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The increasing emergence and dissemination of bacterial pathogens in largemouth bass culture accelerate the desire for new treatment measures. Antimicrobial peptides as the host's antimicrobial source dominate the preferred molecules for discovering antibacterial agents. Here, the potential of Hepcidin-1 from largemouth bass (Micropterus salmoides) (MsHep-1) against bacterial infection is demonstrated. MsHep-1 not only improved the survival rate in infection experiments involving Nocardia seriolae (12 %) and Aeromonas hydrophila (18 %) but also coped with iron overload conditions in vivo. Moreover, the antibacterial activity of MsHep-1 in vitro was identified against both gram-negative and gram-positive bacteria. Mechanistic studies show MsHep-1 leads to bacterial death by changing the bacterial membrane potential and disrupting the bacterial membrane structure. These findings demonstrate that MsHep-1 may play an important role in the host response to bacterial infection. It provides promising strategies in the application of immunosuppression prevention and control in fish. AMPs may be a promising and available reservoir for treating the current bacterial diseases.
Assuntos
Infecções Bacterianas , Bass , Doenças dos Peixes , Hepcidinas , Animais , Hepcidinas/metabolismo , Bass/microbiologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/tratamento farmacológico , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Antibacterianos/farmacologia , Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/patogenicidadeRESUMO
Introduction: Nocardia seriolae adversely impacts a diverse range of fish species, exhibiting significant pathogenic characteristics that substantially impede the progress of aquaculture. N. seriolae infects in fish has a long incubation period, and clinical symptoms are not obvious in the early stages. There is presently no viable and eco-friendly approach to combat the spread of the disease. According to reports, N. seriolae primarily targets macrophages in tissues after infecting fish and can proliferate massively, leading to the death of fish. Interferon-gamma (IFN-γ) is a crucial molecule that regulates macrophage activation, but little is known about its role in the N. seriolae prevention. Methods: IFN-γ was first defined as largemouth bass (Micropterus salmoides, MsIFN-γ), which has a highly conserved IFN-γ characteristic sequence through homology analysis. The recombinant proteins (rMsIFN-γ) were obtained in Escherichia coli (E. coli) strain BL21 (DE3). The inflammatory response-inducing ability of rMsIFN-γ was assessed in vitro using monocytes/macrophages. Meanwhile, the protective effect of MsIFN-γ in vivo was evaluated by N. seriolae infection largemouth bass model. Results: In the inflammatory response of the monocytes/macrophages activated by rMsIFN-γ, various cytokines were significantly increased. Interestingly, interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-a) increased by 183- and 12-fold, respectively, after rMsIFN-γ stimulation. rMsIFN-γ improved survival by 42.1% compared with the control. The bacterial load in the liver, spleen and head kidney significantly decreased. rMsIFN-γ was also shown to better induce increased expression of IL-1ß, TNF-α, hepcidin-1(Hep-1), major histocompatibility complex I (MHCI), and MHC II in head kidney, spleen and liver. The histopathological examination demonstrated the transformation of granuloma status from an early necrotic foci to fibrosis in the infection period. Unexpectedly, the development of granulomas was successfully slowed in the rMsIFN-γ group. Discussion: This work paves the way for further research into IFN-γ of largemouth bass and identifies a potential therapeutic target for the prevention of N. seriolae.
Assuntos
Bass , Nocardiose , Nocardia , Animais , Interferon gama , Escherichia coli , Nocardiose/prevenção & controle , Nocardiose/veterinária , Proteínas RecombinantesRESUMO
The immunosuppression hazard of fish brought by intensive aquaculture needs to be addressed urgently, while chitooligosaccharide (COS) shows the potential application in the prevention the immunosuppression of fish due to its superior biological properties. In this study, COS reversed the cortisol-induced immunosuppression of macrophages and improved the immune activity of macrophages in vitro, promoting the expression of inflammatory genes (TNF-α, IL-1ß, iNOS) and NO production, and increasing the phagocytic activity of macrophages. In vivo, the oral COS was absorbed directly through the intestine, significantly ameliorating the innate immunity of cortisol-induced immunosuppression of blunt snout bream (Megalobrama amblycephala). Such as facilitated the gene expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6) and pattern recognition receptors (TLR4, MR) and potentiated bacterial clearance, resulting in an effective improvement in survival and tissue damage. Altogether, this study demonstrates that COS offers potential strategies in the application of immunosuppression prevention and control in fish.
Assuntos
Infecções Bacterianas , Cyprinidae , Cipriniformes , Animais , Hidrocortisona/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Cyprinidae/genética , Quitina/farmacologia , Quitina/metabolismoRESUMO
Elizabethkingia miricola is a highly infectious pathogen, which causes high mortality rate in frog farming. Therefore, it is urgent to develop a rapid and sensitive detection method. In this study, two rapid and specific methods including recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) and fluorescent probe-based recombinase polymerase amplification (exo RPA) were established to effectively detect E. miricola, which can accomplish the examination at 38 °C within 30 min. The limiting sensitivity of RPA-LFD and exo RPA (102 copies/µL) was ten-fold higher than that in generic PCR assay. The specificities of the two methods were verified by detecting multiple DNA samples (E. miricola, Staphylococcus aureus, Aeromonas hydrophila, Aeromonas veronii, CyHV-2 and Edwardsiella ictaluri), and the result showed that the single band was displayed in E. miricola DNA only. By tissue bacterial load and qRT-PCR assays, brain is the most sensitive tissue. Random 24 black spotted frog brain samples from farms were tested by generic PCR, basic RPA, RPA-LFD and exo RPA assays, and the results showed that RPA-LFD and exo RPA methods were able to detect E. miricola accurately and rapidly. In summary, the methods of RPA-LFD and exo RPA were able to detect E. miricola conveniently, rapidly, accurately and sensitively. This study provides prospective methods to detect E. miricola infection in frog culture.
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CSF-1 and CSF-1R have been well demonstrated in humans, regulating the differentiation, proliferation and survival of the mononuclear phagocyte system. However, the functional study on MaCSF-1 and MaCSF-1R from blunt snout bream (Megalobrama amblycephala) is still unknown. In the present study, we cloned and functionally characterized MaCSF-1 and MaCSF-1R. Multiple sequence alignment and phylogenetic tree analysis showed that both MaCSF-1 and MaCSF-1R were mostly close to the grass carp counterparts. Tissue distribution analysis showed that both MaCSF-1 and MaCSF-1R were widely distributed in all examined tissues, dominantly distributed in spleen, blood and head kidney tissues. Furthermore, confocal microscopy assay and flow cytometry assay showed that MaCSF-1R was the marker on the surface of macrophages. Recombinant MaCSF-1 promoted macrophage proliferation, phagocytosis and the production of IL-10. Through the pull-down experiments and indirect immunofluorescence experiments, the interaction between MaCSF-1 and MaCSF-1R was confirmed. To explore the relationship between MaCSF-1 and its receptor, MaCSF-1R and MaCSF-1R antibody was prepared. Then the MaCSF-1R blockage assay indicated that the role of MaCSF-1 on the macrophages proliferation and phagocytosis was weakened, leading the reduction of IL-10 expression level. In conclusion, MaCSF-1R is the marker on the surface of macrophage membrane; and MaCSF-1 promotes macrophage proliferation, phagocytosis, and significantly increased the expression levels of IL-10 depended on the interacting with MaCSF-1R. This study provides basal data for the biological function of MaCSF-1 and MaCSF-1R, and is valuable for the exploration of MaCSF-1 and MaCSF-1R molecular interactions.
Assuntos
Cyprinidae , Cipriniformes , Proteínas de Peixes/metabolismo , Animais , Proliferação de Células , Proteínas de Peixes/genética , Humanos , Interleucina-10/metabolismo , Macrófagos , Fagocitose , FilogeniaRESUMO
Intensive high-density culture patterns are causing an increasing number of bacterial diseases in fish. Hepcidin links iron metabolism with innate immunity in the process of resisting bacterial infection. In this study, the antibacterial effect of the combination of hepcidin (Cihep) and chitosan (CS) against Flavobacterium columnare was investigated. The dosing regimen was also optimized by adopting a feeding schedule of every three days and every seven days. After 56 days of feeding experiment, grass carp growth, immunity, and gut microbiota were tested. In vitro experiments, Cihep and CS can regulate iron metabolism and antibacterial activity, and that the combination of Cihep and CS had the best protective effect. In vivo experiments, Cihep and CS can improve the growth index of grass carp. After challenge with Flavobacterium columnare, the highest survival rate was observed in the Cihep+CS-3d group. By serum biochemical indicators assay and Prussian blue staining, Cihep and CS can increase iron accumulation and decrease serum iron levels. The contents of lysozyme and superoxide dismutase in Cihep+CS-3d group increased significantly. Meanwhile, Cihep and CS can significantly reduce the pathological damage of gill tissue. The 16S rRNA sequencing results showed that Cihep and CS can significantly increase the abundance and diversity of grass carp gut microbiota. These results indicated that the protective effect of consecutive 3-day feeding followed by a 3-day interval was better than that of consecutive 7-day feeding followed by a 7-day interval, and that the protective effect of Cihep in combination with chitosan was better than that of Cihep alone. Our findings optimize the feeding pattern for better oral administration of Cihep in aquaculture.
Assuntos
Carpas , Quitosana , Infecções por Flavobacteriaceae , Microbioma Gastrointestinal , Animais , Carpas/metabolismo , Hepcidinas/genética , RNA Ribossômico 16S/genética , Proteínas de Peixes/genética , Ferro , Administração OralRESUMO
Aeromonas veronii (A. veronii) is one of the main pathogens causing bacterial diseases in aquaculture. Although previous studies have shown that hepcidin as an antimicrobial peptide can promote fish resistance to pathogenic bacterial infections, but the mechanisms remain unclear. Here, we expressed and purified recombinant yellow catfish (Pelteobagrus fulvidraco) hepcidin protein (rPfHep). rPfHep can up-regulate the expression of ferritin and enhance the antibacterial activity in primary hepatocytes of yellow catfish. We employed berberine hydrochloride (BBR) and Fursultiamine (FSL) as agonists and antagonists for hepcidin, respectively. The results indicated that agonist BBR can inhibit the proliferation of pathogenic bacteria, and the antagonist FSL shows the opposite effect. After gavage administration, rPfHep and the agonist BBR can enhance the accumulation of iron in liver, which may hinder the iron transport and limit the amount of iron available to pathogenic bacteria. Moreover, rPfHep and the agonist BBR can also reduce the mortality rate, bacterial load and histological lesions in yellow catfish infected with A. veronii. Therefore, hepcidin is an important mediator of iron metabolism, and it can be used as a candidate target for prevent bacterial infections in yellow catfish. Hepcidin and BBR have potential application value in preventing anti-bacterial infection.
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Grass carp reovirus (GCRV) is a severe virus that causes great losses to grass carp culture every year, and GCRV-II is the current popular and fatal strain. VP56, fibrin on the outer surface of GCRV-II, mediates cell attachment. In this study, we firstly divided the VP56 gene into four fragments to screen the optimal antigen by enzyme-linked immunosorbent assay and neutralizing antibody methods. The second fragment VP56-2 demonstrates the optimal efficiency and was employed as an antigen in the following experiments. Bacillus subtilis were used as a carrier, and VP56-2 was expressed on the surface of the spores. Then, we performed the oral immunization for grass carp and the challenge with GCRV-II. The survival rate was remarkably raised, and mRNA expressions of IgM were significantly up-regulated in spleen and head kidney tissues in the B. s-CotC-VP56-2 group. Three crucial immune indexes (complement C3, lysozyme and total superoxide dismutase) in the sera were also significantly enhanced. mRNA expressions of four important genes (TNF-α, IL-1ß, IFN1 and MHC-II) were significantly strengthened. Tissue lesions were obviously attenuated by histopathological slide examination in trunk kidney and spleen tissues. Tissue viral burdens were significantly reduced post-viral challenge. These results indicated that the oral recombinant B. subtilis VP56-2 subunit vaccine is effective for controlling GCRV infection and provides a feasible strategy for the control of fish virus diseases.
Assuntos
Bacillus subtilis , Carpas , Doenças dos Peixes/prevenção & controle , Infecções por Reoviridae/veterinária , Reoviridae/imunologia , Vacinas Virais/administração & dosagem , Administração Oral , Animais , Antígenos Virais/imunologia , Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Epitopos , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Imunidade Inata , Intestinos/microbiologia , Reoviridae/fisiologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/prevenção & controle , Infecções por Reoviridae/virologia , Esporos Bacterianos/crescimento & desenvolvimento , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Replicação ViralRESUMO
Hepcidin is an antimicrobial peptide and regulator of iron homeostasis which has two isoforms in most fishes and some mammals. Previous studies have reported that the two hepcidin isoforms have different roles. Hamp type-1 plays a regulatory role in iron metabolism and hamp type-2 mostly performs an antimicrobial role. In this study, we found that Ctenopharyngodon idella (C. idella) have only one hepcidin isoform (hamp type-1), which showed both broad-spectrum antibacterial and iron regulatory functions. C. idella hepcidin mature peptide (hepcidin-25) and truncated peptide (hepcidin-20) exhibited bactericidal activities against both Gram-positive and Gram-negative bacteria in a dose-dependent manner in part through membrane rupture and binding to bacterial genomic DNA. The data from challenge tests demonstrated that the administration of hepcidin-25 significantly reduced mortality rates of C. idella by A. hydrophila infection, probably due to direct bactericidal activities of the peptide and a reduction of iron content in the fish serum. In addition, a comparison between hepcidin-20 and -25 suggests that the N terminal 5 amino acids play a critical role in reducing iron content in fish serum. Our findings revealed an important role of hamp type-1 in maintaining iron homeostasis and fighting against bacterial infections, suggesting the hepcidin has implications for the prevention and control of bacterial infection in aquaculture.
Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hepcidinas/farmacologia , Animais , Antibacterianos/metabolismo , Carpas/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Hepcidinas/metabolismo , Humanos , Ferro/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Hepcidin links iron metabolism with innate immunity during the inhibition of bacterial infection. Our previous studies had shown that recombinant hepcidin can significantly reduce the mortality rate of Ctenopharyngodon idella infected with Flavobacterium columnare under laboratory conditions. Here, we studied the preventive and therapeutic effects of feed supplemented with different doses of recombinant hepcidin on F. columnare-challenged C. idella reared in a cage culture environment. The results showed that in the prevention groups, 30 and 90 mg/kg of added purified and unpurified hepcidin respectively resulted in a higher survival rate in the early post-infection period, while 60 mg/kg of purified hepcidin significantly improved the survival rate in the therapy group (all compared to the control group). In the hepatopancreas, the expression of hepcidin and ferritin was significantly up-regulated, and the levels of ferroportin and serum iron were significantly decreased, especially in the therapy group. In addition, the expression of iron-related genes in spleen and intestine exhibited a similar trend to that in hepatopancreas. Meanwhile, immune genes were up-regulated to varying degrees, and the therapy group exhibited a significantly improved expression of pro-inflammatory cytokines and specific immunity. In summary, our study shows that different doses of recombinant hepcidin had protective effects against bacterial infection by regulating the iron distribution and immune gene expression, which provides a strong foundation for the application of recombinant hepcidin in aquaculture.
Assuntos
Carpas/imunologia , Suplementos Nutricionais , Infecções por Flavobacteriaceae/veterinária , Hepcidinas/administração & dosagem , Imunidade Inata , Ração Animal , Animais , Aquicultura/métodos , Carpas/microbiologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Proteínas de Peixes/administração & dosagem , Proteínas de Peixes/imunologia , Infecções por Flavobacteriaceae/prevenção & controle , Flavobacterium , Hepcidinas/genética , Ferro/sangue , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologiaRESUMO
Yellow catfish (Pelteobagrus fulvidraco) has been an economically important freshwater species in China because of its good meat quality. In present, the high-density breeding industry has suffered great damage from bacterial infections, in especial, the rapid illness and death of fish caused by bacterial septicemia leads to huge economic losses. Therefore, it is urgent and important to identify pathogenic bacteria and study its pathogenicity. In this study, we isolated a bacterial strain from the yellow catfish with typical septicemia and named it E. 719, then, by morphological observations, regression infection, biochemical identification, 16S rDNA sequence analysis and triple PCR identification, E. 719 was determined to be Edwardsiella ictaluri. Further, we infected yellow catfish with E. ictaluri to study its effects on mortality rate, hematological, histopathological disturbances and expression of immune genes. The mortality results showed that E. ictaluri was highly pathogenic, all infected fish died after 14 days post injection, and the distribution of bacteria in body kidney, spleen, liver, head kidney and brain of fish was continuously detected by measuring the amount of bacteria in the tissues. In addition, the number of red blood cells decreased significantly with the time of infection, while the number of white blood cells and thrombocytes increased. In particular, the number of monocytes and neutrophils increased significantly in the differential leucocyte count (DLC). Histopathologic changes observed by HE staining showed similar results, gill, intestine, spleen and head kidney showed obvious inflammation, bleeding and necrosis. Besides, checking by real time quantitative RT-PCR assays, in both spleen and head kidney tissues which were the major immune organs, mRNA expressions of immune gene IL-1ß, TNF-α, and MR significantly increased in the early and middle stages of infection, which suggested that the infection of E. ictaluri caused a strong immune response in yellow catfish. This study provides a preliminary basis for the diagnosis and treatment of pathophysiology septicemia in yellow catfish induced by E. ictaluri.
Assuntos
Peixes-Gato , Edwardsiella ictaluri/fisiologia , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/imunologia , Sepse/veterinária , Animais , Infecções por Enterobacteriaceae/sangue , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/imunologia , Doenças dos Peixes/sangue , Doenças dos Peixes/genética , Sepse/sangue , Sepse/genética , Sepse/imunologiaRESUMO
IFN-γ is an immunomodulatory factor that has been extensively studied in phenotypes of mammalian macrophages and multifarious inflammatory responses. Usually these studies relied on the classical synergistic activation of IFN-γ with LPS (LipoPolySaccharides). However, non-mammalian vertebrates, and in particular fish, are not very susceptible to LPS, and easily acquire tolerance upon repeated exposure. Therefore, for studies in fish, it is necessary to replace the classical IFN-γ+LPS immune system activation method, and find other pathogen-associated molecular patterns (PAMPs) capable of stimulating the fish immune system. Here we used an important farmed fish species, Ctenopharyngodon idella, to study the effects of CiIFN-γ2 (C. idella IFN-γ2) and chitosan (CS) on its immune responses in vivo and vitro. Our results showed that the combination of CS and CiIFN-γ2 significantly enhanced the activation of macrophages, with an activation intensity even stronger than in CiIFN-γ2 and CiIFN-γ2+LPS groups. In vivo, injection of CiIFN-γ2 could improve the survival rate of C. idella infected with Flavobacterium columnare, while a combined injection of CiIFN-γ2+CS only improved protection in the early stages after the challenge. Notably, both injections reduced the bacterial load of viscera and improved the levels of several plasma parameters (TP, T-SOD, LA, and NO). However, a dramatic up-regulation of inflammatory factors, severe inflammatory damage in the intestines and hepatopancreas, and increased mortality in late stages of infection were observed in the CiIFN-γ2+CS group. Our findings provide new insights into the macrophage activation phenotypes and inflammatory responses in fish. They also demonstrate that CiIFN-γ2 could be used as a potential immunopotentiator, but not in combination with CS. This suggests that selection of immunological adjuvants should be carefully tested experimentally.
Assuntos
Carpas , Quitosana/efeitos adversos , Doenças dos Peixes/tratamento farmacológico , Infecções por Flavobacteriaceae/veterinária , Inflamação/veterinária , Interferon gama/farmacologia , Substâncias Protetoras/farmacologia , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Infecções por Flavobacteriaceae/tratamento farmacológico , Infecções por Flavobacteriaceae/imunologia , Infecções por Flavobacteriaceae/microbiologia , Flavobacterium/fisiologia , Inflamação/induzido quimicamenteRESUMO
Prophylactic administration of immunopotentiators has been tested and practiced as one of the most promising disease prevention methods in aquaculture. Chitosan oligosaccharide (COS), as an ideal immunopotentiator, is mainly used as feed additives in aquaculture, and the antimicrobial and immune enhancement effects are highly correlated with molecular weight (MW), but little is known about the mechanisms in teleost. Here, we isolated and purified macrophages in head kidney from blunt snout bream (Megalobrama amblycephala), stimulated them with three different MW (~500 Da, ~1000 Da and 2000~3000 Da) COSs, performed RNA-sequencing, global transcriptional analyses, and verification by quantitative real-time PCR (qRT-PCR) and immunofluorescent staining methods. Differential expression gene (DEG) analysis indicated that gene expression patterns are different and the proportion of unique genes are relatively high in different treatment groups. Biological process and gene set enrichment analysis (GSEA) demonstrated that all three COSs activate resting macrophages, but the degrees are different. Weighted gene co-expression network analysis (WGCNA) reflected gene modules correlated to MW, the module hub genes and top GO terms showed the activation of macrophage was positively correlated with the MW, and larger MW COS activated cell death associated GO terms. Further use of the screening and enrichment functions of STRING and Pfam databases discovered that apoptosis-related pathways and protein families were activated, such as the P53 pathway and caspase protein family. qRT-PCR results showed that as the stimulation time extends, the innate immune-related and P53 pathways are gradually activated, and the degree of activation is positively correlated with the stimulation time. In addition, apoptosis was detected by immunofluorescent staining in three groups. Therefore, the use of COS has two sides-it can activate the immune system against pathogen invasion, but with the increase in stimulation time and MW, macrophage apoptosis is induced, which may be caused by abnormal replication of DNA and excessive inflammation. This study provides a theoretical basis for the rational use of COS as an immunopotentiator in aquaculture.
Assuntos
Apoptose/efeitos dos fármacos , Quitosana/farmacologia , Cipriniformes/metabolismo , Rim Cefálico/citologia , Fatores Imunológicos/farmacologia , Macrófagos/efeitos dos fármacos , Oligossacarídeos/farmacologia , Animais , Quitosana/química , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores Imunológicos/química , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Peso Molecular , Oligossacarídeos/química , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Transcriptoma , Proteína Supressora de Tumor p53/metabolismoRESUMO
The grass carp (Ctenopharyngodon idella), blunt snout bream (Megalobrama amblycephala) and yellow catfish (Pelteobagrus fulvidraco) are economically important fishes in China. Fish hematological features, especially the type and number of peripheral blood cells, are crucial for the evaluation of fish health and the diagnosis of fish diseases. Since the automatic blood cell count equipment for human is not suitable for fishes, the manual method is critical in the quantification of fish blood cells. To make sense of the comparison and interpretation of the blood cell count studies in different articles, the standardization of blood cell classification is necessary. In this study, erythrocytes (red blood cell, RBC), thrombocytes (TC) and leucocytes (i.e. white blood cells, WBC, including lymphocytes, neutrophils and monocytes) were well distinguished in blood smears with Giemsa staining and confirmed by transmission electron microscopy. RBC, TC and WBC were directly counted with an improved Neubauer counting chamber in a modified diluting solution. The differential leucocyte count (DLC) was carried out in blood smears. In view of the labeling characteristics of peroxidase (PO) positivity in neutrophils and non-specific esterase (α-ANAE) positivity in monocytes, PO positive cell percentage and α-ANAE positive cell percentage were also determined in cytochemistry staining smears. No difference was found for the percentages of neutrophils and monocytes between Giemsa staining and cytochemistry staining. The standardized classification, normal count ranges and sizes of the peripheral blood cells by the present systemic studies will provide useful references for monitoring the health status of grass carp, blunt snout bream and yellow catfish.
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Contagem de Células Sanguíneas/veterinária , Carpas/sangue , Peixes-Gato/sangue , Cyprinidae/sangue , Animais , Corantes Azur , Plaquetas/citologia , China , Eritrócitos/citologia , Leucócitos/citologia , Microscopia Eletrônica de Transmissão/veterinária , Valores de ReferênciaRESUMO
Siniperca chuatsi is an economically important fish in China, but infectious spleen and kidney necrosis virus (ISKNV) causes high mortality and significant economic losses. Currently, vaccination is the most promising strategy to prevent infectious diseases, while adjuvant can effectively enhance immune responses. In this study, inactivated ISKNV vaccine was prepared, then poly (I:C), chitosan, anisodamine and ims1312 were used as adjuvants to evaluate the effect on the immune responses and ISKNV replication. Chitosan could strongly boost the protection of liver and spleen tissues by pathological sections. In serum, poly (I:C) and chitosan group had protective effect on catalase, acid phosphatase, blood urea nitrogen. mRNA expressions showed these adjuvants induced the cytokines of early immune responses (TNF-α, Viperin) in both spleen and mesonephron by real time quantitative RT-PCR assays. Meanwhile, poly (I:C), chitosan and anisodamine were significantly improved the antiviral function and inhibited ISKNV replication. Chitosan and anisodamine played a significantly protective role in the immune protective rate test. The results indicated that all the four adjuvants are valid in the inactivated ISKNV vaccine, and chitosan is recommended preferentially. The present study provides reference for other animal vaccine adjuvants.
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Adjuvantes Imunológicos/administração & dosagem , Quitosana/imunologia , Iridoviridae/imunologia , Perciformes/imunologia , Alcaloides de Solanáceas/imunologia , Vacinas Virais/imunologia , Animais , Infecções por Vírus de DNA/imunologia , Enzimas/sangue , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Perfilação da Expressão Gênica/veterinária , Imunidade Inata/efeitos dos fármacos , Perciformes/genética , Poli I-C/imunologia , Replicação Viral/efeitos dos fármacosRESUMO
The yellow catfish (Pelteobagrus fulvidraco) is an economically important fish in China, but Edwardsiella ictaluri, an intracellular pathogenic bacterium, causes great losses to the culture industry. Currently, vaccination is the most promising strategy to combat the infectious diseases, while adjuvant can provide effective assistant for vaccines to enhance immune responses. In the present study, inactivated E. ictaluri vaccine was prepared, then Astragalus polysaccharides (APS), chitosan and poly(I:C) were employed as adjuvants to evaluate the effect on boosting immune responses and protecting yellow catfish against E. ictaluri. The survival rate was obviously improved after vaccination with APS, chitosan or poly(I:C) respectively, in addition, these three adjuvants could clearly protect the target tissue (intestine) by pathological sections in infectious experiments. In sera, total protein levels increased throughout the immunization stages, total superoxide dismutase levels continued to raise after vaccination, and lysozyme activity levels improved at different periods, examining by the commercial kits. Moreover, checking by real time quantitative RT-PCR assays, in both spleen and head kidney tissues which were the major immune organs, mRNA expressions of inflammatory cytokine IL-1ß increased in the early stage of immunity, typical Th1 immune response cytokines IL-2 and IFN-γ2 rose up in the whole immune period, and IgM significantly enhanced in the adjuvant supplementation groups. The results demonstrated the good efficiency of APS, chitosan or poly(I:C) as adjuvant, and provided more options for the fish adjuvants.
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Adjuvantes Imunológicos/farmacologia , Vacinas Bacterianas/imunologia , Peixes-Gato , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/prevenção & controle , Poli I-C/farmacologia , Polissacarídeos/farmacologia , Animais , Astrágalo/química , Quitosana/administração & dosagem , Quitosana/farmacologia , Edwardsiella ictaluri/efeitos dos fármacos , Infecções por Enterobacteriaceae/prevenção & controle , Poli I-C/administração & dosagem , Polissacarídeos/administração & dosagem , Polissacarídeos/química , Potência de Vacina , Vacinas de Produtos Inativados/imunologiaRESUMO
Complement system is an immemorial and pivotal element in innate immunity, protecting individuals from invading pathogens. Due to the emergence of whole genomes and functional researches, systematic identifications of complement system are feasible in many non-model species. In the present study, BLAST analysis was employed to systematically identify and characterize complement system in grass carp (Ctenopharyngodon idella). The results showed that C. idella complement system consists of 64 members, including the complement system pattern recognition, proteases, complement components, receptors and regulators. In which, most genes were well conserved with those in higher vertebrates over the course of evolution. Phylogenetic and syntenic analyses revealed their homologous relationships with other species. mRNA expression analyses of complement system related genes indicated that many members are sustainably expressed in multiple tissues before and after grass carp reovirus (GCRV) or Aeromonas hydrophila infection, which provide in vivo evidence for the response patterns of complement system after viral or bacterial infection. Meanwhile, this study also explored the evolution of complement system from ancestral protists to mammals and then investigated the changes in gene diversification during the evolution. These results will serve the comparative studies on the complement system in evolution and further functional investigations in C. idella.
Assuntos
Carpas , Proteínas do Sistema Complemento/metabolismo , Doenças dos Peixes/metabolismo , Proteínas de Peixes/metabolismo , Infecções por Bactérias Gram-Negativas/veterinária , Infecções por Reoviridae/veterinária , Aeromonas hydrophila , Animais , Proteínas do Sistema Complemento/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reoviridae , Infecções por Reoviridae/metabolismo , Infecções por Reoviridae/virologia , TranscriptomaRESUMO
TLR22 exists in nearly all the poikilothermic vertebrates and plays a central role in the initiation of innate immunity and activation of adaptive immunity. TLR22 signaling pathway has been characterized in detail in fugu (Takifugu rubripes). Here, we thoroughly remold the localization and signaling pathways of TLR22. We characterized TLR22a and TLR22b in grass carp (Ctenopharyngodon idella), designated as CiTLR22a and CiTLR22b, and explored the ligand(s), adaptor(s), and signaling pathway(s). Results show that both CiTLR22a and CiTLR22b localize to lysosome, acidic compartment. Correspondingly, CiTLR22a and CiTLR22b directly bind and respond to dsRNA analog poly(I:C) at pH 5, but not at pH 7.4, the physiological pH. Moreover, CiTLR22a and CiTLR22b exhibit antagonistic function in signal transmission, wherein CiTLR22a facilitates the protein and phosphorylation levels of IRF7 and enhances the promoter activities of major IFNs and NF-κBs, while CiTLR22b downregulates IRF7 phosphorylation and IRF3 protein level and suppresses the IFN and NF-κB pathways. Further investigations revealed that CiTLR22a restrains grass carp reovirus (GCRV) replication and protects cells from GCRV infection, whereas CiTLR22b plays a negative role in response to GCRV infection. This is the first time to systematically clarify the signaling pathways of two isotype TLR22s; especially, subcellular localization and adaptor are different from previous TLR22 report, which results from technical limitations. The results will serve the antiviral immune mechanisms in poikilothermic vertebrates and evolutionary immunology.
Assuntos
Doenças dos Peixes/metabolismo , Proteínas de Peixes/metabolismo , Infecções por Reoviridae/veterinária , Receptores Toll-Like/metabolismo , Animais , Carpas , Doenças dos Peixes/genética , Doenças dos Peixes/virologia , Proteínas de Peixes/genética , Transporte Proteico , Reoviridae/genética , Reoviridae/fisiologia , Infecções por Reoviridae/genética , Infecções por Reoviridae/metabolismo , Infecções por Reoviridae/virologia , Transdução de Sinais , Receptores Toll-Like/genéticaRESUMO
Macrophages are very versatile immune cells, with the characteristics of a proinflammatory phenotype in response to pathogen-associated molecular patterns. However, the specific activation marker genes of macrophages have not been systematically investigated in teleosts. In this work, leukocytes (WBC) were isolated using the Percoll gradient method. Macrophages were enriched by the adherent culture of WBC, then stimulated with lipopolysaccharide (LPS). Macrophages were identified by morphological features, functional activity and authorized cytokine expression. Subsequently, we collected samples, constructed and sequenced transcriptomic libraries including WBC, resting macrophage (Mø) and activated macrophage (M(LPS)) groups. We gained a total of 20.36 Gb of clean data including 149.24 million reads with an average length of 146 bp. Transcriptome analysis showed 708 differential genes between WBC and Mø, 83 differentially expressed genes between Mø and M(LPS). Combined with RT-qPCR, we proposed that four novel cell surface marker genes (CD22-like, CD63, CD48 and CD276) and two chemokines (CXCL-like and CCL39.3) would be emerging potential marker genes of macrophage in grass carp. Furthermore, CD69, CD180, CD27, XCL32a.2 and CXCL8a genes can be used as marker genes to confirm whether macrophages are activated. Transcriptome profiling reveals novel molecules associated with macrophages in C. Idella, which may represent a potential target for macrophages activation.
Assuntos
Carpas/genética , Perfilação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Ontologia Genética , Rim Cefálico/citologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Anotação de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Explosão Respiratória/efeitos dos fármacosRESUMO
The mannose receptor (MR) is a type I transmembrane protein. Its ectodomain has eight C-type lectin-like domains, which are able to recognize and mediate the phagocytosis of a wide range of pathogens. Comprehensive studies have revealed that mammalian MR is widely distributed in the mononuclear phagocyte system (MPS, previously known as the reticuloendothelial system) and play a key role both in the physiological clearance and cell activation. Hitherto, neither the MR distribution, nor the function of clearance and cell activation has been investigated in fish. In the previous study, we have reported the full-length cDNA of blunt snout bream MR, analyzed its structure and relative mRNA expression during embryogenesis and in the liver, head kidney, spleen and intestine of fish after stimulation with killed Aeromonas hydrophila. In the present study, we developed a rabbit polyclonal antibody against MR and undertook a systematic survey of the expression of MR at the protein level by immunohistochemistry. To get more information about MR function, the mRNA expression of MR, pro-inflammatory factor TNF-α and anti-inflammatory factor ARG2 genes was measured by qRT-PCR in the liver, head kidney, and spleen after A. hydrophila challenge. We first observed MR expression in the yolk sac at the fertilized egg stage and possibly MR was expressed by early macrophages. We also showed the MR distribution in head kidney, body kidney, spleen, liver, intestine, muscle, brain, heart, and gills. Following A. hydrophila challenge the MR immunoreactive cells became more widespread in head kidney and spleen, which are the major reticuloendothelial systems of fish. The quantitative studies at mRNA levels showed that there exists a high correlation between MR expression and immune cytokine expressions after bacteria challenge.