Identification of serum immunoglobulin M(IgM)of crucian carp(Carassius auratus gibelio)and immune response to cyprinid herpesvirus 2 infection

Crucian carp(Carassius auratus gibelio),an extensively cultivated freshwater fish,was one of the model species for the study of fish immunology.Polyclonal antibodies were advantageous molecular tools for studying teleost immune system.Specifically,polyclonal antibodies reacting with immunoglobulins(Ig)were used successfully in studies of the teleost fishes.In the present study,we produced polyclonal antibody against CH2 domains of crucian carp IgM,and measured the in vivo dynamics of IgM mRNA caused by CyHV-2 infection.The recombinant protein IgM with relative molecular weight about 53 KD was correctly expressed in prokaryotic cells.The specificity of the polyclonal antibody was evaluated by Western blotting and results revealed that the antibody not only specifically recognized crucian carp serum but also cross-reacted with grass carp serum.Quantitative RT-PCR analysis demonstrated the expression of IgM mRNA changed significantly after CyHV-2 infection.The expression of IgM in the kidney increased and reached a maximum at 6 h post-infection(hpi),while dropped to a low level at 5 days post-infection(dpi).In conclusion,the expression of IgM was significantly upregulated in the kidney of crucian carp infected with CyHV-2,indicating that IgM played a potential role in systemic immunity against viral infection.Polyclonal antibody against crucian carp IgM had certain clinical relevance,which might provide insight into the early stage of virus infection and prevention of the disease.

In vivo effects of neomycin sulfate on non-specific immunity,oxidative damage and replication of cyprinid herpesvirus 2 in crucian carp(Carassius auratus gibelio)

Neomycin belongs to the family of 2-deoxystreptamine-containing aminoglycoside antibiotics.It is widely used for bacterial infections,targeting most gram-negative and some gram-positive bacteria.Neomycin has also been reported to show antiviral activity.Here,we evaluated the toxicity of neomycin sulfate,and investigated its effect on non-specific immunity and viral infection in crucian carp(Carassius auratus gibelio).The safe concentration of neomycin sulfate for crucian carp was determined to be 102.9 mg/kg in vivo.In oxidative damage assays,neomycin sulfate increased superoxide dismutase and catalase activity and decreased malondialdehyde in the liver of crucian carp.In non-specific blood immune assays,the white blood cell count and complement 3 content significantly increased after neomycin sulfate treatment,while no significant changes were observed in antibacterial or lysozyme activity.In a challenge test,neomycin sulfate protected the crucian carp from cyprinid herpesvirus 2(CyHV-2)infection and inhibited CyHV-2 replication.In cytotoxicity assays,low concentrations of neomycin sulfate had no cytotoxicity on cells from the fins of crucian carp.The results of the present study indicate that oral administration of neomycin sulfate reduced oxidative damage,enhanced immunity and provided protection against CyHV-2 in crucian carp.

Astragalus polysaccharide orβ-glucan combined with inactivated vaccine markedly prevent CyHV-2 infection in Carassius auratus gibelio

Herpesviral hematopoietic necrosis disease caused by cyprinid herpesvirus 2(CyHV-2)results in huge economic losses for the gibel carp(Carassius auratus gibelio)aquaculture industry.A vaccination strategy is a feasible method to prevent CyHV-2 infection and the resulting economic losses.In this study,inactivators(includingβ-propiolactone,formaldehyde and binary ethylenimine)were used to prepare an inactivated vaccine.The optimal inactivated CyHV-2 vaccine(0.2%β-propiolactone inactivates CyHV-2 for 48 h)mixed withβ-glucan,anisodamine,chitosan,and astragalus polysaccharide(APS)adjuvants were injected into gibel carp.Theβ-glucan and APS combined with inactivated vaccine significantly improved the relative immune protection rate of gibel carp against CyHV-2 infection.The highest specific antibody levels inβ-glucan and APS groups were found in serum by ELISA assay and antibody neutralization titer.Furthermore,adjuvant addition groups produced better results for lysozyme,total superoxide dismutase,and complement C3 in serum.β-glucan and APS combined with vaccine treatment effectively enhanced mRNA expressions of important immune genes(IL-1β,IgM,IL-2 and IFN-γ2).Minimal tissue lesions(gill,spleen,and head kidney)and virus replication were seen in theβ-glucan and APS groups by histopathological examination and expression analysis of CyHV-2 TK gene.These results indicated that the combination of inactivated vaccine and adjuvantβ-glucan or APS is an effective method against CyHV-2 infection and provided a case study reference for prevention of fish viral diseases using inactivated vaccines.

Combination of iron flocculation and qPCR for quantitative evaluation of virus-shedding intensity of goldfish Carassius auratus infected with cyprinid herpesvirus 2 in the water and the effect of sodium chlorite powder in blocking waterborne horizontal viral transmission

Cyprinid herpesvirus 2(CyHV-2),a member of the Alloherpesviridae family belonging to the genus Cyprinivirus,was initially isolated from goldfish(Carassius auratus)and has been recently emerging as a virulent pathogen for cultured prussian carp(Carassius auratus gibelio)world-wide.In this study,a novel and effective method for concentration and quantification of live CyHV-2 virions from water was successfully established through coupling the iron flocculation with real time qPCR assay.Then,the shedding intensity of CyHV-2 in fish-tank water from artificially-challenged goldfish(25 fish/20 L)was monitored continuously for 7 days on a daily basis through quantitating viral genomic copy numbers by qPCR,and the maximum shedding level was determined to be 105 copies/L.Horizontal transmission research system was established by inoculating healthy goldfish in water spiked with serial dilution of CyHV-2 virions ranging from 107 to 103 copies/L.Our results indicated that water-borne CyHV-2 efficiently caused the infection of tested goldfish even in a concentration of 103 copies/L,and the overall transmission efficacy was not linearly correlated with the level of input virus in the fish tank.Commercial disinfectant Composite Sodium Chlorite Powder(CSCP)has been widely applied in aquaculture to control microbial infection through direct spill in the water,and its effect in inactivating the CyHV-2 infectivity remains unknown.We further determined that the EC50 of CSCP against 3.89 TCID50/mL CyHV-2 was close to 15.625μg/mL in vitro,and application of CSCP in a level as high as 60μg/mL(the safety concentration of CSCP for goldfish)couldn’t protect goldfish from CyHV-2 challenge through immersion.Thus,the disinfectant CSCP was regarded as none-effective for blocking CyHV-2 transmission in water during epidemic.Overall,our data provided quantitative data to demonstrate the shedding intensity of CyHV-2 in water,and CSCP was shown to be not effective in blocking water-borne horizontal transmission of CyHV-2 in goldfish.The virus-concentration protocol and virus-inhibition assay established here also paved the way for evaluating more commercial disinfectants in their effects in blocking water-borne horizontal transmission of CyHV-2.