Blunt snout bream (Megalobrama amblycephala) MaCSF-1 contributes to proliferation, phagocytosis and immunoregulation of macrophages via MaCSF-1R.

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.

Hematological and immune genes responses in yellow catfish (Pelteobagrus fulvidraco) with septicemia induced by Edwardsiella ictaluri.

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.

Administration of dietary recombinant hepcidin on grass carp (Ctenopharyngodon idella) against Flavobacterium columnare infection under cage aquaculture conditions.

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.

Hematological analysis of Ctenopharyngodon idella, Megalobrama amblycephala and Pelteobagrus fulvidraco: Morphology, ultrastructure, cytochemistry and quantification of peripheral blood cells.

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.

The systematic identification and mRNA expression profiles post viral or bacterial challenge of complement system in grass carp Ctenopharyngodon idella.

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.

Astragalus polysaccharides, chitosan and poly(I:C) obviously enhance inactivated Edwardsiella ictaluri vaccine potency in yellow catfish Pelteobagrus fulvidraco.

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.

Chitosan reduces the protective effects of IFN-γ2 on grass carp (Ctenopharyngodon idella) against Flavobacterium columnare infection due to excessive inflammation.

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.

Chitosan and anisodamine improve the immune efficacy of inactivated infectious spleen and kidney necrosis virus vaccine in Siniperca chuatsi.

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.

Chemotactic effect of ß-defensin 1 on macrophages in Megalobrama amblycephala.

Besides their function as a physical barrier against pathogens, ß-defensins possess the ability to induce direct or indirect chemotaxis in leukocytes of mammals. However little is known about the ability of defensins to guide the migration of macrophages in fish. The objective of our study was to investigate whether ß-defensin 1 (maBD1) can recruit leukocytes (specifically macrophages) in vivo and in vitro in a farmed cyprinid fish Megalobrama amblycephala. The M. amblycephala ß-defensin 1 (maBD1) gene was amplified from the head-kidney transcriptome. Synthetic maBD1 polypeptide (as well as its N-terminus half, but not the C-terminus half) was capable of inducing the migration of leukocytes (specifically macrophages) at concentrations from 26.0 µg/mL to 52.0 µg/mL in head kidney tissue in vitro. When injected intraperitoneally in vivo, the number of leukocytes in the peritoneal cavity was in positive correlation with the maBD1 concentration. maBD1 also induced the expression of two proinflammatory cytokines (IL-1beta and TNF-alpha) in spleen, head and body kidney, and hepatopancreas. These results strongly indicate that BD1 has a chemoattractant capacity for macrophages, as well as the ability to modulate the expression of proinflammatory cytokines in fish.

Distribution of mannose receptor in blunt snout bream (Megalobrama amblycephala) during the embryonic development and its immune response to the challenge of Aeromonas hydrophila.

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.

Hepcidin protects grass carp (Ctenopharyngodon idellus) against Flavobacterium columnare infection via regulating iron distribution and immune gene expression.

Columnaris disease (CD) caused by Flavobacterium columnare (F. columnare) is lack of knowledge on effective treatment measures. Bacterial pathogens require iron as an essential nutrient to infect the host. While hepcidin acts as a master regulator in iron metabolism, its contribution to host defense is emerging as complex and multifaceted. In vitro, recombinant Ctenopharyngodon idellus (C. idellus) hepcidin (CiHep) and synthetic CiHep both showed the ability to increase the expression of hepcidin and ferritin in C. idellus kidney cells, especially the recombinant CiHep. In vivo, recombinant CiHep improved the survival rate of C. idellus challenged with F. columnare. In addition, the fish fed diet containing recombinant CiHep (group H-1) had a higher survival rate than other pretreatment groups. The study showed that recombinant CiHep regulated iron metabolism causing iron redistribution, decreasing serum iron levels and increasing iron accumulation in the hepatopancreas. Moreover, the expression of iron-related genes was upregulated in various degrees at a different time except for group H-1. Immune-related genes were also evaluated, showing higher expression in the groups pretreated with CiHep at an early stage of infection. Of note, a clear upregulation of more immune genes occurred in the groups pretreated with recombinant CiHep than that pretreated with synthetic CiHep in the late stage of infection. In conclusion, the recombinant CiHep has a protective effect on the host response to bacterial pathogens. We speculate that hepcidin protects C. idellus against F. columnare infection via regulating the iron distribution and immune gene expression.

Transcriptome Analysis Provides Insights into the Markers of Resting and LPS-Activated Macrophages in Grass Carp (Ctenopharyngodon idella).

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.

Molecular cloning and functional characterization of cyclophilin A in yellow catfish (Pelteobagrus fulvidraco).

Cyclophilin A (CypA) is a ubiquitously expressed protein which involves in diverse pathological conditions including infection and inflammation. In this report, a CypA gene (designated as YC-CypA) was cloned from yellow catfish (Pelteobagrus fulvidraco) which is an important cultured fish species in Asian countries. The open reading frame (ORF) of YC-CypA encoded a polypeptide of 164 amino acids with calculated molecular weight of 17.70 kDa. The deduced amino acid sequences of the YC-CypA shared highly conserved structures with CypAs from the other species, indicating that YC-CypA should be a new member of the CypA family. Full-length YC-CypA protein was expressed in Escherichia coli and specific polyclonal antibody against YC-CypA was generated. The YC-CypA protein showed chemotactic activity by transwell migration assay. The mRNA and protein of YC-CypA could be detected in all examined tissues with relatively higher mRNA level in spleen and higher protein level in head kidney, respectively. The temporal expression patterns of YC-CypA, IL-1ß and TNF-α mRNAs were analyzed in the liver, spleen and head kidney post of Edwardsiella ictaluri infection. By immunohistochemistry assay, slight enhancement of YC-CypA protein was observed in the liver, spleen, body kidney and head kidney of yellow catfish infected with E. ictaluri. In conclusion, YC-CypA of yellow catfish showed chemotactic activity in vitro and might have been involved in cytokines secretion in yellow catfish during the infection of E. ictaluri.

Molecular cloning and expression analysis of mannose receptor in blunt snout bream (Megalobrama amblycephala).

Mannose receptor (MR) plays a significant role in innate immune responses to pathogens in vertebrates. Here we characterized the first teleost MR from Megalobrama amblycephala, named maMR and its expression patterns were investigated. The full-length maMR consists of 5,295 bp encoding a putative protein of 1,433 amino acids. The predicted amino acid sequences showed that maMR contained a signal peptide, a cysteine-rich domain, a single fibronectin type II domain, eight tandemly arranged C-type lectin-like domains, a transmembrane domain and a C-terminal cytoplasmic domain. Phylogenetic analysis revealed the highest similarity of maMR with Danio rerio MR predicted by computational analysis. The maMR-mRNAs were ubiquitously transcribed in different tissues, However the highest transcripts were observed in head kidney. Transcripts of maMR significantly increased at the late stages of embryo and continued to be at the high levels after hatching. The maMR transcripts were significantly increased in M. amblycephala after stimulation with killed Aeromonas hydrophila.

Identification of a gonad-expression differential gene insulin-like growth factor-1 receptor (Igf1r) in the swamp eel (Monopterus albus).

In vertebrate species, the biopotential embryonic gonad differentiation is affected by many key genes and key steroidogenic enzymes. Insulin-like growth factor-1 receptor (Igf1r) has been considered as an important sex-differentiation gene in mammals and could mediate the biological action of Igf1, an important regulator of key steroidogenic enzymes. However, Igf1r gene is still unknown in the swamp eel, an economically important fish. In our study, we identified Igf1r gene in the swamp eel, which was a 2,148-bp open-reading frame encoding a protein of 716 amino acids. The alignment and the phylogenetic tree showed that Igf1r of the swamp eel had a conservative sequence with other vertebrates, especial fishes. Western blotting of Igf1r showed that Igf1r expressed much more in ovotestis and testis than in ovary, indicating an important role of Igf1r during gonad differentiation. We analyzed ubiquitination of Igf1r by co-immunoprecipitation and found the amount of ubiquitinated Igf1r was increased from ovary, ovotestis to testis, which was reversely to the trend of Hsp10 expression during gonadal transformation. It was possible that Hsp10 could suppress Igf1r ubiquitination during gonadal development of the swamp eel.

IFN-γ enhances protective efficacy against Nocardia seriolae infection in largemouth bass (Micropterus salmoides).

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.

Hepcidin contributes to largemouth bass (Micropterus salmoides) against bacterial infections.

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.

Chitooligosaccharide boosts the immunity of immunosuppressed blunt snout bream against bacterial infections.

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.

In vivo studies on the immunotoxic effects of microcystins on rabbit.

Microcystins (MCs) are the toxic molecules produced by common cyanobacterium in freshwater blooms. Their toxicities raise severe health issues in livestock and human beings. In current study, the immunotoxic effects of MC-LR were investigated in rabbit through evaluating the dynamics of white blood cell (WBC) numbers and cytokine production such as interleukin-3 (IL-3), IL-4, IL-6, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α). MCs at the high dose (50 µg MC-LReq kg(-1) ) significantly induced increase in the WBC number but decrease in the Th1 (IFN-γ, TNF-α) and Th2 (IL-3, IL-4, IL-6) production. In the low dose group(12.5 µg MC-LReq kg(-1) ), the number of WBC and the production of IFN-γ, IFN-α, IL-4, IL-3, and IL-6 increased gradually in first 12 h, reach the peaks at 12 h, and dropped after 24 h. Significantly positive correlations were found between the cytokines production of IL-4 and IL-6, IFN-γ and IFN-α, or IL-4 and IFN-γ. In conclusion, MC-LR is able to disturb the rabbit immune system and there exists time-dose response relationship in the MC-LR-eliciting perturbation, which probably give a better insight into investigating the immunotoxicity mechanisms of MCs in vivo. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

Oral administration of hepcidin and chitosan benefits growth, immunity, and gut microbiota in grass carp (Ctenopharyngodon idella).

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.