ß-glucan and anisodamine can enhance the immersion immune efficacy of inactivated cyprinid herpesvirus 2 vaccine in Carassius auratus gibelio.

As one of the most important fish in freshwater aquaculture, gibel carp (Carassius auratus gibelio) is easily susceptible to Cyprinid herpesvirus 2 (CyHV-2). Immersion vaccination has attracted many researchers due to its simple operation in preventing infectious diseases. However, the unavoidable disadvantage is that the immersion vaccine must be used with adjuvants to get a better performance. In this study, gibel carps were vaccinated by a 60 min bath in a ß-propiolactone-inactivated Cyprinid herpesvirus 2, mixed with DTT, ß-glucan, anisodamine and scopolamine, respectively. After immunization, the fishs were challenged by CyHV-2 in 2 weeks. By analyzing pathological section, we found that ß-glucan, anisodamine and scopolamine groups protected the gibel carp compared to the control group, which was consistent with the trend of survival rate. Specifically, ß-glucan group in serum appeared best on lysozyme, TSOD and complement C3. Real time quantitative RT-PCR results demonstrated that in both spleen and head kidney tissues, mRNA expressions of typical Th1 immune response cytokines IL-2 and IFN-γ2 in ß-glucan group and anisodamine group were significantly higher than other groups and the level of immunoglobulins related to systemic immunity (IgM) and mucosal immunity (IgZ) were also enhanced in the immune period. DTT group slightly affected immune gene and serum enzyme activity, while did not show an adjuvant effect on survival rate. In addition, four adjuvant groups could obviously inhibit CyHV-2 replication. This study explored and proved the good efficiency of ß-glucan or anisodamine as immersion immune adjuvant and also provided reference for improving the efficiency of immersion immunity.

Dietary Glycyrrhiza uralensis extracts supplementation elevated growth performance, immune responses and disease resistance against Flavobacterium columnare in yellow catfish (Pelteobagrus fulvidraco).

The present study was conducted to evaluate the influence of Glycyrrhiza uralensis (G. uralensis) extracts on the growth performance, histological structure, immune response and disease resistance against Flavobacterium columnare (F. columnare) of yellow catfish. Fish were fed with two different diets, i.e., basal diet as control group (CG) and diet containing G. uralensis extracts as experimental group (GG). After 60 days feeding, growth performance of GG fish was significantly improved, with increased WG and SGR but decreased FCR compared to CG fish. Fish were then challenged with F. columnare for two times, as fish showed rare mortality after the first infection. GG fish showed significantly lower cumulative mortality during F. cloumnare infection than CG fish after 21 days infection (dpi). Epithelial cell exfoliation and obvious cellular vacuolization in the skin and congestion of gill lamellae were detected in CG fish, while GG fish showed increased width of epidermis and mucous cells number in skin, and increased length of secondary lamina in gill. GG fish also exhibited higher enzyme activity of lysozyme in serum and mRNA expression of lysozyme in head kidney than CG fish at most time points post infection. G. uralensis extracts supplementation also induced earlier serum anti-oxidative responses, with increased superoxide dismutase activity and total antioxidant capacity in GG fish at 1 dpi. Compared to CG fish, GG fish showed increased expression level of genes involved in TLRs-NFκB signaling (TLR2, TLR3, TLR5, TLR9, Myd88, and p65NFκB), resulting in higher expression levels of pro-inflammatory cytokines (IL-1ß and IL-8) in the head kidney post infection. However, these genes showed deviation in the gill of GG fish, which increased at some time points but decreased at other time points. Moreover, G. uralensis extracts supplementation also significantly unregulated the expression levels of IgM and IgD in head kidney, and the expression levels of IgM in the gill of yellow catfish, suggesting the elevated humoral immune response during F. columnare infection. All these results contributed to the elevated disease resistance ability against F. cloumnare infection of yellow catfish after dietary G. uralensis extracts supplementation.

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.

IgM and IgD heavy chains of yellow catfish (Pelteobagrus fulvidraco): Molecular cloning, characterization and expression analysis in response to bacterial infection.

Three different immunoglobulin (Ig) isotypes, namely IgM, IgD, and IgT/IgZ have been described in most teleost, among which IgM and IgT are considered crucial in systematic and mucosal immunity, respectively. However, some teleost have no IgT/IgZ and it is unclear how other Ig isotypes interact to perform immune-protective roles in both systematic and mucosal sites. In this study, the complete cDNA sequences of IgM and IgD heavy chains were cloned and analyzed from yellow catfish (Pelteobagrus fulvidraco). The full-length cDNA of Pf-IgM and Pf-IgD heavy chains contained an open reading frame (ORF) of 1710 and 2991 bp encoding a predicted protein of 570 and 997 amino acids, respectively. Tissue-specific expression analysis indicated that both IgM and IgD were highly expressed in kidney and spleen, and higher expression levels were found at zygote and 13th day post hatching during early development. Multiple sequence alignment and phylogenetic analysis showed IgM and IgD of yellow catfish are closely related to other fish of Siluriformes. Moreover, we also constructed the infection model of yellow catfish with bacteria (Flavobacterium columnare G4) for the first time to study the function of Pf-IgM and Pf-IgD heavy chain genes in immune response. Quantitative real-time PCR (qRT-PCR) showed that significantly up-regulated expression of Pf-IgM was not only detected in liver and spleen, but also in mucosal tissues including skin and intestine, while Pf-IgD was just significantly increased in liver and spleen, which might suggest the main immune-protecting roles of IgM in mucosal tissues of yellow catfish.

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.

Molecular characterization and expression analysis of T cell receptor (TCR) γ and δ genes in dojo loach (Misgurnus anguillicaudatus) in response to bacterial, parasitic and fungal challenge.

In mammalian, T-cell receptors (TCRs) play a key role in recognizing the presented antigen from external to protect organisms against environmental pathogens. To understand the potential roles of TCRγ and TCRδ in dojo loach (Misgurnus anguillicaudatus), Ma-TCRγ and Ma-TCRδ cDNAs were cloned and their gene expression profiles were investigated after bacterial, parasitic and fungal challenge. The open reading frame (ORF) of Ma-TCRγ and Ma-TCRδ cDNAs contained 948 and 867 bp, encoding 316 and 288 amino acid residues, respectively. Structurally, Ma-TCRγ and Ma-TCRδ were consisted of a signal peptide, a variable region, a constant region (IgC), a connecting peptide (CPS), a transmembrane region (TM) and a cytoplasmic domain (CYT), which were similar to those of other vertebrates. Multiple sequence alignment and phylogenetic analysis showed Ma-TCRγ and Ma-TCRδ were closely related to fish of Cyprinidae family. Ma-TCRγ and Ma-TCRδ were widely expressed in all tested organs/tissues, as the highest expressions of Ma-TCRγ and Ma-TCRδ were detected in kidney and gill, respectively. In addition, three infection models of dojo loach with bacteria (F. columnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia sp.) were constructed. The morphological changes of gills and skin after challenged with F. columnare G4 and Ichthyophthirius multifiliis were investigated. Compared to F. columnare G4 infection, mRNA expression of both TCRγ and TCRδ showed higher sensitivity in classical immune organs (kidney and spleen) and mucosal tissues (skin and gill) after challenge with Ichthyophthirius multifiliis and Saprolegnia sp. Our results first indicated that TCRγ and TCRδ of dojo loach might function differently in response to challenge with different pathogens.

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.

Transcriptomic analysis of Mandarin fish brain cells infected with infectious spleen and kidney necrosis virus with an emphasis on retinoic acid-inducible gene 1-like receptors and apoptosis pathways.

Infectious spleen and kidney necrosis virus (ISKNV) has caused significant economic losses in the cultured Mandarin fish (Siniperca chuatsi) industry. The molecular mechanisms that underlie the pathogenesis of the viral infection remain poorly understood. In this study, deep RNA sequencing technique was used to analyze the transcriptomic profiles of Mandarin fish brain cells (CPB) at progressive time points after ISKNV infection. A total of 96,206,040 clean data from 98,235,240 sequence reads were obtained. These raw data were assembled into 66,787 unigenes. Among these unigenes, 33,225 and 29,210 had significant hit the Nr and SwissProt databases where they matched 27,537and 19,638 unique protein accessions, respectively. In the samples harvested at 24 or 72 h post of the infection, a total of 10,834 or 7584 genes were differentially expressed in infected CPB cells compared to non-infected cells, including 5445 or 3766 up-regulated genes and 5389 or 3818 down-regulated genes, respectively. In addition, 12 differentially expressed genes (DEGs) were validated by quantitative PCR. These DEGs were involved in many pathways of viral pathogenesis. Further analysis of the major DEGs genes involved in the RLRs and apoptosis pathways revealed some interesting findings. In the RLRs pathway, ISKNV infection inhibited the activation of NF-κB via over expression of the IKKB-α and IKKB-ß and lessened expression of interleukin-1 receptor-associated kinase 4 (IRAK4). In the apoptosis pathway, ISKNV infection could induce apoptosis mainly via tumor necrosis factor (TNF) mediated extrinsic pathway. The cellular apoptosis induced by ISKNV infection was confirmed using annexinV-FITC/PI and DAPI staining methods.

Analysis of the transcriptomic profilings of Mandarin fish (Siniperca chuatsi) infected with Flavobacterium columnare with an emphasis on immune responses.

Flavobacterium columnare (FC) is the causative pathogen of columnaris which has caused great economic loss in fish culture worldwide, including in Mandarin fish (Siniperca chuatsi) culture. In the present study, the transcriptomic profiles of the head kidneys from FC-infected and non-infected Mandarin fish were obtained using HiSeq™ 2000 (Illumina). Totally 31,168 unigenes with high quality were obtained. Genes involved in protein folding, metabolism and energy, immune responses, oxidoreductase activity, cell growth and death were identified as enriched classes. 1019 differently expressed genes between the two groups were identified, including 603 up-regulated and 416 down-regulated genes. 27 differently expressed immune related genes were scrutinized, including 17 up-regulated and 10 down-regulated genes. Six of the differently expressed genes were further validated by qRT-PCR. The roles of the immune related genes were discussed. Identification of the host genes in response to FC infection will shed a new light on the prevention of columnaris.

Abortive infection of snakehead fish vesiculovirus in ZF4 cells was associated with the RLRs pathway activation by viral replicative intermediates.

Snakehead fish vesiculovirus (SHVV) is a negative strand RNA virus which can cause great economic losses in fish culture. To facilitate the study of SHVV-host interactions, the susceptibility of zebrafish embryonic fibroblast cell line (ZF4) to the SHVV was investigated in this report. The results showed that high amount of viral mRNAs and cRNAs were detected at the 3 h post-infection. However, the expressions of the viral mRNAs and cRNA were decreased dramatically after 6 h post-infection. In addition, the expressions of interferon (IFN) and interferon-induced GTP-binding protein Mx were all up regulated significantly at the late stage of the infection. Meanwhile, the expressions of Retinoic acid-inducible gene I (RIG-I) and Melanoma differentiation-associated gene 5 (MDA5) were also all up-regulated significantly during the infection. Two isoforms of DrLGP2 from zebrafish were also cloned and analyzed. Interestingly, the expression of DrLGP2a but not DrLGP2b was significantly up-regulated at both mRNA and protein levels, indicating that the two DrLGP2 isoforms might play different roles during the SHVV infection. Transfection experiment showed that viral replicative intermediates were required for the activation of IFN-α expression. Taken together, the abortive infection of SHVV in ZF4 cells was associated with the activation of RLRs pathway, which was activated by viral replicative intermediates.

Gut mucosal immune responses and protective efficacy of oral yeast Cyprinid herpesvirus 2 (CyHV-2) vaccine in Carassius auratus gibelio.

Cyprinid herpesvirus 2 (CyHV-2) causes herpesviral hematopoietic necrosis (HVHN) disease outbreaks in farmed Cyprinid fish, which leads to serious economic losses worldwide. Although oral vaccination is considered the most suitable strategy for preventing infectious diseases in farmed fish, so far there is no commercial oral vaccine available for controlling HVNN in gibel carp (C. auratus gibelio). In the present study, we developed for the first time an oral vaccine against CyHV-2 by using yeast cell surface display technology and then investigated the effect of this vaccine in gibel carp. Furthermore, the protective efficacy was evaluated by comparing the immune response of a single vaccination with that of a booster vaccination (booster-vaccinated once 2 weeks after the initial vaccination). Critically, the activities of immune-related enzymes and genes expression in vaccine group, especially in the booster vaccine group, were higher than those in the control group. Moreover, strong innate and adaptive immune responses could be elicited in both mucosal and systemic tissues after receipt of the oral yeast vaccine. To further understand the protective efficacy of this vaccine in gibel carp, we successfully developed the challenge model with CyHV-2. Our results showed the relative percent survival was 66.7% in the booster vaccine group, indicating this oral yeast vaccine is a promising vaccine for controlling CyHV-2 disease in gibel carp aquaculture.

Oral Administration of Bacillus subtilis Subunit Vaccine Significantly Enhances the Immune Protection of Grass Carp against GCRV-II Infection.

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.

Hepcidin Protects Yellow Catfish (Pelteobagrus fulvidraco) against Aeromonas veronii-Induced Ascites Disease by Regulating Iron Metabolism.

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.

The Combination of Molecular Adjuvant CCL35.2 and DNA Vaccine Significantly Enhances the Immune Protection of Carassius auratus gibelio against CyHV-2 Infection.

Cyprinid herpesvirus 2 (CyHV-2) infection results in huge economic losses in gibel carp (Carassius auratus gibelio) industry. In this study, we first constructed recombinant plasmids pcORF25 and pcCCL35.2 as DNA vaccine and molecular adjuvant against CyHV-2, respectively, and confirmed that both recombinant plasmids could be effectively expressed in vitro and in vivo. Then, the vaccination and infection experiments (n = 50) were set as seven groups. The survival rate (70%) in ORF25/CCL35.2 group was highest. The highest specific antibody levels were found in ORF25/CCL35.2 group in major immune tissues by qRT-PCR, and confirmed in serum by ELISA assay, antibody neutralization titer, and serum incubation-infection experiments. Three crucial innate immune indices, namely C3 content, lysozyme, and total superoxide dismutase (TSOD) activities, were highest in ORF25/CCL35.2 group in serum. pcORF25/pcCCL35.2 can effectively up-regulate mRNA expressions of some important immune genes (IL-1ß, IL-2, IFN-γ2, and viperin), and significantly suppress CyHV-2 replication in head kidney and spleen tissues. The minimal tissue lesions can be seen in ORF25/CCL35.2 group in gill, spleen, and trunk kidney tissues by histopathological examination. The results indicated that the combination of DNA vaccine pcORF25 and molecular adjuvant pcCCL35.2 is an effective method against CyHV-2 infection, suggesting a feasible strategy for the control of fish viral diseases.