RESUMO

Toll-like receptor 19 (Tlr19) is a fish-specific TLR that plays a critical role in innate immunity. In the present study, we aimed to identify tlr19 from common carp (Cyprinus carpio L.) and explored its expression profile, localization, adaptor, and signaling pathways. A novel tlr19 cDNA sequence (Cctlr19) was identified in common carp. Phylogenetic analysis revealed that CcTlr19 was most closely related to Danio rerio Tlr19. Subcellular localization analysis indicates that CcTlr19 was synthesized in the free ribosome and then transported to early endosomes. Cctlr19 was constitutively expressed in all the examined tissues, with the highest expression in the brain. After poly(I:C) and Aeromonas hydrophila injection, the expression of Cctlr19 was significantly upregulated in immune-related organs. In addition, the expression of Cctlr19 was upregulated in head kidney leukocytes (HKL) upon stimulation with different ligands. Immunofluorescence and luciferase analyses indicate that CcTlr19 recruited TRIF as an adaptor. Furthermore, CcTlr19 can activate the expression of ifn-1 and viperin. Taken together, these findings lay the foundation for future research to investigate the mechanisms underlying fish tlr19.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aeromonas hydrophila , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas , Interferons/genética , Filogenia , Poli I-C , Análise de Sequência de DNA/veterinária , Transdução de Sinais , Receptores Toll-Like/química
14.
Fish Shellfish Immunol ; 115: 189-197, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34147613

RESUMO

The present study examines the effectiveness of DNA vaccine against Aeromonas hydrophila through oral route using chitosan-tripolyphosphate (Cs-TPP) nanoparticles encapsulation. The virulent gene of outer membrane protein (OMP) and hemolysin (hly) related to pathogenicity of A. hydrophila was used to construct a DNA vaccine using pVAX1, and the construct was named as pVAX-OMP and pVAX-hly DNA vaccines. The pVAX-OMP and pVAX-hly DNA vaccines were encapsulated by Cs-TPP nanoparticles and size measured by field emission scanning electron microscopy (FE-SEM). The encapsulation efficiency of Cs-TPP nanoparticles was found to be 79.6% for pVAX-OMP DNA and 82.3% for pVAX-hly DNA binding with Cs-TPP nanoparticles. The stability and invitro release profile of plasmid DNA was also determined after encapsulation using DNase and chitosanase. DNA vaccines distribution in tissues was investigated in fish fed with the pVAX-OMP, pVAX-hly and pVAX-OMP+pVAX-hly encapsulated in Cs-TPP nanoparticles and confirmed by PCR and multiplex PCR. The results suggest that Cs-TPP nanoparticles encapsulated DNA vaccine delivered into fish by feeding. After oral vaccination of Labeo rohita were challenged with A. hydrophila by intraperitoneal injection. Relatively, gene expression of c- and g-type lysozyme followed by pro- and anti-inflammatory cytokines (Interlukin-10 and Tumor Growth Factor ß) was up-regulated in heart and kidney for pVAX-OMP+pVAX-hly vaccinated group. Moreover, fish fed with pVAX-OMP+pVAX-hly encapsulated in Cs-TPP nanoparticles had a significantly higher survival rate (76.2%) against A. hydrophila. This study concludes that pVAX-OMP and pVAX-hly DNA vaccines can be delivered orally using Cs-TPP nanoparticles for protection against A. hydrophilainfection.


Assuntos
Vacinas Bacterianas/administração & dosagem , Quitosana/análogos & derivados , Cyprinidae , Doenças dos Peixes/prevenção & controle , Infecções por Bactérias Gram-Negativas/veterinária , Vacinação/veterinária , Vacinas de DNA/administração & dosagem , Administração Oral , Aeromonas hydrophila/fisiologia , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Quitosana/administração & dosagem , Quitosana/imunologia , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/prevenção & controle , Proteínas Hemolisinas/imunologia , Nanopartículas/administração & dosagem
15.
Fish Shellfish Immunol ; 115: 160-170, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34147614

RESUMO

C-type lectins (CTL) are a large group of pattern-recognition proteins and to play important roles in glycoprotein metabolism, multicellular integration, and immunity. Based on their overall domain structure, they can be classified as different groups that possess different physiological functions. A typical C-type lectin (named as OmLec1) was identified from the fish, Onychostoma macrolepis, an important cultured fish in China. Open reading frame of OmLec1 contains a 570 bp, encoding a protein of 189 amino acids that includes a signal peptide and a single carbohydrate-recognition domain. The phylogenetic analysis showed that OmLec1 could be grouped with C-type lectin from other fish. OmLec1 was expressed in all the tissues in our study, and the expression level was highest in liver. And its relative expression levels were significantly upregulated following infection with Aeromonas hydrophila. The recombinant OmLec1 protein (rOmLec1) could agglutinate some Gram-negative bacteria and Gram-positive bacteria in vitro in the presence of Ca2+, showing a typical Ca2+-dependent carbohydrate-binding protein. Furthermore, rOmLec1 purified from E. coli BL21 (DE3), strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-dependent manner. These results indicate that OmLec1 plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis.


Assuntos
Cyprinidae/genética , Cyprinidae/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Lectinas Tipo C/genética , Lectinas Tipo C/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 , Infecções por Bactérias Gram-Negativas/veterinária , Lectinas Tipo C/química , Filogenia , Alinhamento de Sequência/veterinária
16.
J Int Med Res ; 49(5): 3000605211012201, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33942635

RESUMO

We herein review and analyze the diagnosis, treatment, and outcome of a severe infection caused by a human bite. A 68-year-old man was bitten on the forearm by a 3-year-old child. Rapid progression of infection, severe local and systemic poisoning, and diverse clinical manifestations were observed at presentation. Based on the medical history, physical signs, imaging examinations (X-ray films, color Doppler ultrasound, and computed tomography), laboratory examinations, and multidisciplinary consultation, the patient was diagnosed with gas gangrene or gas gangrene-like changes. Twenty-four hours after the injury, an emergency amputation was performed (open amputation with wound closure after 1 week). After the operation, the patient was sent to the intensive care unit for isolation and further anti-infection and anti-shock treatments. His condition gradually improved after treatment and he was discharged without further complications. Bacteriological and pathological examinations indicated Aeromonas hydrophila infection leading to extensive necrotizing fasciitis of the limb and severe systemic poisoning. In addition, pre-existing myelodysplastic syndrome progressing to acute myeloid leukemia was identified as a possible predisposing factor. Human bites can cause serious infections requiring timely treatment, particularly in patients with predisposing comorbidities.


Assuntos
Mordeduras Humanas , Fasciite Necrosante , Aeromonas hydrophila , Idoso , Amputação , Pré-Escolar , Serviço Hospitalar de Emergência , Fasciite Necrosante/diagnóstico por imagem , Fasciite Necrosante/cirurgia , Humanos , Masculino
17.
Microb Pathog ; 156: 104896, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33965506

RESUMO

Aquatic Pathogens are expected to encounter tremendous levels of variation in their environment - both abiotic and biotic. Here we examined the change in innate immune parameters and mortality pattern of Carassius auratus during the interaction of co-infection due to an ectoparasite, Argulus and bacteria Aeromonas hydrophila, along with a temperature gradient. Experimental fish were assigned randomly to six treatment groups (T1-T6). Fish of groups T1, T3 and T5 are assigned for healthy fishes kept at 23, 28 and 33°c temperature and served as control. T2, T4 and T6 groups are the co-infected groups kept at temperature gradient. For the haematological and enzyme parameter analysis, sampling was done at 24 h, 72 h and 168 h post challenge from 4 fish in all experimental groups. A temperature dependent increase in intensity of Argulus was observed in the experimental group. Both in control group and co-infected group a temperature dependent mortality pattern was observed, showing an increased mortality of 60% in T6 and 20% in T5 group. A significant decrease of RBC, Hb, and PCV values was observed in co-infected group when compared with control fish in each of the experimental group. Also a temperature dependent increase in WBC, neutrophil and monocyte value was observed in control fish. Whereas, a significant reduction in WBC, neutrophil and monocyte was observed in co-infected fish exposed to 33 °C during the progression of infection. Furthermore, T4 group showed a significantly higher Nitroblue tetrazolium test, Myelo peroxidase and lyzozyme activity compared to other co-infection group. A significantly increased activity of Superoxide dismutase, Glutathione peroxidise and catalase activity was recorded in control fish exposed to 33 °C (T5) whereas, there was no significant difference observed in the activity of catalase and Glutathione peroxidise in the other control fish (T1 and T3 group). This result implies that increase in temperature not only accelerates the intensity of co-infection but also imbalance the health status of the fish by hampering the immunological and physiological parameters towards more detrimental side.


Assuntos
Coinfecção , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Aeromonas hydrophila , Ração Animal/análise , Animais , Coinfecção/veterinária , Resistência à Doença , Carpa Dourada , Infecções por Bactérias Gram-Negativas/veterinária , Imunidade Inata , Temperatura
18.
Biochem Biophys Res Commun ; 562: 1-8, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34030039

RESUMO

Protein lysine propionylation (Kpr) modification is a novel post-translational modification (PTM) of prokaryotic cells that was recently discovered; however, it is not clear how this modification regulates bacterial life. In this study, the protein Kpr modification profile in Aeromonas hydrophila was identified by high specificity antibody-based affinity enrichment combined with high resolution LC MS/MS. A total of 98 lysine-propionylated peptides with 59 Kpr proteins were identified, most of which were associated with energy metabolism, transcription and translation processes. To further understand the role of Kpr modified proteins, the K168 site on malate dehydrogenase (MDH) and K608 site on acetyl-coenzyme A synthetase (AcsA) were subjected to site-directed mutation to arginine (R) and glutamine (Q) to simulate deacylation and propionylation, respectively. Subsequent measurement of the enzymatic activity showed that the K168 site of Kpr modification on MDH may negatively regulate the MDH enzymatic activity while also affecting the survival of mdh derivatives when using glucose as the carbon source, whereas Kpr modification of K608 of AcsA does not. Overall, the results of this study indicate that protein Kpr modification plays an important role in bacterial biological functions, especially those involved in the activity of metabolic enzymes.


Assuntos
Aeromonas hydrophila/enzimologia , Regulação Enzimológica da Expressão Gênica , Lisina/metabolismo , Propionatos/metabolismo , Aeromonas hydrophila/genética , Aeromonas hydrophila/metabolismo , Proteínas de Bactérias/metabolismo , Carbono/farmacologia , Glucose/farmacologia , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Modelos Moleculares , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo
19.
J Proteomics ; 244: 104275, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34044167

RESUMO

SlyA is a well-known transcription factor that plays important roles in the regulation of diverse physiological functions including virulence and stress response in various bacterial species. The biological effects of slyA have species-specific characteristics. In this study, a phenotype assay showed that slyA gene deletion in Aeromonas hydrophila (ahslyA) decreased biofilm formation capability but did not affect bacterial hemolytic activity or acid stress response. The differentially expressed proteins between ΔahslyA and wild-type strains were compared by label-free quantitative proteomics to further understand the effects of AhSlyA on biological functions. Bioinformatics assays showed that ΔahslyA may be involved in the regulation of several intracellular metabolic pathways such as galactose metabolism, arginine biosynthesis, and sulfur metabolism. A further phenotypic assay confirmed that AhSlyA plays an important role in the regulation of sulfur and phosphate metabolism. Moreover, ahslyA also directly or indirectly regulated at least eight outer membrane proteins involved in the maintenance of cell permeability. Overall, the results provide insights into the functions of ahslyA and demonstrate its importance in A. hydrophila. BIOLOGICAL SIGNIFICANCE: In this study, we compared the DEPs between the transcriptional regulator slyA-deleted and the wild-type A. hydrophila strains using a label-free quantitative proteomics method. The bioinformatics analysis showed that slyA may be involved in the regulation of several metabolic pathways. Subsequent phenotype and growth assays confirmed that ΔahslyA affected sulfur and phosphate metabolism, and OM permeability. Finally, a ChIP-PCR assay further confirmed that AhSlyA directly binds to the promoters of several candidate genes, including sulfur metabolism-related genes. These results indicated that slyA plays an important regulatory role in pleiotropic physiological functions of A. hydrophila, and these functions may be different from those identified in previous reports from other bacterial species.


Assuntos
Aeromonas hydrophila , Proteômica , Aeromonas hydrophila/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Redes e Vias Metabólicas , Fatores de Transcrição/genética
20.
Fish Shellfish Immunol ; 114: 293-300, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34004271

RESUMO

Functional additives of natural origin included as dietary supplements have become an alternative to synthetic antibiotics to improve health and resistance to ecologically correct pathogenic diseases in fish farming. We tested whether incorporating a mixture of phytobiotics such as volatile oils of thyme, red thyme and pepper rosemary into the diet improves growth performance, oxidative stress, immune and hematological responses and resistance of juvenile Nile tilapia when subjected to a challenge with Aeromonas hydrophila compared to a synthetic antibiotic (enrofloxacin). The experimental design was completely randomized with three experimental groups: control diet, diets containing a mixture of thyme phytobiotic essential oils, red thyme and pepper rosemary (FTB) and the synthetic antibiotic enrofloxacin (ATB), with four replicates (14 fish per repetition/experimental unit). Plasma glucose levels, leukocyte respiratory activity, serum lysozyme levels, number of circulating erythrocytes and leukocytes, levels of lipid peroxidation (LPO), catalase (CAT) and glutathione S-transferase (GST) activity at the end of 20 days of feeding (phase) were evaluated and 24 h after exposure to bacteria (phase II). The supplementation of FTB and ATB did not change the performance parameters, but it was sufficient to increase lysozyme, leukocytes, neutrophils and monocytes after the bacterial challenge, reduction of CAT and LPO activity and the highest GST activity (P < 0.05). The results of the present study suggest that FTB as a dietary supplement has benefits and can replace synthetic ATB, including supplementation with FTB for 20 days to provide greater antioxidant protection in Nile tilapia, mitigate the impacts of stressors and modulate immunity, providing to fish greater resistance and protection against diseases.


Assuntos
Aeromonas hydrophila , Ração Animal/análise , Ciclídeos , Dieta/veterinária , Suplementos Nutricionais , Doenças dos Peixes/microbiologia , Fenômenos Fisiológicos da Nutrição Animal , Animais , Antibacterianos/uso terapêutico , Enrofloxacina/uso terapêutico , Doenças dos Peixes/prevenção & controle , Infecções por Bactérias Gram-Negativas/prevenção & controle , Infecções por Bactérias Gram-Negativas/veterinária , Lippia/química , Fitoterapia , Óleos Vegetais/administração & dosagem , Óleos Vegetais/farmacologia , Thymus (Planta)/química
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